Matrix protein from influenza A virus and its role in cross-protection in mice. The matrix (M) protein of influenza A virus, one of the two group-specific internal proteins of the virion, was isolated in a pure form, and its immunogenicity was stable to heating at 100 degrees C for 2 min. Mice immunized with isolated M protein in complete Freund adjuvant and subsequently infected with influenza virus cleared virus more rapidly from their lungs than did unimmunized mice. Despite the rapid clearance of virus, the mice developed pneumonia that was at least as severe as in unimmunized mice. Preliminary studies suggest that the rapid clearance of influenza virus from the lungs of mice immunized with M protein may be initiated by a cell-mediated rather than a humoral response. The mechanism by which a cross-reactive internal virion protein can initiate clearance of the different subtypes of influenza is not clear. Perhaps the M protein is exposed on the surface of the virus-infected cell and is responsible for the cross-reactivity at the cytotoxic T-cell level recently detected between influenza A virus subtypes.


Processing of streptococcal cell walls by rat macrophages and human monocytes in vitro. Phagocytosis and degradation of cell walls by peritoneal macrophages obtained from Fischer 344 or Buffalo rats was measured in tissue culture. Group A cell wall antigen, detected by immunofluorescence, persisted in cultured rat macrophages for at least 40 days, whereas group D cell wall material was eliminated by 6 to 8 days. This same pattern of persistence of group A cell walls and elimination of group D cell walls was observed in cultures of human monocytes followed for 24 days in culture. Group A streptococcal cell walls labeled with either [14C]alanine or [14C]glucose were handled in a similar manner by macrophages from either Fischer 344 or Buffalo rats. In contrast, [14C]glucose-labeled group D cell walls were degraded at a much faster rate. Buffalo macrophages were more efficient than Fischer 344 macrophages in degrading group D cell walls. The inability of macrophages to degrade group A cell walls was not due to a failure of lysosomes to fuse with phagosomes. Neither serum lysozyme in the culture medium nor cell wall-associated autolysin contributed to the degradation of group D cell walls by macrophages. Neither immune serum nor macrophages obtained from specifically immunized rats influenced phagocytosis or persistence of group A cell walls.


Cytotoxicity of rat macrophages activated by persistent or biodegradable bacterial cell walls. Peritoneal macrophages from Fischer 344 rats were cytotoxic for mouse fibroblasts after in vitro phagocytosis of non-biodegradable group A streptococcal cell walls. In contrast, macrophages from Buffalo rats were not activated to cytotoxicity by bacterial cell walls. Activation to cytotoxicity was only achieved by the macrophages after an interval of at least 3 days in culture after ingestion of group A cell walls. If macrophages were cultured only 24 h after ingestion of cell walls, a cytostatic effect on target cells was observed. Phagocytosis of biodegradable group D streptococcal cell walls did not activate macrophages from either rat strain to become cytotoxic. No direct toxicity of cell walls for macrophages or mouse L-cells was observed. The cytotoxicity of Fischer 344 macrophages appears to require direct cell contact with target cells.


Rickettsial hemolysis: adsorption, desorption, readsorption, and hemagglutination. The energy-dependent adsorption of radioiodinated rickettsiae to sheep erythrocytes was demonstrated. The iodination procedure, however, decreased the hemolytic activity of the rickettsiae. No desorption of rickettsiae from isolated rickettsia-erythrocyte complexes (prevented from lysing by NaF) could be measured. On the other hand, rickettsiae desorbed from this complex during or after lysis and readsorbed and lysed other erythrocytes. Thus, the usual hemolytic assay measures multiple rounds of adsorption and lysis. Although lysis of the rickettsia-erythrocyte complex was insensitive to anti-rickettsial rabbit serum, adsorption and readsorption were completely inhibited by such antiserum. Hemagglutination of erythrocytes by rickettsiae was observed (in the presence of NaF to prevent lysis) and was sensitive to the same inhibitors as adsorption.


Host defenses in experimental scrub typhus: role of spleen and peritoneal exudate lymphocytes in cellular immunity. Lymphocytes obtained from spleens or peritoneal exudates of immune donor mice were evaluated for their ability to passively confer protection on recipients subsequently challenged with virulent scrub typhus rickettsiae. Peritoneal exudate lymphocytes (PELs) injected intraperitoneally were able to transfer complete protection against rickettsial challenge by 5 days after immunization, whereas splenic lymphocytes (SpL's) required 15 days to exhibit similar resistance. When immune lymphocytes were transferred intravenously, cells from both anatomical compartments required 15 days after immunization before they were able to completely protect recipients. PELs maintained this protective capacity for 2 weeks, but the passive immunity induced by intravenously transferred SpL's rapidly diminished to insignificant levels. It was particularly interesting that the protective effect of SpL's could be dramatically reduced by the concomitant presence of a mineral oil-induced peritoneal exudate. Almost total abrogation of resistance was observed when SpL's obtained from exudate-bearing mice were transferred intravenously. The protective capacity of both PELs and SpL's was resistant to 1,200 rads of gamma radiation at 7 to 10 days after immunization, but resistance was transient and by 3 weeks was undetectable. It was not possible to determine from this study whether the transferred lymphocytes were proximate mediators of protection in scrub typhus infection of mice or whether they served to recruit the host's own defenses, or both. However, it was possible to conclude that PEL's and SpLs exhibited functional heterogeneity and that PELs were more efficient mediators of protection.


Virulence and immunogenicity of a temperature-sensitive dengue-2 virus in lower primates. Clones of dengue-2 virus were tested for virulence by inoculation of rhesus monkeys and chimpanzees. Although primates showed no overt signs of illness, inoculation with the parent virus or a subline of a large-plaque clone resulted in a viremia lasting 1 to 7 days. By these criteria, sublines of a small-plaque clone were significantly less virulent and produced little or no viremia in primate hosts. Although they had a substantially reduced viremia, primates inoculated with the small-plaque sublines showed stimulation of complement-fixing, hemagglutination-inhibiting, and neutralizing antibodies. The protection afforded rhesus monkeys 3 months after inoculation with two of the small-plaque sublines was demonstrated by a lack of viremia and a failure to escalate preexisting antibody levels after challenge with the parent virus. Both the S-1 subline and the parent virus had a limited capacity to produce central nervous system pathology in monkeys inoculated intrathalamically and intrathecally. Evidence thus far accumulated for primates indicates that the S-1 subline of dengue-2 virus has potential value as a candidate vaccine virus.


Impairment of cell-mediated immune responses by infection with Mycobacterium lepraemurium. The effect of chronic infection with Mycobacterium lepraemurium upon cell-mediated immune responses was studied in Lewis rats. Rats infected for 40 to 175 days were completely protected from attempted induction of experimental adjuvant disease, and the severity of experimental allergic encephalomyelitis in leprous rats was markedly attenuated. Full manifestations of each autoimmune disease were expressed in littermate control groups. Skin homograft rejection by infected rats was significantly impaired (P less than 0.001) as was the delayed-type hypersensitivity response to sheep erythrocytes (P less than 0.02). It is suggested that chronic infection with M. lepraemurium exerts a nonspecific inhibitory effect on cell-mediated immunity by perturbation of normal lymphocyte recirculation and by induction of immuno-suppressor cell activity.


Genetic studies on Neisseria gonorrhoeae from disseminated gonococcal infections. Isolates from uncomplicated and disseminated gonococcal infections were analyzed by using deoxyribonucleic acid-mediated transformation. Most pairs of auxotrophs could recombine, producing independent transformants. When the constellation of arginine (Arg), hypoxanthine (Hyx), and uracil (Ura) requirements was present in donor and recipient, no recombination for these traits could be detected. Except for Arg to Hyx, no linkage between Arg, Hyx, Ura, penicillin G sensitivity, and serum resistance could be demonstrated. Some distant linkage of Ura to nalidixic acid and rifampin resistances was found. The data show that the traits associated with disseminated gonococcal infection strains are not closely linked but are identical in all strains, indicating a common origin.


Isolation from corynebacterium diphtheriae C7(beta) of bacterial mutants that produce toxin in medium with excess iron. Five mutants that produce toxin in medium with excess iron were isolated from strain C7(beta). The iron content of bacteria grown on this medium was considerably higher than that of C7(beta) cells grown in medium containing the minimum amount of iron needed to inhibit toxin production. When the nonlysogenic, nontoxinogenic strain C7(-) was lysogenized with phages from each of the mutants, toxin production by all of the resulting lysogens, like that by parent strain C7(beta), ceased upon iron addition. When the mutants were superinfected with beta45 phage, both toxin and CRM45 were produced in medium with excess iron. One of the mutant strains lost its prophage as a result of treatment with ultraviolet light. When the cured strain was lysogenized with phage carrying a mutation in the tox structural gene, the lysogen produced the mutant protein at the maximum rate in medium with excess iron. These findings show that the mutant strains are not phage mutants, but are bacterial host mutants, and that a host factor(s) is involved in the inhibition of toxin production by iron.


Purification of Neisseria gonorrhoeae surface L-antigen. A purified preparation of the L-antigen of Neisseria gonorrhoeae with a 40-fold increase of antigenic activity over the crude extract was prepared. The antigen was extracted from the cell envelope by mild treatment with sodium dodecyl sulfate and purified by Sepharose 4B, diethylaminoethyl-cellulose, and diethyl-(2-hydroxypropyl)aminoethyl (QAE)-Sephadex column chromatography. The purified antigen was heat labile and trypsin sensitive. The smallest antigenically active subunit appeared to have a molecular weight of 38,500, as shown by polyacrylamide gel electrophoresis of the product incubated with sodium dodecyl sulfate.


Binding of sodium [195Au]aurothiomalate to Mycoplasma arthritidis. The binding of sodium [195Au]aurothiomalate (ATM) to whole cells of Mycoplasma arthritidis has been measured within the temperature range of 4 to 53 degrees C. The time course (kinetics) and the effect of varying the total concentration of free ATM in the suspension at 37 degrees C were also measured. The extent of binding at 37 degrees C leveled off after 30 min at approximately 65 nCi of [195Au]ATM per mg of membrane protein. The amount of ATM bound per cell appeared to be directly proportional to the concentration of free ATM in the range of 0.25 to 0.60 muCi of [195Au]ATM per ml. An Arrhenius plot showed two distinct regions with slopes of -0.56 degrees K/mg of protein (5 to 22 degrees C) and -2.78 degrees K/mg of protein (22 to 53 degrees C). The break in the Arrhenius plot corresponds to a temperature known to be related to a sudden change in mycoplasma membrane fluidity. Estimations of Scatchard binding constants and number of binding sites per membrane protein molecule resulted in 0.8 to 1.4 sites per molecule in the intact organisms compared to 1.4 to 1.8 sites in membrane fragments. The latter were purified by repeated washings in phosphate-buffered saline after alkaline lysis of the cells. It is suggested that ATM reacts with available protein sulfhydryl groups in the mycoplasmal membrane.


Detection and differentiation of iron-responsive avirulent mutants on Congo red agar. Agar medium containing Congo red dye differentiates virulent and avirulent colonies of Shigella, Vibrio cholerae, Escherichia coli, and Neisseria meningitidis. Like virulent plague bacilli, wild-type cells of these species absorb the dye and produce red colonies. Mutants or colonial variants have been isolated that fail to absorb the dye and produce colorless colonies. These mutants exhibit reduced virulence in the chicken embryo model, but their virulence is enhanced by supplementation with iron. Of those species tested, only Neisseria gonorrhoeae isolates failed to grow in the presence of this dye. Inhibition of growth by Congo red may thus provide a simple means for differentiating gonococci from other Neisseria.


Photosensitivity of DNA-bound 7,12-dimethylbenz(alpha)anthracene. Structural information about the products formed when 7,12-dimethylbenz(alpha)anthracene (DMBA) is bound to DNA in mammalian cell cultures has been sought through studies of the photosensitivities of these products and of various model compounds. Under conditions of light exposure in which the DNA-DMBA products were highly photosensitive, 8,9,10,11-tetrahydro-DMBA and 5,6-dihydro-DMBA were stable, whereas 9,10-dimethyl-anthracene and DMBA itself were highly photosensitive. This indicates that in the binding reaction with DNA, DMBA retains either the aromatic benz(alpha)anthracene nucleus or is metabolically activated in the 1,2,3,4-ring.


Biologic and antigenic characteristics of Epstein-Barr virus-related Herpesviruses of chimpanzees and baboons. Leukocyte-transforming agents were isolated in baboon leukocytes inoculated with oral excretions from immunosuppressed chimpanzees. The transformed lymphoblasts had B cell surface markers and harbored herpes-type virus particles; 5-10% of the cells contained cytoplasmic antigens reactive with Epstein-Barr virus (EBV)-antibody-positive chimpanzee, human and baboon sera. These sera also neutralized the transforming activity of the chimpanzee virus. Long-term lymphoid cell lines were established from circulating lymphocytes of normal baboons: two from Papio cynocephalus and three from P. hamadryas. The cells had B cell surface markers, contained herpes-type virus particles and produced virus with leukocyte-transforming activity. No virus-associated nuclear antigen was detectable with reference baboon and chimpanzee sera; however, the cells reacted with selected human sera containing antibodies to EBV nuclear antigen (EBNA). Absorption experiments confirmed the specificity of this reaction. Baboon lymphoblasts produced baboon virus-associated soluble complement-fixing (CF/S) antigen. Baboon sera had CF antibodies to viral (CF/V) antigen derived from EBV but failed to react with EBV-associated CF/S antigen. Chimpanzee and baboon herpesviruses had similar in vitro host cell ranges but were different from those of EBV. Inoculation of baboons, rhesus monkeys and cottontop marmosets failed to produce detectable illness or palpable tumors.


Reflection of light by small areas of the ocular fundus. Light reflected from 50 micrometer spots on the fundi of two rhesus monkeys and three human volunteers was measured as a function of wavelength in 10 nm. jumps from 400 to 900 nm. The areas measured were a retinal artery and vein, areas of the disk, macula, and retina devoid of visible blood vessels, and the fovea.


Effect of oral estramustine phosphate on pituitary, gonadal, and adrenal function in the green monkey (Cercopithecus aethiops sabaeus). We studied the effects of estramustine phosphate on pituitary, gonadal, and adrenal function in the green monkey. A 2-week course of estramustine phosphate (75 mg per day) reversibly suppressed the luteinizing hormone and testosterone response to luteinizing hormone-releasing hormone but did not affect pituitary-adrenal function assessed by the 11-deoxycorticosterone response to metyrapone. Treatment with oral estramustine phosphate also resulted in a significant increase in corticosteroid-binding globulin but did not affect a series of liver enzymes nor hematology. The results are consistent with a pure estrogen effect produced by hydrolysis of the carbamate-ester bona at a site distant from estrogen target tissues and could explain most of the reported in vivo effects of this compound.


A study of alleged leprosy bacillus strain HI-75. Subcultures of strain HI-75 of Skinsnes leprosy bacillus received in Antwerp and London have been studied bacteriologically and compared. Both contained moderately large acid-fast bacilli readily subcultured and maintained on ordinary mycobacteriologic media. These organisms were found to be a variety of Mycobacterium marianum (syn. serofulaceum) and were considered likely to be a laboratory contaminant. The earlier subculture studied also contained numbers of a much smaller mycobacterium (of a similar size to M. leprae) which appeared to be dead and which did not grow on the ordinary media. Skin tests and immunodiffusion analyses performed with extracts of the earlier subculture failed to demonstrate the presence of the specific antigens of leprosy bacilli. Similar studies on other cultures of Skinsnes bacillus must be performed to confirm or refute its identity as M. leprae.


Compartmentation of enzymes in the rabbit parotid salivary gland. A study by enzyme histochemical, tissue fractionation and morphometric techniques. Rabbit parotid gland was chosen as a suitable model tissue for studying the role of membranes in enzyme secretion by acinar cells. The study was initiated using subcellular fractionation techniques. During these experiments the effects of various tissue disruption conditions such as the medium and the duration and vigour of homogenization were explored and the results assessed by enzyme and chemical assays and both quantitatively and qualitatively by electron microscopy. A series of basic fractions was isolated and marker enzyme or chemical assays selected for each of the relevant membrane types (rough endoplasmic reticulum, Golgi apparatus, zymogen granule, plasmalemma). A parallel study was effected using enzyme histochemical methods applied to frozen sections. Interesting comparisons could then be made between histochemical and biochemical methods of enzyme demonstration. These comparisons are discussed. The basic fractions provide the material from which specimens of purified membranes of the four species can be obtained. The isolation and characterization of such purified membranes is the subject of another report.


Localization of neurotensin-immunoreactive cells in the small intestine of man and various mammals. Antibodies against synthetic bovine neurotensin were raised in rabbits and used to demonstrate neurotensin-immunreactive cells by immunohistochemical methods. In the jejunum and ileum of all species investigated (man, dog, monkey, cat, rabbit, sheep, rat, mouse, hamster, chinese hamster, gerbil, pig and guinea pig) cells were present in the mucosa, which reacted specifically with antineurotensin serum using the indirect immunofluorescence and peroxidase-antiperoxidase methods. In the monkey Tupaia the distribution of neurotensin-immunoreactive cells was examined by investigating serial sections through  the entire gastro-entero-pancreatic (GEP) endocrine system, again showing most neurotensin-immunoreactive cells in the jejunum and ileum. The functional role of the presence of neurotensin immunoreactivity in the gut is discussed.


[Inhalation therapy in respiratory diseases (author's transl)]. The various techniques for aerosol therapy are described, as applied to inflammations of the upper and lower airways. Advances in treatment include dinatrium cromoglicicum and beclomethasone-dipropionate, with the latter medication improving results in patients with allergic reactions of the nasal and bronchial mucosa.


Practical methods of determining serum immunoglobulin M and immunoglobulin G concentrations in foals. Serum concentrations of immunoglobulin M (IgM) and immunoglobulin G (IgG) can be determined in the horse with a satisfactory degree of accuracy, using commercially available reagents. Selected lots of anti-human IgM can be used in precipitation tests to detect and quantitate equine IgM. Commercially available anti-equine IgG tended to overestimate the amount of IgG in single radial immunodiffusion tests. Even with these limitations, commercial reagents can be used to differentiate immunodeficiency disorders of foals, including combined immunodeficiency and failure of passive transfer of colostral antibody from mare to foal.


Ribosomal ribonucleic acids from Streptomyces griseus. Nucleic acids from Streptomyces griseus 178 were isolated during cultivation. After their fractionation on a column of methylated serum albumin adsorbed on Kieselguhr, the 16 S and 23 S RNA were isolated. To characterize RNAs their sedimentation coefficients, Tm and nucleotide composition were determined. During cultivation of S. griseus 178 rRNA level reaches two maximum peaks and the production of streptomycin influences nucleic aicds of the producer organism.


Renal vascular response to heat stress in baboons--role of renin-angiotensin. To determine if hyperthermia in the baboon caused a reduction of renal blood flow (RBF) similar to that reported in man, we repeatedly exposed six unanesthetized male baboons (Papio cynocephalus) to ambient temperatures of 42.5-49.0 degrees C for 55-175 min. Internal temperatures rose 1.0-2.0 degrees C. On the average, RBF fell 23.7% per degrees C, renal vascular resistance (RVR) rose 34.0% per degrees C, and mean arterial pressure (MAP) fell by only 2.9 Torr. Plasma renin activity (PRA), measured in four baboons, rose 97.5% per degrees C. To investigate the role of the renin-angiotensin system in this renal response, we infused propranolol or saralasin (1-sar-8-ala-angiotensin II), an angiotensin II antagonist, systemically in 14 experiments on three baboons. Both propranolol and saralasin infusions prevented most of the reduction in RBF during hyperthermia. Propranolol prevented the increase in PRA. We conclude that renal vasoconstriction accompanies moderate hyperthermia in the awake baboon, and much of this response is mediated by a beta-adrenergic release of renin.


Structure-function relationships in the interaction of alpha-thrombin with blood platelets. Highly purified alpha-thrombin has been chemically modified in an attempt to determine which features of the molecule are important for normal platelet-thrombin interactions. Modifying agents included diisopropylphosphorofluoridate and 1-chloro-3-tosylamido-7-amino-L-2-heptanone, which modify serine and histidine, respectively, at the catalytic site, as well as N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide, which modify a single tryptophan at or near the fibrinogen-binding site. Active site-directed modification did not appreciably affect the binding characteristics, but prevented platelet activation. In contrast, modification of tryptophan at the macromolecular substrate-binding site resulted in the loss of high affinity binding of thrombin to platelets, while low affinity binding was apparently unaffected. This modification altered but did not abolish the ability of thrombin to effect platelet aggregation and release of [14C]serotonin. These results suggest that residues at the catalytic site are not involved in binding and that the macromolecular substrate-binding site of alpha-thrombin participates in high affinity binding to platelets. These data are also consistent with the existence of at least two types of binding sites for thrombin on the platelet surface as well as more than one platelet-binding region on the thrombin molecule.


Retention of amylase in the secretory granules of parotid gland after extensive release of Ca++ by ionophore A-23187. The effect of the ionophore A-23187 was tested on isolated secretory granules of rat parotid gland. The ionophore caused extensive release of calcium from the granules without effecting release of amylase or other secretory proteins. It is therefore concluded that the role of calcium in the granules is probably not that of a stabilizing agent.


Membrane interactions between adjacent mucols secretion granules. In primate goblet cells, the membranes of adjacent mucous granules from contact areas which appear as extensive pentalaminar fusion sites in thin sections. In freeze-fracture replicas, the same membrane areas are smooth, except for a few 6-8-nm particles which adhere to the E face. These protein-poor membrane interaction sites are relatively long-lived, and it is proposed that further stimulus may be required to trigger membrane fission.


Effect of insulin on ultrastructure and glycogenesis in primary cultures of adult rat hepatocytes. Insulin in the presence of high concentrations of glucose has a beneficial trophic effect on the development of primary cultures of hepatocytes. Compared to the situation observed in hormone-free control cultures, the flattening of the reaggregated hepatocytes is enhanced, and the reconstituted cell trabeculae are enlarged and tend to form a confluent monolayer after 3 days; the survival time is prolonged from 3 to 5 or 6 days. Ultrastructural modifications are also initiated by insulin; numerous glycogen particles appear after 24 h, in between the cisternae of the proliferated smooth endoplasmic reticulum. After 48 h, large amounts of glycogen are stored, and numerous polysomes are present. A small number of cells showed an increased synthesis of lipid droplets in the lumen of the smooth endoplasmic reticulum and form liposomes at the same time. After 72 h, cytolysomes filled with glycogen develop, simulating glycogenosis type II. Simultaneously, microtubules and microfilaments, closely related to numerous polysomes, appear in cytoplasmic extensions constituting undulating membranes. The biochemical data demonstrate that, in the absence of insulin, a high concentration of glucose stimulates glycogenesis and hinders glycogenolysis. This effect of glucose on polysaccharide synthesis is progressively lost. The addition of insulin to the culture induces after 48 and 72 h, a three- to fivefold increase of the glucose incorporation into glycogen, as compared to the controls. The presence of insulin is required to maintain the hepatocyte's capacity to store glycogen. Glycogen synthetase is converted into its active form under the influence of glucose. Insulin increases the rate of activation.


Diminished prolactin reserve: a case report. A 17 year old male patient presented with short stature and delayed puberty. Investigations showed normal thyroid function and intact TSH response to TSH-releasing hormone (TRH). Although basal levels of LH were low, both LH and FSH rose following the administration of LH-releasing hormone (LHRH). ACTH secretion assessed indirectly by the cortisol response to insulin hypoglycemia was normal. Growth hormone levels increased following the onset of sleep, as well as after the administration of insulin, L-dopa and L-arginine. Basal levels of prolactin were low (2-5 ng/ml) compared with 5-12 ng/ml in controls. There was a markedly impaired prolactin response to TRH (maximum rise above basal values of 3 ng/ml compared to a rise of 12-29 ng/ml in controls). Prolactin levels did not rise after the administration of chlorpromazine or L-arginine. There was some suppression of prolactin levels after L-dopa. Similar patterns of prolactin are seen in panhypopituitarism, where they are usually associated with other hypophyseal hormonal deficiencies. The diminished prolactin reserve demonstrated in this subject in the presence of intact function of the remainder of the anterior pituitary is compatible with the diagnosis of diminished prolactin reserve.


Elevated basal growth hormone levels and growth hormone response to TRH in alcoholic patients with cirrhosis. Basal plasma growth hormone (GH) and GH responses after the administration of thyrotropin releasing hormone (TRH) were studied in 9 male alcoholic patients with cirrhosis. Basal GH was significantly higher in cirrhotic patients than in normal men. Intravenous injection of synthetic TRH (400 microgram) caused a significant increase in plasma GH in 7 out of the 9 cirrhotic patients examined, while it did not increase GH levels in normal subjects.


Rapid method for auxotyping multiple strains of Neisseria gonorrhoeae. A rapid method for auxotyping strains was developed that uses microtiter plates. This miniplate technique enables rapid identification of major auxotypes present in clinical strains. Additional growth requirements can be identified by adding individual amino acid supplements to complete gonococcal genetic medium. Analysis of 8 clones from 40 patients revealed that 10 had more than 1 auxotype. Deoxyribonucleic acid-mediated transformation can be used to establish whether the strains with apparently more than one auxotype are defective in the same locus in each of the involved biosynthetic pathways. Selection of more than one clone is required in precise epidemiological studies.


Imferon agar: improved medium for isolation of pathogenic Neisseria. Imferon, an iron-dextran complex, enhances the growth of Neisseria gonorrhoeae and N. meningitidis. The use of Imferon as a replacement for ferric nitrate, in a defined supplement for GC agar significantly increased the average colony sizes of both gonococci and meningococci. In comparison with Thayer-Martin medium, Imferon agar increased the speed and rate of isolation of gonococci from clinical specimens.


Radiometric detection of carbohydrate catabolism by pathogenic Neisseria. A liquid scintillation procedure for the catabolism of D-[1-14C]glucose and [U-14C]maltose by pathogenic Neisseria was tested. Definitive results were obtained within a 30-min incubation period.


Hereditary C7 deficiency. Diagnosis and HLA studies in a French-Canadian family. The serum of a 44-yr-old woman of French-Canadian descent having a B-27 positive ankylosing spondylitis was deficient in the seventh component of complement (C7) as determined by hemolytic and immunochemical methods. No inhibitor against C7 was detected, and the levels of all other complement components were normal. No deficiency in the opsonic activity of the serum was found, and the results of basic coagulation studies of the plasma were normal. On investigation of the patient's family, two sisters were found to have the same deficiency but were otherwise in good health. The seven other siblings were heterozygous for C7 deficiency, while the paternal aunt had a normal C7 level. In the third generation, six children of the three homozygous sisters and five children of heterozygotes were available for testing. Studies of the HLA antigens in all the 22 subjects and in three spouses indicated no close linkage between the CM deficienty and the HLA system. In addition, the simultaneous occurrence of two hereditary complement deficiencies (C2 and C7) was discovered in one family of this remarkable kindred.


Characterization of gonococcal antigens responsible for induction of bactericidal antibody in disseminated infection. The role of gonococcal antigens in serum bactericidal activity was determined for an isolate of Neisseria gonorrhoeae from a patient with disseminated gonococcal infection (DGI). Gonococcal outer membranes were purified by differential ultracentrifugation of sheared organisms treated with EDTA. The membranes were solubilized in an endotoxin-disaggregating buffer, and the proteins were separated from the endotoxin by molecular sieve chromatography. Chemical characterization of the endotoxin from the DGI strain revealed the presence of heptose (7.8% of carbohydrate composition) and 2-keto-3-deoxyoctonate (6.1%, wt/wt) in concentrations similar to rough endotoxins of gram-negative enteric bacteria. Dermal Schwartzman reactions were positive for this endotoxin (4 mug) and the corresponding outer membrane (139 mug), but negative for the protein fraction (>500 mug). The patient's whole serum or the IgG fraction, each with complement, reduced the number of the infecting organisms by greater than 1 log(10) in a bactericidal assay. Outer membrane and its protein and endotoxin fractions (0.8-500 mug) from the DGI strain were separately preincubated with the patient's convalescent serum and specific inhibition of bactericidal activity occurred with the endotoxin fraction (25 mug) and the outer membrane (100 mug); the protein (500 mug) exhibited no inhibition. Similar results were obtained by inhibiting the bactericidal activity of rabbit antiserum, prepared by intravenous inoculation of an isolate from a patient with pelvic inflammatory disease, with antigen purified from that strain. That this was specific immune inhibition and not anticomplementary activity was shown by the failure of these antigens to inhibit other complement-dependent bactericidal systems.


Glucagon release from rat pancreatic islets. A combined morphological and functional approach. The release of glucagon induced in isolated rat islets by arginine or by calcium deprivation has been subjected to combined functional and morphological quantifications. Arginine-stimulated glucagon release was associated with a significant increase of morphological events linked to exocytosis. By contrast, the paradoxical events linked to exocytosis. By contrast, the paradoxical release of glucagon provoked by calcium deprivation, although accompanied by a significant loss of granule stores, was not associated with an increase of morphologically detectable exocytosis.


The effect of thrombocytopenia on experimental arteriosclerotic lesion formation in rabbits. Smooth muscle cell proliferation and re-endothelialization. This study was designed to investigate the mechanisms involved in fibromusculoelastic lesion formation produced by selective de-endothelialization by the intra-arterial balloon catheter technique in thrombocytopenic rabbits. Thrombocytopenia was induced and maintained for up to 30 days by daily injections fo highly specific sheep anti-rabbit platelet sera (APS). Evidence for re-endothelialization was obtained by i.v. Evans blue dye 30 min before sacrifice. Rabbits received daily injections of APS, which reduced the mean platelet count to 5,600/cm3; control animals received identically treated normal sheep sera on the same schedule, and had mean daily platelet counts of 363,000/cm3. Evaluation of intimal thickness was assessed by counting cell layers in semithin sections. Intimal thickening in aortae from rabbits treated with APS was strikingly suppressed, in contrast to those from normal sheep sera-treated animals which showed a mean intimal thickness of 18 cell layers within 28 days often after de-endothelialization. Re-endothelialization was not affected by APS treatment. These results indicate that the proliferation of smooth muscle cells is dramatically inhibited by reduction of platelets.


Unconjugated bilirubin and an increased proportion of bilirubin monoconjugates in the bile of patients with Gilbert's syndrome and Crigler-Najjar disease. Bilirubin pigments were studied in the bile of 20 normal adults, 25 patients with Gilbert's syndrome, 9 children with Crigler-Najjar disease, and 6 patients with hemolysis, to determine how a deficiency of hepatic bilirubin UDP-glucuronosyltransferase would affect the end products of bilirubin biotransformation. In the bile from patients with Gilbert's syndrome, a striking increase was found in the proportion of bilirubin monoconjugates (48.6+/-9.8% of total conjugates) relative to that in normal bile (27.2+/-7.8%). This increase was even more pronounced in children with Crigler-Najjar disease, in whom, even in the most severe cases, glucuronide could always be demonstrated in the bile. Furthermore, unconjugated bilirubin-IXalpha was unquestionably present in the bile of these children and amounted to 30-57% of their total bilirubin pigments (<1% in the controls). It was not possible to predict from the biliary bilirubin composition whether a child would respond to phenobarbital therapy or not. Bile composition was normal in patients with hemolysis, except when there was associated deficiency of hepatic glucuronosyltransferase. Therefore, the observed alterations were not a simple consequence of unconjugated hyperbilirubinemia. The present findings suggest that Crigler-Najjar disease represents a more pronounced expression than Gilbert's syndrome of a common biochemical defect. Hepatic bilirubin UDP-glucuronosyltransferase deficiency leads to decreased formation of diconjugates with an ensuing increase in the proportion of bilirubin monoconjugates in bile; in the most severe cases, an elevated content of biliary unconjugated bilirubin is also found.


Complementary laminar terminations of afferents to area 17 originating in area 18 and in the lateral geniculate nucleus in squirrel monkey. The projections from area 18 and the lateral geniculate nucleus onto area 17 of the squirrel monkey (Saimiri) were investigated with retrograde (horseradish peroxidase) and anterograde (tritiated proline) labelling techniques, and the (Fink-Heimer) silver impregnation method for degenerating axons and their terminals. The association fibers from area 18 terminated in all layers of area 17 except in layer IV and in the lower aspect of layer IIIc. The greatest number of terminals were in layers I, V and VI. The bulk of geniculocortical fibers terminated in layer IV and the lower aspect of layer IIIc; a minority of the geniculocortical fibers terminated in layer VI and the lower aspect of layer IIIb. Thus, the majority of fibers from the two sources investigated terminate in a complementary laminar fashion in area 17. The portion of area 17 on the lateral surface of the hemisphere, where the central visual field is represented, received a less dense projection from the geniculate nucleus than the striate cortex in the calcarine fissure, where the peripheral visual field is represented. Ocular dominance columns were not apparent in the striate cortex. No evidence was found that the lateral geniculate nucleus projects to area 18. The results of combined injections of horseradish peroxidase and tritiated proline in area 17 indicated a point-to-point reciprocity between area 17 and the ipsilateral lateral geniculate nucleus.


Antiplaque potential of topical stannous fluoride. Sterile enamel samples treated by topical application of 10% stannous fluoride were incubated with S mutans and S sanguis respectively and with a mixed culture to which L casei had been added. The results were compared with sterile nonfluoridated enamel samples subjected to the same procedures. The parameters compared were postincubation pH values, bacterial concentrations on the surfaces, and enamel surface alterations. Stannous fluoride treated samples consistently yielded higher pH readings than nonfluoridated samples; the concentration of microorganisms adhering to the surfaces was conspicuously reduced on the pretreated enamel when compared by scanning electron microscopy to untreated samples, as were enamel surface alterations. These findings lend support to other recent investigations which have shown that fluorides act in several ways to inhibit cariogenesis in addition to reducing hydroxyapatite solubility.


Evaluation of intracytoplasmic particles in histiocytes after endodontic therapy with a hydrophilic plastic. The presence of granules within the cytoplasm of periapical tissue histiocytes had been observed in teeth which had a root canal overfilled with a hydrophilic plastic polymer. SEM examinations revealed these granules to be barium sulfate, which was added to the polymer for radiopacity. X-ray spectra revealed strong calcium peaks in bone adjacent to the histiocytes, which showed strong barium peaks. In addition, between the histiocytes and the bone a greater concentration of calcium was seen. Because the histiocytes and not the plastic polymer contained barium sulfate, the evidence for this material's biocompatibility is greatly enhanced.


Effectiveness of glutaraldehyde  as a chemosterilizer used in a wrapping technique on simulated metal instruments. Chemosterilization utilizing glutaraldehyde-moistened gauze as a wrap on simulated metal instruments was evaluated. Non-sporeforming test organisms were killed within five minutes. Two sporeforming test organisms were killed in six hours. The sporicidal effect obtained would indicate that glutaraldehyde used in a wrap technique could sterilize objects.


Indium-111-labeled autologous leukocytes in man. Autologous leukocytes have been isolated, labeled with indium-111, and administered to 15 patients suspected of inflammatory disease. The stability of the label has been demonstrated and the in vivo kinetics and distribution of the labeled cells studied. The distribution is influenced by the type and viability of the cells separated by three different techniques. Generally, there was initial accumulation of radioactivity in the lungs; approximately half of this cleared in 15 min and the remainder slowly. Twenty-five to 50 percent of the radioactivity subsequently distributed in the spleen, liver, and bone marrow, and these did not show significant change with time up to 48 hr post injection. The In-111 radioactivity administered as labeled leukocytes free from erythrocytes cleared from the circulating blood with a half-time of 7.5 hr. In three of 15 patients, the suspicion of inflammatory disease could not be confirmed, and in these a normal distribution of radioactivity was observed. In the remaining 12 patients, focal accumulation of radioactivity was detectable within 4 to 24 hr after administration, and subsequent confirmation of sepsis was obtained. From three such patients, samples of abscesses were recovered which showed markedly higher radioactivity than that in the same weight of blood.


Indium-111-labeled cellular blood components: mechanism of labeling and intracellular location in human neutrophils. Human neutrophils were labeled with In-111 oxine by incubation at room temperature, and the fate of the oxine and the intracellular location of the In-111 were determined. Neutral (1:3) In-111 oxine complex diffuses rapidly across the cell membrane and then dissociates. Some of the oxine leaves the cell and the In-111 binds intracellularly. After short periods of incubation the label is distributed mostly to four soluble components, a small proportion of which had a distribution similar to that of specific and azurophil granules. After longer incubation periods there was relatively less radioactivity with the soluble components and probably more attached to the particulate material. A small peak of radioactivity also appeared in the region of DNA distribution, but no confirmation could be obtained for the association of the In-111 radioactivity with DNA.


A quantitative evaluation of rheumatoid arthritic activity with Tc-99m HEDP. In an attempt to develop a quantitative method of evaluating rheumatoid arthritic activity, Tc-99m HEDP joint uptake values and joint-to-bone ratios were studied in ten adult rheumatoid arthritic patients and 17 nonarthritic patients. A joint-to-bone activity ratio of 1.8 at the fourth hour after injection (RA Index) discriminated clinically active rheumatoid arthritic joints from control joints with 95% accuracy. Serial studies on five patients during drug trial demonstrated a positive correlation between RA Index and the clinical manifestations of rheumatoid arthritic activity. The RA Index may be a useful quantitative parameter for evaluation of rheumatoid arthritic activity following therapy.


Lenticular complications of long-term steroid therapy in children with asthma and eczema. Forty-two chronic asthmatic children, including 10 with associated eczema, were examined for lenticular complications. The children had received oral corticosteroids for a minimum of 3 years. Twelve were treated with beclomethasone inhalant therapy, and the children with eczema had had topical corticosteroids. Only one child was found to have cataracts commensurate with corticosteroid therapy. Possible reasons for the low incidence of cataracts in this study are discussed.


Variability of extracts used in immunoprecipitin tests. Immunodiffusion reactions of several different commercial fungal extracts from organisms were compared to those obtained from patients' environments. Sera were obtained from patients suspected of having hypersensitivity lung disease (HLD). There was a difference in reactivity among the sources of the antigen and in some lots of antigen from the same commercial supplier. The results demonstrate a wide variation in the reactivity of different antigenic extracts and emphasize the need for uniform standardization of preparations used in precipitin tests.


Incidence of IgG short-term sensitizing antibodies in an allergic population. Heat-stable, immunoglobulin G, short-term sensitizing antibodies (IgG S-T S) were sought in serum from 149 allergic patients who had strongly positive immediate skin tests to inhalant allergens. The sera were tested by passive cutaneous anaphylaxis (PCA) in monkeys. No IgG S-T S antibodies were demonstrated in 169 tests with a variety of allergens. Antibody with the characteristics of IgE was demonstrated in 47% of monkey PCA tests and, in an additional 34% of sera. IgE antibody to the same allergen was demonstrated by radioallergosorbent testing (RAST).


Failure of anti-gamma 3 antibodies to detect some gamma3 chains by an agglutination inhibition test. Four of eight anti-gamma3 antibodies that detect gamma3 by double diffusion were found to test for Gm allotypes when they were used in the agglutination inhibition test. One tested for Gm (26), two for Gm (11), and one for both allotypes. So far as the present data show, the remaining four test for a gamma3 isotype in the agglutination inhibition test. We suggest that a sample that is negative by the agglutination inhibition test should be tested by double diffusion before it is concluded that the sample lacks gamma3.


Platelet immuno-assay (PIA) as a simple and sensitive technique for the detection of antigens. The platelet immuno-assay (PIA) is a simple and inexpensive technique to detect antigens with ng/ml sensitivity using micro-titer equipment and commercially available antisera. It can be easily adapted for virtually any antigen to which rabbit antiserum containing specific IgG-antibodies cna be obtained. The assay is inhibited by IgG and C1q in amounts greater than or equal to 125 microgram/ml and 12.5 microgram/ml respectively. Its application is thus limited to those fluids where IgG and C1q are present in less than these concentrations.


[Grippe and pregnancy. Relation of birth weight to placenta weight]. The different results that have been published concerning the problem of the final outcome for patients who have had influenza during pregnancy reported in numerous works might be explained by differences in methodology used by the different authors. A study was carried out in the Haguenau Maternity Hospital (Bas-Rhin) when an epidemic of influenza occurred in 1972-173. The virological diagnosis was made by carrying out separate serological estimations on 1940 pregnant women. It can be shown that the mean birthweight dropped in infants of mothers who had contracted influenza during pregnancy even when corrections were made for sex and the duration of the pregnancy. The drop in weight of the placenta (37.3 g as a mean) was more obvious and can totally explain the drop in fetal weight. It was not possible to demonstrate any increase in the number of congenital malformations that were found in the neonatal period in the infants born to women who had had influenza. These results suggest that there is no direct passage of the influenza virus across the placenta which, however, is itself modified by the infection.


In vivo chemotaxis induced by polyunsaturated fatty acids. Intradermal injection of as little as 500 ng of arachidonic acid or the metabolites from arachidonic acid incubated with soybean lipoxygenase produced infiltration of the upper dermis by polymorphonuclear leukocytes 18 hours after injection. In experiments comparing the chemotactic properties of four fatty acids in varying concentrations, oxidative products of arachidonic acid by soybean lipoxygenase were the most powerful followed by free arachidonic acid and free linoleic acid while stearic acid did not produce significant infiltration. These findings suggest that the elevated levels of free arachidonic acid and an arachidonic acid metabolite (12L-hydroxy-5, 8, 10, 14-eicosatetraenoic acid), recently found in psoriatic epidermis, may at least in part be attracting polymorphonuclear leukocytes into psoriatic skin.


Immunization with Shigella dysenteriae type 1: evaluation of antitoxic immunity in prevention of experimental disease in rhesus monkeys (Macaca mulatta). The role of serum antitoxic antibody in protection against the dysentery caused by Shigella dysenteriae type 1 (Shiga's bacillus) was studied in monkeys fed 10(10) virulent organisms after parenteral immunization with a formalin-inactivated Shiga toxoid preparation standardized in mice. Two 125-microgram doses of toxoid adsorbed on aluminum hydroxide adjuvant and given 14 days apart provided mice with a 54-fold increase in resistance to parenteral toxin. In rhesus monkeys (Macaca mulatta), the same regimen of toxoid permitted the safe parenteral administration of toxin in incremental doses ranging from 100 to 1,000 mouse 50% lethal doses and resulted in correspondingly high titers of antitoxin in serum. Nevertheless, the immunized monkeys responded to orally administered Shiga bacilli by development of diarrhea and dysentery that was as severe as (or more severe than) the response of unimmunized controls. The failure of extraordinarily high levels of circulating antibody to protect against experimental shigellosis suggests that the intestinal mucosal sites of toxinmediated response are beyond the reach of systemic antitoxin.


Rapid bioassay for clindamycin alone and in the presence of aminoglycoside antibiotics. A rapid bioassay for determination of concentrations of clindamycin in serum was developed with use of a strain of Lancefield group B Streptococcus (Streptococcus agalactiae) that is uniformly resistant to aminoglycoside antibiotics, tetracycline, and polymyxin. An agar diffusion assay system was used that included the addition of patient's sera and three standard concentrations of clindamycin to 5-mm wells cut in the seeded agar. Pretreatment of serum with penicillinase allowed measurement of clindamycin in the presence of penicillins and cephalosporins by the same assay method. Assays of clindamycin in serum using this system could be read routinely in as little as 4 hr and allowed determination of levels of drug in serum of 2.5-40 microgram/ml. Linear regression analyses indicated that values obtained by this assay compared favorably with the results obtained with use of Bacillus subtilis strain ATCC 6633 or Sarcina lutea strain ATCC 9341 (American Type Culture Collection, Rockville, Md.). Repetitive measurement of sera with known concentrations of clindamycin indicated the average deviation to be +/- 10%. Seeded bioassay plates could be prepared in advance and stored at 2 C-8 C for up to one week before use. This assay may also be used for measurement of lincomycin, erythromycin, vancomycin, and certain beta-lactam antibiotics in the presence of aminoglycosides.


Host-parasite relationships in experimental airborne tuberculosis. VI. Influence of vaccination with Bacille Calmette-Guérin on the onset and/or extent of hematogenous dissemination of virulent Mycobacterium tuberculosis to the lungs. Guinea pigs vaccinated with bacille Calmette-Guérin (BCG) and unvaccinated guinea pigs were challenged by the respiratory route six weeks or six months after vaccination and sacrificed at various intervals after challenge. The six lobes of the lung were cultured separately, and the percentage of culture-positive lobes was calculated, as well as the log10 number of virulent bacilli recovered. The latter was subjected to an analysis of variance, which compared the fate of bacilli in the four largest lobes with the fate of those in the two smallest lobes. The results indicated no difference between the six-week and six-month intervals between vaccination and challenge. In the longer intervals between challenge and sacrifice, small numbers of secondary lesions could be seen on the lobes of the BCG-vaccinated animals. It was concluded that vaccination with BCG retarded the onset and/or reduced the extent of hematogenous dissemination of virulent mycobacteria to the lungs.


Patterns and mechanisms of emergence of resistance to amikacin. Emergence of gram-negative bacilli resistant to amikacin occurred in five of 96 patients treated. Three of the five instances were associated with clinical failure and arose during therapy for an infection caused by a pathogen also susceptible to gentamicin. The other two episodes were associated with colonization. The enzyme aminoglycoside-6'-acetyltransferase, which inactivates amikacin, was found in Serratia marcescens. Decreased permeability to amikacin was shown in four isolates of Pseudomonas aeruginosa. The emergence and existence of these organisms is of great epidemiologic importance. Judicious use of amikacin and adherence to general principles of infection control are advised.


Rifampin in the treatment of experimental brucellosis in mice and guinea pigs. Rifampin, a broad-spectrum antibiotic able to penetrate intracellularly, was used for treatment of infections with Brucella melitensis in mice and Brucella abortus in guinea pigs. Treatments were administered for seven, 14, or 21 days; mice were given 25 mg of rifampin/kg per day, and guinea pigs 100 mg/kg per day. Efficacy of the drug was determined by comparison of rifampin-treated animals with saline-treated controls and with tetracycline-treated mice (200 mg/kg per day) according to the following criteria: (1) primary infections of the spleen and (in guinea pigs) of the lymph nodes; (2) residual infections of the spleen, i.e., infections shown after complementary treatment with suspensions of killed Corynebacterium parvum or with cortisone; (3) splenomegaly; and (4) serological response (in guinea pigs). Treatment with rifampin, even for one or two weeks, drastically reduced the number of infections by all of these criteris, and treatment for three weeks cured nearly all mice; the incidences of primary and residual infections in rifampin-treated mice after three weeks were 0 and 8.5%, respectively, as compared with 70.3% and 73.5%, respectively, in tetracycline-treated mice. Of 25 guinea pigs treated with rifampin for three weeks, spleen infection was shown in one, and lymph node infections in 10.


Kinetics of phagocytosis and bacterial killing by human polymorphonuclear leukocytes and monocytes. The kinetics of phagocytosis and bacterial killing by normal human polymorphonuclear leukocytes (PMNLs) and by monocytes (MNs) were compared by use of [3H]thymidine-labeled Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes. The rate of phagocytosis by PMNLs was approximately twice that by MNs for all three bacterial species. Although a marked difference was found in opsonic requirements for phagocytosis of S. aureus, E. coli, and L. monocytogenes, phagocytosis by PMNLs and MNs was mediated via the same serum factors. All three species were killed rapidly once they were associated with leukocytes; however, the rate of killing by MNs was slower than that of PMNLs. The slower rate of killing appeared to be secondary to slower ingestion of attached bacteria by MNs. Thus, PMNLs and MNs appear to possess receptors with specificity for the same bacterial opsonins; however, PMNLs are capable of more efficienct bacterial phagocytosis (attachment and ingestion) than are MNs.


Experimental studies on the pathogenesis of infections due to Pseudomonas aeruginosa: direct evidence for toxin production during Pseudomonas infection of burned skin tissues. Direct evidence is presented for the production of an exotoxin by Pseudomonas aeruginosa multiplying at the burned site in an infected mouse. Pseudomonas toxin was assayed by measurement of its ability to catalyze the transfer of radioactivity from [14C]adenine-labeled nicotinamide adenine dinucleotide to elongation factor 2 (adenosine diphosphate ribosylation activity). This enzyme activity was found in saline extracts of burned infected skin but was not present in similar extracts of burned uninfected skin. It was detected in the serum of infected animals by 26 hr after infection. The level of active elongation factor 2 in the livers of infected mice was reduced significantly after infection. These data suggest that pseudomonas exotoxin, produced by bacteria multiplying at the burn site, enters the circulation and is disseminated to different organs where it acts by depletion of elongation factor 2 and thus causes a reduction in protein synthesis.


Application of the indirect enzyme-labeled antibody microtest to the detection and surveillance of animal diseases. The rapid, indirect enzyme-labeled antibody (ELA) microplate test has been developed as a diagnostic and surveillance tool to aid in the control of animal disease. The test has been applied to viral (hog cholera), parasitic (trichinosis), and bacterial (brucellosis) diseases of animals. A correlation of greater than 95% was observed between the hog cholera ELA test and the serum neutralization test for hog cholera in greater than 2,000 field samples obtained during the 1976 epizootic in New Jersey. Serum samples from all of 56 swine naturally infected with Trichinella spiralis at a level considered dangerous to humans were ELA-positive, whereas only one of 360 packinghouse sera negative for T. spiralis was ELA-positive. Preliminary experiments with bovine brucellosis (Brucella abortus) indicate that the ELA test is more sensitive than other test methods currently in use. ELA procedures should soon become tests of choice for the detection of antibodies to animal disease agents.


The enzyme-linked immunosorbent assay and its application to parasitic infections. The enzyme-linked immunosorbent assay (ELISA) was used for the serodiagnosis of four parasitic infections. ELISA was performed as a macromethod in polystyrene tubes and/or as a micromethod in polystyrene microplates. Conventionally raised pigs were infected orally with different numbers of Trichinealla spiralis larvae, and the pigs' sera were tested for antibody to T. spiralis. Results were compared with those from sera of uninfected conventionally raised pigs. Cynomolgus monkeys were infected orally and immunized with Toxocara canis. The results of macro-ELISA and micro-ELISA were compared with those of immunofluorescence. Micro-ELISA proved to be more sensitive than macro-ELISA, the latter being more sensitive than immunofluorescence. The applicability of ELISA for seroepidemiology was demonstrated by results obtained with sera from patients with proved African trypanosomiasis. With both macro-ELISA and micro-ELISA it was possible to discriminate between the positive and negative sera. ELISA was used for study of the seroepidemiology of toxoplasmosis. Again, positive sera could be distinguished from negative sera. The examples presented show that ELISA can be used as an additional serologic tool for the detection of both helminthic and protozoal infections.


Screening for primary immunodeficiencies associated with purine nucleoside phosphorylase deficiency or adenosine deaminase deficiency. A screening method has been established for PNP deficiency. This enzyme activity in dried blood absorbed on filter paper can be detected by the formation of a blue insoluble formazan with a gel containing inosine, xanthine oxidase, MTT-tetrazolium, and phenazine methosulfate. The color change is very clear and definite and no false results have been obtained in the testing of 256 enzyme-positive and 107 enzyme-negative samples. The enzyme activity in dried blood on filter paper is so stable at room temperature that samples can be mailed. A screening method for ADA deficiency was developed, also depending on the color change of MTT-tetrazolium. Although the test is slightly more expensive than that developed by Moore and Meuwissen, its accuracy is greater. A common screening method for detecting deficiencies of either PNP or ADA is described.


Serum lysozyme in multiple myeloma.

Serum lysozyme in multiple myeloma. SLL's were tested by turbidometric assay on 33 male patients with multiple myeloma with three to 58 tests (mean 11) for each patient over a 7-year period. The age of the patients ranged from 31 to 83 years, with a median age of 58 years. SLL's in the normal controls were 14.4 +/- 3.5 microgram/ml. Patients with myeloma had a median lysozyme level of 16 microgram/ml and mean of 16.5. The SLL's in patients with lgG1,2,3,4, Iga, and kappa and lambda light-chain myeloma were similar. Slightly higher mean SLL'S were noted in older patients. Patients with severe renal disease also had higher SLL'S. No significant changes in SLL's were seen during infections or during mild granulocytopenia (granulocyte count greater than 500 cells/mm3). In severe granulocytopenia (granulocyte count less than 500 cells/mm3) the SLL's decreased and returned to normal levels when the white blood cell counts improved. In eight patients surviving for more than 5 years, the SLL's were not different from those of the other patients. SLL values in patients with multiple myeloma did not differ significantly by statistical test from those of controls when those patients with impaired renal function are excluded.


Electric shock produced drinking in the squirrel monkey. Squirrel monkeys were periodically exposed to brief electric tail shocks in a test environment containing a rubber hose, response lever, and a water spout. Shock delivery produced preshock lever pressing and postshock hose biting. Additionally, all subjects displayed licking responses following postshock biting-attack episodes. Further experiments showed that licking was: (1) influenced by hours of water deprivation; (2) drinking behavior; (3) the direct result of shock delivery; and (4) developed spontaneously in naive subjects with or without opportunities for hose biting or lever pressing. Removing the opportunity to attack increased postshock drinking. A noxious environmental stimulus that causes aggression can also produce drinking.


Preliminary evidence for the intracellular localization of luteinizing hormones in mammalian oocytes. Preliminary autoradiographic and immunofluorescent evidence is presented which suggests the localization of hLH beta, hCG and oLH within the oocytes of several mammalian species. Specificity of the response is supported by negative observations in the presence of cold hormone or antibodies to other proteins. The mechanism of action of these gonadotrophins in such a site remains to be defined.


Treatment of epilepsy with clonazepam and its effect on other anticonvulsants. Clonazepam was added to the treatment of patients with poorly controlled epilepsy in a double-blind trial and an open trial. Considerable improvement occurred with patients with myoclonic jerks and tonic-clonic convulsions, and with photosensitive epilepsy. Patients with atypical petit mal and focal epilepsies also improved. Drowsiness was initially common but lasted only a short time. No evidence was found for an action of clonazepam on the metabolism of other drugs, but treatment with phenobarbitone lowered serum concentrations of clonazepam. We conclude that clonazepam is particularly valuable in epilepsy with associated myoclonsu and in photosensitive epilepsy.


Regeneration distal to a prolonged conduction block. In 6 baboons a tourniquet round the knee was used to produce a prolonged local conduction block. This was followed, within a few days, by a surgical crush of the tibial or deep peroneal nerve at the ankle, in order to produce Wallerian degeneration distally. Electrophysiological recordings from small foot muscles were then used to study the time-course of regeneration in motor fibres. When the results were compared with those from crushed but unblocked nerves of the opposite leg, there was no evidence that either reinnervation of muscles or the subsequent maturation of the regenerating motor nerve fibres was delayed by the prolonged proximal conduction block.


Transoral, transsphenoidal microsurgical exposure of the pituitary gland and infundibulum in the rhesus monkey. Description of operative technique. A transoral, transsphenoidal microsurgical technique for exposing the pituitary stalk and gland of the rhesus monkey is described and illustrated. The technique has proved to be a safe and practical method for performing a variety of pituitary operations in a total of 37 animals. The procedures have included complete hypophysectomy, posterior lobe resection, pituitary stalk section, collection of portal venous blood, collection of pituitary venous blood, and placement of lessions in the organum vasculosum of the lamina terminalis.


Xylitol associated changes in amylase and protein content of monkey parotid saliva. The purpose of this study was to examine amylase activity in the parotid saliva of monkeys fed a high sugar diet supplemented for 3-day periods with either sucrose or xylitol. All monkeys fed xylitol displayed a significant elevation of amylase activity and total protein content in their parotid saliva. The specific activity of amylase (units/mg protein) did not differ between the two diets. The possible importance of this observation to the purported cariostatic action of xylitol is discussed.


Sparing effect of folic acid deficiency on the development of vitamin B12 deficiency in baboons fed a vitamin B12 deficient diet. The effect of a vitamin B12 and folic acid deficient diet on juvenile and adolescent baboons (Papio cynocephalus anubis) was studied. The baboons developed clinical and hematological signs characteristics of folacin deficiency, although they were less severe in juvenile baboons. The signs disappeared when folic acid was replaced in the diet. The serum vitamin B12 levels increased in all baboons fed the vitamin B12 and folic acid deficient diet. When folic acid was added to the diet, the levels gradually decreased in adolescent baboons, liver vitamin B12 levels decreased to a lesser extent when fed a vitamin B12 and folic acid deficient diet than when fed a vitamin B12 deficient diet. In juvenile baboons fed a vitamin B12 and folic acid deficient diet, for 7 months and a vitamin B12 deficient diet for a further 11 months, liver vitamin B12 levels did not decrease at any time but were similar to those in baboons fed a vitamin B12 and folic acid supplemented diet.


Short- and long-term effects of nitrogen metabolism of feeding protein during mild or severe energy restriction. The complex relationship between protein intake and energy restriction was studied in acutely and chronically restricted adult rats. Rats were fed either 12 or 24 kcal/day from protein alone or protein plus carbohydrate from 3 to 16 days in one experiment. In another, groups were fed either 0.75 or 1.5 g lactalbumin/day in diets supplying 90%, 80% or 70% of the ad libitum energy intake for 8 or 104 days. Body composition and nitrogen of various tissues were determined. In experiment 1, feeding protein conserved tissue and body nitrogen as long as sufficient adipose stores were maintained, but thereafter had no advantage over lower protein intakes supplemented with carbohydrate. In experiment 2, all rats gained weight in proportion to energy intake. Protein level was protective of carcass nitrogen, water and liver nitrogen in both the short- and long-term at the 80% and 90% level of energy intake. Energy intake was more important than protein in conserving muscle nitrogen. Effects of energy and protein restriction are complex and dependent upon the total amount of protein and energy provided, body reserves and duration of restriction.


Accumulation of orthochromatophilic normoblasts in bone marrow of vitamin E-deficient monkey, Macaca fasicularis. Crab-eating monkeys, Macaca fasicularis, fed a vitamin E-depleted diet for over 15 months, showed signs of anemia manifested by a lowering in hematocrit, hemoglobin concentration and red blood cell number, and by an increased susceptibility of erythrocytes to hydrogen peroxide lysis. Bone marrow pictures indicated the presence of multinucleated erythroid precursor cells and an accumulation of orthochromatophilic normoblasts. Supplementation with vitamin E resulted in reticulocytosis and a return of the normal bone marrow picture.


Evaluation of single-pigment shift model of anomalous trichromacy. The spectral sensitivity of the visual photopigments, the interobserver variability in color judgments, and the spectral locus of unique yellow provide three major problems for accounts of X-chromosomal-linked anomalous trichromacy. According to the single-pigment hypothesis, the primary defect in anomalous trichromacy is a wavelength shift in the peak sensitivity of one of the three visual photopigments. We show that this shift results in reduction of the anomalous trichromat's r-g opponent chromatic channel. The distribution of response variability in Rayleigh equation match widths due to factors other than the spectral characteristics of the photopigments is similar in normal and anomalous trichromats. When normal and anomalous trichromats make hue estimations of sets of stimuli designed to contain similar chromatic information, their judgments show similar variability. Calculation of the r-g opponent chromatic channel can provide correct predictions of the spectral loci for unique yellow for anomalous trichromats.


The ultrastructure of oral neuromas in multiple mucosal neuromas, pheochromocytoma, medullary thyroid carcinoma syndrome. The ultrastructure of oral neuromas from 2 patients with multiple mucosal neuromas, pheochromocytoma, medullary thyroid carcinoma syndrome reveals numerous hypertrophic unmyelinated and myelinated axons, hyperplastic neurilemmal cells and associated collagen fiber formation. These tumors are described and compared ultrastructurally with neurilemmomas and neurofibromas as described by other authors. On the basis of this comparison, these tumors are not considered to be of neurilemmal origin. On the basis of light and electron microscopic histology it is postulated that these tumors represent hypertrophy of axons similar to that noted in amputation neuromas.


Estimation of the volumes of multinucleate giant cells. An estimation of the volumes of three multinucleate giant cells has been attempted by measurements of the areas of enlarged tracings of serial paraffin sections which was estimated at approximately 5 mum in scanning electron-micrographs. In addition, three-dimensional models of these tracings constructed in wax were used to estimate the volumes of the same cells by means of Archimedes' Principle. The results indicate a close correlation between the estimates derived from the two procedures and suggest that the three cells measured have a volume of the order of magnitude of 50,000 to 100,000 mum(3).


The effect of turpentine on hamster cheek pouch mucosa: a model of epithelial hyperplasia and hyperkeratosis. Hamster cheek pouches were painted thrice weekly with 50% v/v turpentine in liquid paraffin over periods ranging from 1 day to 16 weeks. Pouch mucosa was examined histologically for changes and measurements of epithelial thickness were made with a calibrated eyepiece micrometer. An increase in epithelial thickness was first observed 48 hrs. after a single painting and was accompanied by inflammatory changes in both epithelium and connective tissue. Maximal epithelial thickening occurred after 9 weeks of thrice weekly painting. Cheek pouches of animals left without further treatment for up to 1 year following 9 weeks painting were indistinguishable from controls. The epithelial response to treatment with turpentine is that of a reversible benign epithelial hyperplasia with hyperkeratosis.


Pulpal response to a new visible light-cured composite restorative material: Fotofil. A new visible light-cured composite resin, Fotofil, has been developed for the esthetic restoration of anterior teeth. The pulpal responses elicited by this material in unlined cavities and cavities lined with Dycal were compared with those elicited by a negative control consisting of modified zinc oxide-eugenol cement Notebec and a silicate cement Syntrex. The pulpal responses were evaluated in the permanent maxillary and mandibular incisors in vervet monkeys using labial Claterial in each monkey. Groups of 10 monkeys were killed at intervals of 2 days, and 2 and 8 weeks after restoration of the teeth. The most severe pulpal responses were elicited by the unlined Fotofil and Syntrex restorations at all three time intervals.  The Dycal lined Fotofil restorations elicited a milder pulp response than the Nobetec at 2 days and 2 weeks. No pulpal response was elicited by the latter two materials at 8 weeks. These results clearly showed that Fotofil and Syntrex are irritant to the dental pulp up to 8 weeks. The placement of a Dycal liner beneath Fotofil restorations reduced the pulpal irritation elicited by this material to levels which were lower than those elicited by the Nobetec control at 2 days and 2 weeks.


Immunopathology of Sjögren-like disease in NZB/HZW mice. In vitro responsiveness of spleen cells from NZP/NZW (B/W) female mice, aged 2 through 40 weeks, to the mitogens phytohemagglutinin (PHA), concanavalin-A (Con-A) and lipopolysaccharide (LPS) was determined. An age-related decline in proliferative response of splenic cells was found; this decline correlated with the age of onset and progression of lymphoid infiltration in lacrimal and salivary glands in the mice. The worsening with age of this lymphoid infiltrate, coupled with the decline in responsiveness to mitogens, suggests a complex immunopathologic process with destruction of glands terminating in a disease similar to the Sjögren syndrome in humans.


The influence of traumatic intrusion of primary teeth on their permanent successors in monkeys. A macroscopic, polarized light and scanning electron microscopic study. Primary maxillary and mandibular central incisors were experimentally intruded on 13 monkeys, in order to examine changes of the enamel in the permanent successors. To study the effect of removal of the displaced tooth, the left primary incisor was extracted 1 h after the trauma, whereas that on the right was left in the jaw. Six weeks later the animals were sacrificed. Macroscopically, circumscribed areas of opacities on labial enamel were noted. The SEM examination showed that non-affected parts of enamel had reached anatomical form, whereas inhibition studies and microradiographs demonstrated that the maturation process was far from complete. Arrest of enamel maturation was noted as corresponding to the extent of the surface opacity. In the SEM small areas displaying rupture of the surface enamel were seen. No differences were observed either macroscopically or histologically in relation to immediate removal or preservation of the intruded primary incisor.


Calcification mechanisms: roles for cells and mineral. Recent findings slggest that cellular activity and mineral chemistry have important roles in initiating mineral formation in calcifying tissues. Under biological conditions, hydroxyapatite (HA), the predominant and most stable form of insoluble calcium phosphate, forms only through a more soluble, noncrystalline precursor, amorphous calcium phosphate (ACP). The conversion of ACP to HA can be extensively inhibited by many biologically ubiquitous small ions at their typical tissue concentrations. The earliest mineral deposits in the matrix of several calcifying tissues are associated with tiny membranous vesicles. Intramitochondrial calcium phosphate granules are also seen within intact cells of certain calcifying tissues. Current hypotheses for initial mineral deposition, based on these findings, are reviewed.


Effectiveness of low-dose continuous intravenous insulin infusion in diabetic ketoacidosis. A prospective comparative study. Twenty pediatric patients with diabetic ketoacidosis were randomly assigned in equal numbers to receive insulin either as a low-dose continuous intravenous infusion or as high-dose intermittent subcutaneous injections. Blood was obtained hourly for determinations of total CO2, plasma glucose, and osmolality, and, in previously untreated patients, plasma insulin. Serum values of beta hydroxybutyrate, electrolytes, and acetone were monitored every two hours. Plasma insulin levels were in the therapeutically effective range with each method of administration. There were no statistically significant differences in rate of correction of ketoacidosis, rate of reduction of plasma glucose, or decline in plasma osmolality. The incidence and the severity of hypokalemia were increased in the patients receiving subcutaneous insulin. There was less variation in the rate of reduction of plasma glucose in the infusion group. Low-dose continuous intravenous infusion of insulin is at least as effective in treating diabetic ketoacidosis as the traditional high-dose intermittent subcutaneous injection of insulin and offers some definite advantages.


Studies on the biology of the periodontium of marmosets. XIII. Histopathology of niacin deficiency stomatitis in the marmoset. Adult cotton top marmosets made niacin deficient by long-term dietary deprivation, developed a syndrome characterized by anorexia, weight loss, weakness, diarrhea, dermatitis, enterocolitis and stomatitis. The stomatitis was highlighted by a necrotizing gingivitis and periodontitis and by an ulcerative and atrophic glossitis.


Polymer sorption of nitroglycerin and stability of molded nitroglycerin tablets in unit-dose packaging. The sorption of nitroglycerin by thermoplastic polymers and the stability of molded nitroglycerin tablets in strip packaging were studied. The polymers investigated varied greatly in their affinity for nitroglycerin, the order of decreasing affinity being: vinyls greater than low density polyethylene greater than ionomers greater than high density polyethylene. With the proper choice of packaging, molded nitroglycerin tablets stabilized with povidone maintained acceptable potency for up to 2 years at 26 degrees when strip packaged in unit doses. Chemical decomposition (hydrolysis) of nitroglycerin also was investigated. Povidone accelerated the decomposition of nitroglycerin; at high temperature, decomposition was a significant factor in tablet stability for tablets containing povidone.


The mode of action of bumetanide: inhibition of chloride transport across the amphibian cornea. The diuretic drug bumetanide (10(-7 M) reduced the transmural potential difference and short-circuit current across the amphibian cornea in vitro. This effect reflected a decline in the outflux (endothelial to epithelial, or tear, side) of CI, which is the direction of its active transport. There was no change in the influx. The effect was slowly reversible at 10(-4) M and was about two times as great when the drug was on the endothelial as compared to the epithelial side. Furosemide had a similar effect on CI transport, but the dose-response curves of the two drugs were not parallel. Furosemide was about 60 to 200 times less potent than bumetanide. The effect of furosemide was about five times as great when the drug was on the endothelial rather than when it was placed on the epithelial side. Thiocyanate (2 x 10(-2) M) also inhibited the active CI transport across the cornea, but it was equally effective on either side of the membrane. Bumetanide had no effect on the passive CI movements across the toad lens or frog skin in vitro. The response of the skin differs from that of furosemide and thiocyanate. These observations of the effects of bumetanide on CI transport may be relevant to the mechanism of its diuretic actions in the kidney tubule.


Ketamine self-administration by the rhesus monkey. Intravenously administered ketamine served as a reinforcer of self-administration behavior in rhesus monkeys during daily 2-hour sessions. When the dose of ketamine was varied over a wide range at fixed-ratio schedules of reinforcement of 1, 8 and 64, the response rate was an inverted U-shaped function of the dose. Maximal response rates occurred at progressively higher doses as the fixed-ratio size was increased; drug intake per session was positively related to the dose. When the ketamine dose was held constant and the fixed-ratio was increased in a geometric series, the response rate increased as the fixed-ratio was increased to FR 128 or FR 256; additional increases in fixed-ratio size produced abrupt decreases in response rates. Since the response rate increased linearly as the fixed-ratio was increased geometrically, drug intake was a decreasing function of the response requirement. With respect to magnitude of reinforcement and fixed-ratio response requirement, the data show that ketamine maintains self-administration behavior in a manner similar to that of a number of other drugs and conventional reinforcers.


Quantitative analysis of the velocity characteristics of optokinetic nystagmus and optokinetic after-nystagmus. 1. Velocity characteristics of optokinetic nystagmus (OKN) and optokinetic after-nystagmus (OKAN) induced by constant velocity full field rotation were studied in rhesus monkeys. A technique is described for estimating the dominant time constant of slow phase velocity curves and of monotonically changing data. Time constants obtained by this technique were used in formulating a model of the mechanism responsible for producing OKN and OKAN.2. Slow phase velocity of optokinetic nystagmus in response to steps in stimulus velocity was shown to be composed of two components, a rapid rise, followed by a slower rise to a steady-state value. Peak values of OKN slow phase velocity increased linearly with increases in stimulus velocity to 180 degrees /sec. Maximum slow phase eye velocities in the monkey are 2-3 times as great as in humans.3. At the onset of OKAN, slow phase velocity falls by about 10-20%, followed by a slower decline to zero. Peak OKAN slow phase velocities were linearly related to optokinetic stimulus velocities up to 90-120 degrees /sec. Above 120 degrees /sec OKAN slow phase velocity saturated although OKN slow phase velocity continued to increase.4. The charge and discharge characteristics of OKAN were studied. The OKAN mechanism charged in 5-10 sec and discharged over 20-60 sec in darkness. The time constants of decay in OKAN slow phase velocity decreased as stimulus velocities increased. They also decreased on repeated testing. In several monkeys there was a consistent difference in the rate of decay of OKAN slow phase velocity to the right and left.5. Extended visual fixation discharged the activity responsible for producing OKAN. Short fixation times caused only a partial discharge of the OKAN mechanism. Following brief periods of fixation, OKAN resumed but with depressed slow phase velocities.6. A model based on a state realisation of a peak detector was formulated which approximately reproduces the salient characteristics of OKN and OKAN. This model predicts the three dominant characteristics of OKAN: (1) charge over 5-7 sec, (2) slow discharge in darkness, and (3) rapid discharge with visual fixation. With the addition of direct fast forward pathways, it also correctly predicts the rapid and slow rise in OKN. We postulate that OKAN is produced by a central integrator which is also active during OKN. Presumably this integrator acts to maximize velocities during OKN and to smooth and stabilize ocular following during movement of the visual surround.


Effects of calcium and calcium-chelating agents on the inward and outward current in the membrane of mollusc neurones. 1. Effects of internal and external Ca and Ca-chelating agents, EGTA and EDTA on transmembrane ionic currents were studied in isolated, internally dialysed neurones from the molluscs, Helix pomatia and Limnea stagnalis.2. The possible pharmacological effect of internally applied EGTA was investigated on the background of constant free Ca concentration (5.3 x 10(-9)M). EGTA had no effect on Ca and Na inward currents but considerably depressed the delayed K outward current. No effective removal of this action could be achieved by the elevation of intracellular free Ca.3. In the absence of divalent cations in the external medium, EGTA (as well as EDTA) applied either intra- or extracellularly caused the appearance of a very large Na inward current with kinetics similar to those of Ca inward current and with the reversal potential around 10 mV. Effective concentrations of chelating agents were 0.1 mM (extracellular) and 1.0 mM (intracellular).4. Increase in intracellular Ca in the absence of EGTA (by dialysis of the cell with Ca-saturated solutions) did not produce any significant effect on the delayed K outward current. The small change observed in this current could be evaluated as a depression of maximum slope conductance and a shift to more negative membrane potential.5. Ca inward current has been found extremely sensitive to internal Ca. 5.8 x 10(-8)M of internal free Ca produced its complete depression. This effect was reversible. Na inward current could be inhibited with 3.5 x 10(-7)M intracellular Ca.


Evaluation of radiotherapists' reports for treatment of prosthodontic patients after irradiation. A simplified explanation of gaining insight into the radiotherapist's report in order to deal with a patient who has received radiation treatment has been discussed. The best method for increasing understanding of this report is to work in conjunction with the radiotherapist before and after the patient is given radium. Five factors are important in the treatment of the irradiated patient. These are: (1) initially recognizing the radiation patient, (2) understanding the radiotherapist's report, (3) following good prosthodontic principles, (4) integrating treatment with the radiotherapist's report to prevent future problems, and (5) maintaining extremely close follow-up to detect any sore spots that could lead to major complications such as bone or soft-tissue necrosis. A poor candidate for dentures is normally a difficult candidate following radiation. Not all patients are capable of tolerating a prosthesis following radiation therapy. However, for many, function and esthetics can be restored.


Effect of colloidal association on the measured activity of alkylbenzyldimethylammonium chlorides against Pseudomonas aeruginosa. The antibacterial activities of a homologous series (C8-C18) of alkylbenzyldimethylammonium cholorides (ABDAC) against Pseudomonas aeruginosa have been measured using both a minimum inhibitory concentration (MIC) procedure and a sterilization kinetics test carried out in deionized water. There was a log-linear relationship between activity measured by kinetics and carbon number. With MIC there was a log-linear relationship up to C14, when there was a turndown in activity. Consideration of the colloidal association of ABDAC in deionized water and in a simple salts growth media leads us to suggest that use of high concentrations of nutrient salts in MIC tests will lower the effective concentration of the surface active agents. This change may be responsible for the turndown in activity observed in MIC tests, and that in such circumstances the MIC test does not give a true reflection of the intrinsic activity of the compounds. Literature reports of parabolic relationships between ABDAC alkyl chain length and antimicrobial activity are reinterpreted on this basis.


Methotrexate analogues. 9. Synthesis and biological properties of some 8-alkyl-7,8-dihydro analogues. A series of 8-alkyl-7,8,-dihydromethotrexate analogues was prepared by direct alkylation of 7,8-dihydromethotrexate, after pilot studies were performed with simpler pteridines. These compounds are tested for in vitro inhibitory activity against Lactobacillus casei and as enzyme inhibitors against dihydrofolate reductase and thymidylate synthetase derived from this organism. All of the analogues were less inhibitory toward dihydrofolate reductase than was methotrexate but were more inhibitory toward thymidylate synthetase. The analogues were also evaluated for in vitro inhibitory activity against the CCRF-CEM human lymphoblastic leukemia cells. In vivo against the L-1210 leukemia in mice, several of the analogues exhibited some antileukemic activity.


Synthesis of 2-benzyloxy and 2-benzylthio analogues of primaquine as potential antimalarials. A series of 2-benzyloxy and 2-benzylthio analogues of primaquine has been synthesized and evaluated against Plasmodium berghei in the mouse and Plasmodium cynomolgi in the rhesus monkey. 8-Aminoquinoline toxicity, as measured in the Rane mouse screen, was reduced, and these compounds showed significant blood schizonticidal antimalarial activity in mice. In monkeys, significant tissue-schizonticidal activity was observed.


Urea uptake and translocation in toad urinary bladder: the effect of antidiuretic hormone. The uptake of C14-urea into everted and noneverted bladder sacs was compared, over short time periods (up to 2 min), with the transepithelial urea fluxes. This method allowed the study of the time course of urea uptake and distribution, while previously this problem was only studied in steady-state conditions. When mucosal uptake was studied no accumulation of C14-urea inside the tissue was observed, indicating that the mucosal border could be the limiting step. Comparative studies of urea and inulin uptake from the serosal side showed that urea equilibrated with the water epithelial cells in less than 30 sec. This accumulation suggested again that the mucosal border is an effective barrier for urea translocation. The kinetics of the increase in urea permeability induced by antidiuretic hormone was also studied and it was similar (T1/2:4.3 min) to the kinetics of the increase in water permeability induced by the hormone (T1/2:5.6 min). A strong parallelism was also observed between the time course of the increases in water and urea permeabilities induced by medium hypertonicity (T1/2 25 and 26 min, respectively). The values obtained for the permeability coefficient ktrans), either at rest or under ADH were similar to those previously reported employing steady-state techniques (28+/-8 and 432+/-25 cm-sec-1-10(-7), respectively).


Cell and organelle shrinkage during preparation for scanning electron microscopy: effects of fixation, dehydration and critical point drying. The critical point drying method of preparing samples for scanning electron microscopy is associated with a variable amount of specimen shrinkage. We studied  the causes of this phenomenon is isolated mouse hepatocyte nuclei and in human erythrocytes and found that the critical point drying process itself caused most of the shrinkage that we observed (a 25-30% reduction in diameter in both specimens). Glutaraldehyde fixation and ethanol dehydration caused only minimal size reduction, prior to critical point drying. Substitution of an inert (ethylene glycol-ethylene glycol monethyl ether) dehydration technique did not alter the final result. Previous studies in our laboratory using high resolution SEM and correlative transmission microscopy of isolated nuclei have demonstrated that the shrinkage represents a miniaturization of the organelles in which all structural components retain their usual relationships.


Partial enzyme digestion studies on Escherichia coli, Pseudomonas, Chlorella, Drosophila, HeLa and yeast 5S RNAs support a general class of 5S RNA models. Fox and Woese (1975a) have shown that a model of 5S RNA secondary structure similar to the one originally derived for Chlorella 5S RNA can be generalized with relatively minor variations to all sequenced 5S RNA molecules, i.e. that corresponding base paired regions can be formed at approximately the same positions. We present experimental data in favour of this hypothesis and show that the points at which ribonucleases T1, T2 and pancreatic ribonuclease cleave six different 5S RNA molecules under 'mild' conditions (high ionic strength, low temperature, low RNAase concentration) nearly always fall in the proposed single-stranded regions. We conclude that this model is a good approximation to the conformation of 5S RNA in solution.


In vivo growth advantage of the MOPC-315 mouse myeloma over its immunoselected variants. Myeloma cells of the "wild type" that produce complete immunoglobulin molecules and those of the more usual variant type that display only one kind of chain [either light (L) or heavy (H)] were cocultivated ip and sc in syngeneic BALB/c mice. With each of six deliberately selected variants, a progressive increase in the proportion of wild-type cells was observed; the rate of change suggested that these variants had an approximately 10% slower growth rate than that of the wild-type tumor. In contrast, a variant that arose spontaneously overgrew the wild-type cells. The results may account for a) the stable capacity of most wild-type tumors to produce complete immunoglobulin molecules (L- plus H-chains) over many years, even though they frequently generate variant cells that produce only L- or only H-chains; and b) the occasional spontaneous change of myeloma cell populations from predominantly wild-type to variant cells.


Secondary structure of RNA from bacteriophages f2 Qbeta, and PP7. Electron microscopy of RNA-protein monolayers prepared under partial denaturing conditions has been used to compare the secondary structure of coliphage f2 and Qbeta and Pseudomonas aeruginosa phage PP7 RNAs. The secondary structure map of f2 RNA contains a central open loop and four symmetrically placed hairpins, which is similar to the pattern reported by Jacobson (A. B. Jacobson, Proc. Natl. Acad. Sci. U.S.A. 73:307-311, 1976) for the closely related phage MS2. With the same denaturing conditions, Qbeta RNA, which is 20% larger than f2 or PP7 RNA, has a central open loop and a smaller terminal loop. PP7 RNA has two large, closed secondary structures, one of which is nearly central. The base composition of PP7 RNA was determined and is similar to that of the group I coliphage RNAs. Thus, the greater amount of large base-paired structure is not related to an increased guanine-plus-cytosine content of PP7 RNA. With increased denaturing conditions, the central, closed structure of PP7 RNA is converted into an open loop. The central structures of all three phages include about 700 nucleotides. The relevance of these findings to the genetic maps of the coliphage RNAs is discussed.


Bacteriophage PMB12 conversion of the sporulation defect in RNA polymerase mutants of Bacillus subtilis. The pseudotemperate phage PMB12 was isolated from soil on the basis of its ability to enhance the rate of sporulation of Bacillus subtilis 168. PMB12 was subsequently shown to convert the sporulation defect in two genetically distinct classes of sporulation mutants. One class includes those rifampin-resistant mutants that are also spore-negative (mutated at the rif locus). The other class includes a strain carrying the sporulation mutation spoCM-1. The spoCM-1 mutation is linked to cysA15 by PBS1 transduction but is distinct from the rif locus. Several other sporulation mutants were not converted by PMB12. PMB12 is related to phage PBS1. However, PBS1 did not convert the above sporulation mutants. The replication of PBS2, a clear-plaquing derivative of PBS1, is rifampin insensitive, apparently due to a phage-induced rifampin-insensitive RNA polymerase. PMB12 replication is also rifampin insensitive.


Morphogenesis of bacteriophage phi 29 of Bacillus subtilis: mapping and functional analysis of the head fiber gene. A set of mutants of Bacillus subtilis bacteriophage phi29 unable to synthesize the head fiber protein has been identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Infectious phage are produced during restrictive infection. We have focused on mutant sus 8.5(900) because the mutation is suppressible by both the su(+3) and su(+44) hosts, and it can be mapped by three- and four-factor crosses. After restrictive infection with mutant sus 8.5(900), a fragment about 70% of the size of the normal fiber is produced as well as particles that are fast-sedimenting in sucrose gradients relative to phi29(+). These particles have the buoyant density of particles with the fibers removed and have the absolute plating efficiency of phi29(+). Fiber protein is absent from prohead as well as virion. A second set of mutants produces fiber protein with a slightly altered electrophoretic mobility. This type of fiber protein is either present or absent on both prohead and virion. A third class of mutants, typified by 914, produces a "normal" fiber, but a major head protein of altered electrophoretic mobility. After infection by this mutant, the fiber is absent from both prohead and virion, and the biological and physical properties of the 914(-) particle are similar to those of particles produced after infection of the su(-) host by sus8.5(900). These observations suggest that the head fiber is not an essential component of the prohead or virion and that the assembly process is efficient in the absence of fiber protein. Three- and four-factor genetic crosses have established the order sus8(769)-8(914)-sus8.5(900)-sus9(756) and indicate that cistrons 8 and 8.5 code for the major head protein and head fiber protein, respectively.


Order of the two major head protein genes of bacteriophage phi 29 of Bacillus subtilis. Bacteriophage phi 29 mutation sus8(22) has been mapped by two-factor crosses between markers sus8(769) and ts8(93). Whe sus8(22) infects Bacillus subtilis su- proteins, HP1 (major head protein) and HP3 (fiber protein) are not synthesized; instead, a fragment with a molecular weight of 25,000 is produced. The tryptic peptides of the fragments overlap with corresponding peptides in protein HP1, but not with the peptides of protein HP3, showing that cistron 8 codes for the major head protein HP1.