Regulation of hydrogenase in Rhizobium japonicum. Factors that regulate the expression of an H2 uptake system in free-living cultures of Rhizobium japonicum have been investigated. Rapid rates of H2 uptake by R. japonicum were obtained by incubation of cell suspensions in a Mg-phosphate buffer under a gas phase of 86.7% N2, 8.3% H2, 4.2% CO2, and 0.8% O2. Cultures incubated under conditions comparable with those above, with the exception that Ar replaced H2, showed no hydrogenase activity. When H2 was removed after initiation of hydrogenase derepression, further increase in hydrogenase activity ceased. Nitrogenase activity was not essential for expression of hydrogenase activity. All usable carbon substrates tested repressed hydrogenase formation, but none of them inhibited hydrogenase activity. No effect on hydrogenase formation was observed from the addition of KNO3 or NH4Cl at 10 mM. Oxygen repressed hydrogenase formation, but did not inhibit activity of the enzyme in whole cells. The addition of rifampin or chloramphenicol to derepressed cultures resulted in inhibition of enzyme formation similar to that observed by O2 repression. The removal of CO2 during derepression caused a decrease in the rate of hydrogenase formation. No direct effect of CO2 on hydrogenase activity was observed.


Simultaneous loss of multiple differentiated functions in aerial mycelium-negative isolates of streptomycetes. Germination and outgrowth of spores of Streptomyces alboniger, Streptomyces scabies, and Streptomyces violaceus-ruber in the presence of intercalating dyes resulted in a high frequency (2 to 20%) of occurrence of aerial mycelium-negative (Amy-) isolates. Coincident with the appearance of the Amy- trait was the loss of several differentiated functions, including the characteristic pigments and earthy odor of the wild types. All S. alboniger, 27% of S. scabies, and 39% of the S. violaceus-ruber Amy- isolates were arginine auxotrophs. The missing enzyme step was identified as argininosuccinate synthetase by using a sensitive microassay for estimation of enzyme activity. The remainder of the S. scabies and S. violaceus-ruber isolates were prototrophs. In addition, S. alboniger Amy- isolates failed to produce or respond to the stimulator of aerial mycelium formation isolated from the wild type. The Amy- isolates did not revert to either Amy+ of Arg+. The lack of any detectable reversion, coupled with the high frequency of curing, supports the idea that a deletion of genetic material, possibly a plasmid, has occurred.


Nitrite reduction in Veillonella alcalescens. Nitrite reduction was examined in Veillonella alcalescens C-1, and obligate anaerobe with an ATP-yielding nitrate-reducing system. Hydrogen donors for nitrite reduction included hydrosulfite, hydrogen gas, and pyruvate, but not pyridine nucleotides, in the presnece or absence of flavins. Pyruvate-linked nitrite reduction was not inhibited by 4,4,4-trifluoro-1-(2-thienyl) 1,3-butanedione, dicoumarol, or 2-heptyl-4-hydroxy-quinoline-N-oxide. The noninvolvement of membrane-bound factors was supported by the fact that 100% of pyruvate-linked activity remained in the soluble fraction after fractionation of crude extracts by ultracentrifugation. Using DEAE-cellulose column chromatography, however, the participation of ferredoxin in nitrite reduction was demonstrated. The product of nitrite reduction appeared to be ammonia, as determined from H2-to-NO2- ratios. Nitrite reductase was induced by nitrate or nitrite and was repressed by increased levels of reduced nitrogenous compounds.


Physiological responses of bacteria to cytochalasin A: effects on growth, transport, and enzyme induction. Cytochalasin A at 5 to 25 microgram/ml (1.0 x 10(-5) to 5.2 x 10(-5) M) inhibited the growth of three gram-positive bacteria, Arthrobacter sialophilus, Staphyloccus aureus, and Bacillus amyloliquifaciens, but had little or no effect on the growth of three gram-negative bacteria, Excherichia coli, Pseudomonas maltophilia, and Aeromonas proteolytica. A. sialophilus and S. aureus recovered spontaneously from cytochalasin A-mediated growth inhibition after a considerable lag period, which was dependent on the drug dose. It was demonstrated that this long-term recovery did not involve selection of resistant variants. Cytochalasin A had no detrimental effect on cell viability in A. sialophilus or S. aureus, but caused lysis of B. amyloliquifaciens. The drug prevented enzyme inductions and inhibited transport of valine, uridine, and glucose in the gram-positive organisms. It had little or no effect on these processes in the gram-negative organisms. In studies with A. sialophilus, the drug inhbitied respiration of exogenous substrates, but did not depress endogenous respiration. These results constitute the first unequivocal evidence for the bacteriostatic properties of this class of compounds and indicate that cytochalasin A halts various physiological processes in gram-positive bacteria primarily by inhibiting solute transport.


Synthesis of guanosine tetra- and pentaphosphates by the obligately anaerobic bacterium Bacteroides thetaiotaomicron in response to molecular oxygen. Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) were detected in formic acid extracts of air-exposed culutres of Bacteroides thetaiotaomicron. The identification of ppGpp and pppGpp in B. thetaiotaomicron was based on the following results: (i) cochromatography of 32P-labeled hyperphosphorylated nucleotides in two different two-dimensional solvent systems with authentic ppGpp and pppGpp; (ii) incorporation of [3H]guanosine into the putative ppGpp and pppGpp; (iii) alkaline lability; and (iv) resistance, to periodate oxidation. There was a marked increase in the concentration of ppGpp and pppGpp after shift from anaerobic to aerobic conditions, and accumulation of both ppGpp and pppGpp was blocked under these conditions by pretreatment of the culture with rifampin or tetracycline. Growth and incorporation of [3H]guanosine, [3H]tymidine, [14C]succinate, and L-[35S]methionine into macromolecules were inhibited immediately upon exposure to air. The accumulation of ppGpp and pppGpp in B. thetaiotaomicron upon exposure to air may represent a novel signal for synthesis of these compounds.


650 private psychiatric patients.

650 private psychiatric patients. 650 psychiatric patients seen for the first time in San Diego on a private basis received a research interview that allowed a study of demographic and clinical characteristics. They were compared with 100 psychiatric patients seen in similar circumstances in Wisconsin. The 2 populations were remarkably similar in several respects.


Acute brain syndromes.

Acute brain syndromes. Four hundred and nine cases of acute brain syndrome, encountered in psychiatric consultations among 54,942 general hospital patients over a 5 year period, fell into 4 groups according to symptom picture: lethargic, bewildered, agitated and psychotic. Treatment included reality orientation, protection and pharamacologic measures.


The wish to be held and hold in couples. Information pertaining to the wish to be held and to hold was gathered from 56 couples to answer the following questions: 1) What sex difference is there in the preference for being held or holding? 2) Which of the 2 meanings for the wish to be held is usually described? 3) Are men able to separate the wish for body contact as an end in itself from it as prelude to sexual intercourse? 4) Is disparity in body contact desires a cause of marital disharmony?


Physician marriages.

Physician marriages. A questionnaire study of physicians' ratings of various aspects of their marriage, was sent to 100 randomly selected physicians in Ramsey County (St. Paul, Minnesota). Eighty per cent of the sample responded. All 80 had been married at least once. There were fewer divorces reported compared with the general population. The group of 80 rated their marriages above average. Spouses who were considered extroverted correlated very significantly with a good marriage rating. The educational level of the spouse did not correlate with the marriage rating. Working long hours was not associated with poor marriage ratings or divorce. No significant relationship was revealed in examining the study variables for grouped medical specialties.


Parental involvement as a requisite for successful adolescent therapy. The effect of parental involvement in the treatment program of 66 consecutive adolescent patients is analyzed. Adolescents whose parents participated regularly in a parents' group or in individual parental consultation and therapy achieved a 64% success rate, using narrow success criteria. For those adolescents whose parents were not involved in the treatment process, the failure rate was virtually 100% (chi2=21.07, p less than 0.001). There was no statistically significant difference in treatment outcome between the cohorts of parental involvement. However, a parents' group is recommended where feasible in lieu of individual parent consultation alone for reasons of catharsis, universality, mutual support, and collective experience.


Visceral larva migrans and eosinophilia in an emotionally disturbed child. Visceral larva migrans, Entamoeba coli, evidence of latent toxoplasmosis and a history of plumbism were found in an emotionally disturbed, retarded child. Patients with pica should be screened for parasitism and other diseases transmitted orally.


Fantasy and reality in patients reports of incest. This paper explores some of the complex difficulties facing clinicians trying to assess whether a patient's report of incest is fantasy or reality. The role of sexualized family interactions, the age of the child, the nature of the act, and the quality of the reports are discussed as relevant variables. Cases are used to illustrate various points. In conclusion, we provide 8 questions the clinician should review before making any judgments about a report of incest being reality, especially when legal procedures are involved.


Motion sickness--key to neurobiologic variation. Individual variation in motion sickness susceptibility correlates with a wide variety of perceptual, cognitive, and personality variables suggesting that it may be a unique index of individual difference in fundamental mechanisms of the orientation process. Data from a large ongoing clinical study involving over 1,500 psychiatric inpatients indicate a clear-cut association between a number of the MMPI scales (including Social Isolation, Ego Strength and Schizophrenia) and motion sickness susceptibility. Marked difference across the motion sickness susceptibility spectrum in the condition on discharge of lithium and antidepressant treated patients was also found, when compared to other drug therapies, suggesting specific drug responses by the motion sick group.


The clinical characteristics of personality disorder subtypes in Naval service. The clinical importance of personality disorder subtypes was evaluated through an analysis of the demographic characteristics and outcomes for 11,549 navy enlisted men who received a primary diagnosis of a character and behavior disorder. Men with similar demographic characteristics serving in similar environments and demonstrating grossly similar clinical pictures received different diagnoses and dispositions in the various hospitals studied. Within any 1 hospital over the 4 study years, while demography of character and behavior disorder patients remained stable, diagnostic breakdowns and administrative and outcome characteristics varied markedly. The data are thus consistent with the hypothesis that diagnostic subtypes for personality disorders have limited clinical and prognostic meanings. The label may reflect the idiosyncratic preference of a particular physician at 1 point in time as much as the clinical picture or prognosis of the patient.


Life events associated with adolescent pregnancies. A retrospective study of the life events reported by 121 pregnant adolescents and 261 controls has disproved the null hypothesis that those 2 groups are 2 random samples from the same population. The pregnant girls differ markedly in terms of the events that occurred within their families. Most striking among these events was the greater number of deaths of family members reported by the pregnant girls. Clearly, a prospective study is needed to confirm the findings.


Narcolepsy and automatic behavior: a case report. Narcolepsy is characterized by excessive daytime sleepiness and cataplexy, which may be accompanied by hypnogogic or hypnopompic hallucinations and sleep paralysis. Automatic behavior is a relatively newly recognized symptom of the narcolepsy syndrome. This case report describes a particularly troublesome sort of automatic behavior--shoplifting--in a narcoleptic patient. It illustrates how a sleep-laboratory evaluation was used to confirm the diagnosis of narcolepsy and considers aspects of the treatment of the problem.


Adapted melodic intonation therapy: a case study of an experimental language program for an autistic child. In order to develop a useful communication system, a 3-year-old, non-verbal autistic boy was treated for 1 year with a Simultaneous Communication method involving signed and verbal language. As this procedure proved not useful in this case, an adaptation of Melodic Intonation Therapy (signing plus an intoned rather than spoken verbal stimulus) was tried. With this experimental language treatment, the patient produced trained, imitative and, finally, spontaneous intoned verbalizations which generalized to a variety of situations.


Glucose dehydrogenase (hexose 6-phosphate dehydrogenase) and the microsomal electron transport system. Evidence supporting their possible functional relationship. The ability of a microsomal enzyme, glucose dehydrogenase (hexose 6-phosphate dehydrogenease) to supply NADPH to the microsomal electron transport system, was investigated. Microsomes could perform oxidative demethylation of aminopyrine using microsomal glucose dehydrogenase in situ as an NADPH generator. This demethylation reaction had apparent Km values of 2.61 X 10(-5) M for NADP+, 4.93 X 10(-5) m for glucose 6-phosphate, and 2.14 X 10(-4) m for 2-deoxyglucose 6-phosphate, a synthetic substrate for glucose dehydrogenase. Phenobarbital treatment enhanced this demethylation activity more markedly than glucose dehydrogenase activity itself. Latent activity of glucose dehydrogenase in intact microsomes could be detected by using inhibitors of microsomal electron transport, i.e. carbon monoxide and p-chloromercuribenzoate (PCMB), and under anaerobic conditions. These observations indicate that in microsomes the NADPH generated by glucose dehydrogenase is immediately oxidized by NADPH-cytochrome c reductase, and that glucose dehydrogenase may be functioning to supply NADPH.


Characterization of Ustilago Ribonuclease U2. Effects of chemical modification at glutamic acid-61 and cystine-1 and of organic solvents on the enzymatic activity. RNase U2 was purified and crystallized from the enriched culture medium (ammonium sulfate-urea-corn meal) of Ustilago sphaerogena and its characteristics were investigated. Chemical modification of RNase U2 was conducted with monoiodoacetic acid to carboxymethylate Glu-61 and with 2-methoxy-5-nitrotropone to nitrotroponylate the amino terminal residue. The amino terminal residue was modified reversibly by this reagent. Comparison of the 2'-AMP binding in the modified enzyme and the native one showed that Glu-61 is essential for the formation of the enzyme-substrate complex, while the amino terminal residue plays no important role in the enzymatic activity. The enzymatic activity and the structure of RNase U2 in aqueous organic solution were also investigated. The affinity of the enzyme for 2'-AMP, the inactivation by monoiodoacetic acid and the fluorescence intensity were examined. The profiles of the changes in the properties of the enzyme protein were consistent with those in the enzymatic activity. Fluorescence studies of the enzyme suggest that the tryptophan residue is closely related to the activity.


Biochemical studies on the muscle microsomes of Ascaris lumbricoides var. suum. I. Biochemical characterization and electron transport of Ascaris microsomes. Two subcellular fraction, P-1 and P-2, were isolated by differential centrifugation from 0.25 M sucrose muscle homogenates of the parasitic roundworm, Ascaris lumbricoides suum. Morphological studies indicated that P-1 fraction consisted of intact mitochondria, whereas P-2 fraction consisted almost exclusively of vesicular components. The difference spectrum of Ascaris microsomes showed a characteristic b-type cytochrome spectrum with three distinct absorption peaks at 560, 525, and 424 nm. However, the alpha-peak at 560 nm was asymmetric with a shoulder at 555 nm. This microsomal b-type cytochrome was reduced by NADH, which was inhibited by rotenone and HgCl2. The reduced b-type cytochrome was easily reoxidized by shaking. NADH-oxidase activity observed in Ascaris microsomes was inhibited by rotenone, but not by KCN, NaN3, and antimycin A. On the other hand, NADH-cytochrome c and NADH-neotetrazolium (NT) reductase activities in Ascaris microsomes were not inhibited by antimycin A and rotenone, but were inhibited by HgCl2. Further observations indicated that neither HgCl2 nor rotenone inhibited Ascaris microsomal NADH-ferricyanide (FC) reductase activity, but rabbit antibody prepared against the purified NADH-FC reductase inhibited the NADH-cytochrome c reductase activity, the reduction of b-type cytochrome and the NADH-oxidase activity, as well as microsomal NADH-FC reductase activity.


Absorption, fluorescence, and linear dichroism spectra of fluorescein mercuric acetate (FMA) bound to F-actin. Two kinds of effects of divalent cations. Two kinds of F-actin were prepared; one binds magnesium at the unique divalent cation binding site of actin protomer and the other binds calcium at this site. They were designated as F(Mg)-actin and F(Ca)-actin, respectively. The binding of fluorescein mercuric acetate (FMA) to F(Mg)-actin and F(Ca)-actin was studied spectroscopically. The absorption and fluorescence spectra of bound FMA differed slightly but distinctly between F(Mg)-actin and F(Ca)-actin. Moreover, FMA bound to F(Mg)-actin showed linear dichroism in the presence of 2 mM MgCl2 (or 2mM CaCl2) in the solvent, while the dichroism was abolished by the removal of divalent cations from the solvent. In contrast, FMA bound to F(Ca)-actin did not show any appreciable linear dichroism irrespective of the presence (or absence) of divalent cations in the solvent. These results suggest that the structure of F-actin is characteristically regulated by divalent cations in a dual mode.


Phospholipase A of sea snake Laticauda semifasciata venom. Isolation and properties of novel forms lacking tryptophan. The venom gland extracts of the sea snake Laticauda semifasciata contained at least four forms of phospholipase A separable on a CM-cellulose column. They were designated as phospholipases A I-IV in the order of elution from the column. Phospholipases A I, III, and IV were isolated in a homogeneous state. They were similar to one another in amino acid composition and molecular weight (14,000) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phospholipase A I contained one tryptophan residue. whereas III and IV did not. Although all these forms had the same A2-type positional specificity, they were classified into two groups (I, and III and IV) on the basis of enzymic properties. Phospholipase A I had a higher specific activity and showed normal kinetics, whereas III and IV had approximately one-tenth of the specific activity of I and showed biphasic kinetics due to their activation by the reaction products. Phospholipase A I, the major form, seems to be identical with phospholipase A reported previously (Tu, A.T., Passey, R.B., & Toom, P.M. (1970) Arch. Biochem. Biophys. 140, 96-106), whereas the other two, III and IV, are new. Phospholipase A I became more like III and IV in enzymic properties on modification with N-bromosuccinimide.


Studies on rat liver catalase. X. Effect of hemin and an inhibitor on the translation of catalase messenger RNA1. Rat liver catalase mRNA was translated in a rabbit reticulocyte lysates and wheat germ cell-free system in the presence or absence of hemin and/or a translational inhibitor prepared from reticulocytes, liver cells, and wheat germs. Failure to add hemin to the lysates, or the addition of a hemin-regulated translational inhibitor (HRI) to the hemin-supplemented lysates caused a repressed translation. A preparation of inhibitor from rat liver showed activity similar to that of HRI for this translating system. The translation repression by rat liver inhibitor was reversed by eIF-2 (initiation factor) or GTP, but ATP enhanced the repression. The translation of catalase mRNA in the wheat germ system was not affected by the addition of hemin. An inhibitor prepared from wheat germ extracts, as well as the rat liver inhibitor, markedly decreased the rate of translation. eIF-2, GTP, and ATP behaved in the manner described above. Catalase synthesis in a cell-free system derived from rat liver (using endogenous mRNA) was not influenced by either hemin or the inhibitor. The possibilities are discussed that the synthesis of catalase in liver cells is controlled by a translational inhibitor at the level of chain initiation, and that the formation of the inhibitor from its inactive proinhibitor is regulated by the amount of heme.


Effect of treatment with hemin on rat liver catalase. Rats were injected with a single or repeated doses of hemin intraperitoneally, and the effect on liver catalase [EC 1.11.1.6] was studied. A single administration of hemin caused a reduction in the concentration of liver catalase, both in enzymatic activity and in catalase protein determined immunochemically. The reduction occurred a few hours after the hemin injection, and is probably due to stimulated degradation. Disappearance of radioactivity from liver catalase prelabelled with [14C]leucine was enhanced following the administration of hemin. No evidence for a repression in vivo incorporation of [14C]leucine and [3H]sigma-aminolevulinic acid into liver catalase was obtained with hemin-treated rats. When the hemin was given repeatedly at 12-h intervals, the level of liver catalase decreased considerably. However, the impairment in catalase-synthesizing activity of liver cells of rats thus treated was rather slight, when examined in a cell-free system. Some differences were noted between the results in the present study and those in previous investigations with Sedormid-treated rats.


The identification of aminoalkylphosphonyl cerebrosides in the marine gastropod, Monodonta labio. From Monodonta labio a mixture of aminoalkylphosphonyl cerebrosides was isolated in a yield of 1.0% of the total complex lipids. The aminoalkylphosphonyl cerebrosides were found to be composed of a major portion of 1-O-[6'-O-(N-methylaminoethylphosphonyl)galactopyranosyl]ceramide and a minor portion of 1-O-[6'-O-(aminoethylphosphonyl)galactopyranosyl]ceramide. Palmitic (49.7%) and 2-hydroxy palmitic (12.2%) acids were the main fatty acid constituents and a small amount of oleic acid was also detected. The long chain bases had a characteristic pattern with 16-22 carbon chain lengths, and were classified into four groups: normal dihydroxy monounsaturated bases (40.6%), branched dihydroxy monounsaturated bases (34.0%), dihydroxy diunsaturated bases (22.0%), and trihydroxy bases (2.2%). Among them dihydroxy 18 : 1 and 18 : 2 and branched 19 : 1 bases were predominant.


Microtubule assembly in the presence of adenosine triphosphate. Microtubule (MT) assembly was investigated in the presence of ATP and Ca ions using both crude extract (CE) and purified microtubular proteins (PMP) prepared from porcine brains. ATP inhibited MT assembly from CE prepared with the reassembly buffer containing 1 mM GTP. Half-maximal inhibition occurred at an ATP concentration of 0.4-0.5 mM. Calcium ions, on the contrary, cancelled the ATP-induced inhibition, 1-2 microM calcium ions supporting maximal MT assembly. The ATP-induced inhibition in PMP was not so prominent as in CE, but occurred significantly in the presence of RNA. In PMP dissolved in the reassembly buffer containing ATP and yeast tRNA, the content of the ring fraction decreased significantly as compared with PMP containing only RNA. Furthermore, microtubule-associated proteins were found to be capable of binding ATP. The significance of the ATP-induced inhibition of MT assembly and the release of the inhibition by Ca2+ was discussed.


Formation of lysosulfatide, 3',6'-anhydropsychosine, ceramide, and sphingosine by saponification of cerebroside sulfate. Effect of the sulfate group on the hydrolysis. Saponification of cerebroside sulfate (sulfatide) by refluxing with 1 N KOH in 90% n-butanol for 1 h yielded ceramide, sphingosine, lysosulfatide (psychosine-3'-sulfate ester) and a hitherto unknown compound. The latter compound was identified as 3,6-anhydrogalactosyl sphingosine (3',6'-anhydropsychosine) from its mass spectrum. The structure of lysosulfatide was confirmed by reacylating it to sulfatide by condensing it with lignoceroyl chloride. The resulting sulfatide, which was chromatographically identical to control sulfatides, was not oxidized by periodate. The sulfatide was also permethylated and methanolyzed. The sugar moiety obtained was identified as methyl 2,4,6-tri-O-methylgalactoside by gas-liquid chromatography and thin-layer chromatography. The presence of the sulfate group in lysosulfatide was further confirmed by IR spectroscopy and the presence of radioactivity when it was prepared from [35S]sulfatide. The effect of the sulfate group on cleavage of the galactoside linkage and on the formation of the 3,6-anhydro derivative is discussed.


The effect of aging on the synthesis of hexosamine-containing substances from rat costal cartilage. A decrease in sulfation of chondroitin sulfate with aging. The amount of glycosaminoglycan (GAG) in dry costal cartilage tissue of rats decreased with aging, while the GAG content in mg DNA (unit cartilage cell) remained the same with aging. These results can be explained by the finding that the total number of cartilage cells decreased with aging. Electrophoretic analysis showed that chondroitin 4-sulfate was the major GAG in rat costal cartilage of various ages. Rat costal cartilage of different ages was incubated with radioactive precursors, and newly synthesized GAG was prepared and the radioactivity analyzed to determine the biosynthetic activity. As to changes in the radioactivity uptake with aging per mg dry cartilage tissue, aging influenced [35S]sulfate incorporation into GAG more significantly than [3H]glucosamine incorporation into GAG. There was a significant decrease in the specific radioactivity of [35S]sulfate per mg DNA (unit cartilage cell), whereas the specific radioactivity of [3H]glucosamine per mg DNA did not change significantly with aging. Both the total sulfotransferase activity and the specific activity per mg DNA decreased significantly with aging. Analysis of disaccharide units formed after chondroitinase ABC digestion of labeled GAG isolated from young and old cartilage showed that the percentage of incorporation of [3H]glucosamine into deltaDi-OS increased significantly with aging. These results suggested that the appearance of nonsulfated positions in the structure of the chondroitin sulfate chain increased with aging. On the basis of gel chromatography on Bio-Gel A-1.5 m no significant difference in the approximate molecular size of chondroitin sulfate was observed between the young and old GAG samples. The present study indicated that the sulfation of chondroitin sulfate chains from rat costal cartilage decreased with the process of aging.


Extraction and functional reformation of thick filaments in chemically skinned molluscan catch muscle fibers. A method for the almost complete extraction of myosin from smooth muscle fibers of the anterior byssal retractor muscle (ABRM) of Mytilus edulis was developed, and functional reformation of thick filaments in the fibers was achieved. Complete removal of myosin from the glycerol-extracted ABRM fibers with a solution containing 600 mM KCl, 5 mM MgCl2, and 5 mM ATP was difficult. However, successive treatments of the ABRM fibers with glycerol and saponin made the plasma membrane permeable to Mg-ATP and myosin. The extraction of myosin completely eliminated the tension induced by the addition of Mg-ATP. Partial recovery of tension development was observed by irrigation of myosin into fibers from which myosin had been extracted. Similar results were obtained using rabbit myosin instead of ABRM myosin. Addition of heavy meromyosin, on the other hand, had a suppressive effect on the tension development, as is the case in glycerinated rabbit psoas muscle fibers.


Purification of cat submaxillary kallikrein. The cat submaxillary gland contains 1,000--2,400 kallikrein units (KU)/g of tissue. The submaxillary kallikrein was purified to homogeneity by acetone fractionation, DEAE-Sephadex A-50 chromatography, Sephadex G-75 gel filtration, and Ampholine isoelectric focusing. The kallikrein was separated by isoelectric focusing into 6--7 forms with pI's between 4.2 and 5.1. One mg of the purified kallikrein contained 930--1,260 KU in the dog vasodilator assay, and hydrolyzed 15--25 and 9--12 mumol of N-alpha-benzoyl-L-arginine ethyl ester (BAEE) and N-alpha-toluenesulfonyl-L-arginine methyl ester (TAME), respectively, in 1 min at 25 degrees C and pH 8.0. The Km's of the purified kallikrein with BAEE and TAME were 0.67 and 0.34 mM, respectively. Hydrolysis of N-alpha-benzoyl-L-tyrosine ethyl ester (BTEE), N-alpha-benzoylarginine-p-nitroanilide (BApNA), and casein was small or negligible. The apparent molecular weight of the kallikrein was estimated to be 5 X 10(4) by Sephadex G-100 gel filtration and 4.7 X 10(4) by polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS). The kallikrein was found to contain 18.5% carbohydrate by weight. Trasylol and soybean trypsin inhibitor were not specific inhibitors of this kallikrein.


Fluorometric studies on conformational changes in tropomyosin associated with depolymerization. Spectrofluorometric studies on the conformational changes in tropomyosin associated with depolymerization of the molecules were carried out using 1-anilino-8-naphthalene sulfonate (ANS). When ANS-probed tropomyosin was depolymerized to its monomer, the fluorescence intensity markedly increased, with a decrease in fluorescence polarization. On the other hand, the emission maxima of the ANS-tropomyosin complexes of both forms were the same. The temperature dependence of the polarization of the complexes at various KCl concentrations suggested that the segmental motion of a moiety containing the fluorophore was considerably activated by depolymerization of tropomyosin. In the polymerized and oligomeric forms, a thermal transition in the polarization was observed with a transition temperature of 30 degrees C. Titration curves of tropomyosin with ANS showed simple saturation kinetics with both monomer and polymer, and the apparent dissociation constants were estimated to be 9.93 X 10(-5) M (monomer) and 7.43 X 10(-5) M (polymer). On the other hand, the number of the ANS-binding sites increased from 0.5 to 2.0 per tropomyosin monomer on depolymerization of the molecules. Based on these results, the conformational state of tropomyosin in the polymerized form is discussed.


Polypeptides synthesized in vitro by poly(A)+ and poly(A)- mRNAs of rat ascites hepatoma AH 7974 cells. Polysomal RNA of rat ascites hepatoma AH 7974 cells was fractionated into poly(A)+ and poly(A)- mRNAs and these RNAs were translated into polypeptides in a protein-synthesizing system derived from wheat germ. The analysis of polypeptides synthesized in vitro by two-dimensional gel electrophoresis revealed that there are three classes of polypeptides. The first group can be synthesized equally by both poly(A)+ and poly(A)- mRNAs; the second and third groups are synthesized predominantly by poly(A)+ and poly(A)- mRNAs, respectively. These results suggest that the three classes of polypeptides can be characterized by the presence or absence of poly(A) tails of the corresponding mRNAs.


Human spleen histone H2B. Isolation and amino acid sequence. The amino acid sequences of human histones have been investigated for studies of histone evolution. The whole histone was prepared from human spleen and was separated into 3 fractions, H4+H3+H2A, H2B, and H1, by our technique of CM-cellulose chromatography. The H2B fraction was further purified by Bio-Gel P-60 chromatography. For sequence determination, the H2B molecule was first split into 4 major fragments I to IV, by limited chymotryptic digestion at pH 5.0 and 15 degrees C, followed by Sephadex G-50 chromatography. Fragments I and III were then digested with trypsin, yielding 18 and 16 peptides, respectively, on column and paper chromatographies. Sequence analyses of these tryptic peptides, as well as chymotryptic fragments II and IV, showed no differences from the corresponding parts of calf thymus H2B sequence, making it possible to locate fragments I to IV at residues 1--40, 41--42, 43--121 and 122--125 of the total sequence. The only new findings were microheterogeneities at residues 39 (75% valine and 25% isoleucine) and 124 (70% serine and 30% alanine). The sequence of the most basic cluster at residues 27--24, -Lys-Lys-Arg-Lys-Arg-Ser-Arg-Lys-, was confirmed with a peptide obtained from fragment I by staphylococcal protease digestion. Thus, it is concluded that the H2B sequence of lower mammals was conserved during the evolutionary process leading to man.


Nuclear magnetic relaxation of 19F as a novel assay method of superoxide dismutase. The 19F longitudinal nuclear magnetic relaxation rate (T-1(1p)) of F- solutions is greatly enhanced by copper, zinc or manganese superoxide dismutase. The measured T-1(1p) values are at least an order of magnitude, and in most cases 2 orders of magnitude, higher than those of other metal-containing proteins and low molecular weight complexes. This property is suitable for a direct, specific, and sensitive assay of superoxide dismutase, free of interference by other molecules. For mixtures of the copper, zinc and manganese enzymes, addition of CN- permits quantification of the relative amounts of each enzyme. In the case of copper, zinc enzyme, the method proved exquisitely sensitive to the native state of the active site. Results are reported to show that the 19F relaxation method can be used to assay biological fluids and crude homogenates for copper, zinc and manganese superoxide dismutases, under conditions that offer considerable advantages in comparison to other assay methods.


The physical state of membrane lipids modulates the activation of the first component of complement. Activation of the first component of human complement (C1) by bilayer-embedded nitroxide spin label lipid haptens and specific rabbit antinitroxide antibody has been measured. The nitroxide spin label hapten was contained in host bilayers of either dimyristoyl phosphatidylcholine or dipalmitoyl phosphatidylcholine in the form of both liposomes and vesicles. At a temperature of 32 degrees C, which is intermediate between the hydrocarbon chain-melting temperatures of the two phospholipids, activation of C1 in such vesicles and liposomes is more efficient in the fluid membrane. Studies of C1 activation in binary mixtures of cholesterol and dipalmitoyl phosphatidylcholine indicate that the activation of C1 is not limited by the lateral diffusion of the lipid haptens in these membranes.


The binding of ribosomal protein S4 does not change the gross conformation of the 16 S RNA. The binding of ribosomal protein S4 to the 16 S RNA does not result in a large shape or conformational change in the 16 S RNA under the conditions of reconstitution. The sedimentation coefficient, frictional coefficient ratio, and effective hydrodynamic radius of the 16 S RNA.protein S4 complex are very similar to those obtained for the 16 S RNA free in solution. Only subtle conformational differences were obtained in the comparison of the complex and free 16 S RNA by circular dichroism. Thus, extensive organization of the 16 S RNA by ribosomal protein S4 is not a step in the process of self-assembly of the 30 S subunit.


Photoactivation of the nematicidal compound alpha-terthienyl from roots of marigolds (Tagetes species). A possible singlet oxygen role. The nematicidal compound alpha-terthienyl from roots of Tagetes species generates upon irradiation with near ultraviolet light reactive oxygen species on which the in vitro nematicidal activity depends. This system was studied by following the inhibition of glucose-6-phosphate dehydrogenase by photoactivated alpha-terthienyl and protection of the enzyme activity in the absence of oxygen and by various additions. Addition of mannitol, benzoate, superoxide dismutase or catalase did not have any effect nor did H2O2. This suggests that OH., O-.2, and H2O2 are not the reactive oxygen species involved. The enzyme was protected against photoactivated alpha-terthienyl in air-saturated solutions by singlet oxygen quenchers such as histidine, methionine, tryptophan, bovine serum albumin, and NaN3. Furthermore, inactivation of the enzyme was about 3.5 times faster in D2O than in H2O. When alpha-terthienyl in CH2Cl2 was irradiated in the presence of the olefin adamantylideneadamantane, a stable dioxetane was formed which decomposed to adamantanone when heated above its melting point. These results indicate a singlet oxygen-mediated process.


Rate-limiting steps in metabolic pathways. A method is proposed to detect whether a given enzyme catalyzes a rate-limiting step in a metabolic pathway. With the use of a range of concentrations of specific inhibitors of an enzyme, the finding of a biphasic response with an initial null effect indicates the non-rate-limiting nature of the enzyme. With this method, phosphoenolpyruvate carboxykinase is indicated to catalyze a rate-limiting step in lactate gluconeogenesis in hepatocytes from fasted rats.


Error induction and correction by mutant and wild type T4 DNA polymerases. Kinetic error discrimination mechanisms. The fidelity of DNA synthesis as determined by the misincorporation of the base analogue 2-aminopurine in competition with adenine has been measured as a function of deoxynucleoside triphosphate substrate concentrations using purified mutator (L56), antimutator (L141), and wild type (T4D) T4 DNA polymerases. Although the rates of both incorporation and turnover of aminopurine and adenine decrease as substrate concentrations are decreased, the ratio of turnover/polymerase activity is increased. Thus, the nuclease/polymerase ratio of each of these three DNA polymerases can be controlled. The misincorporation of aminopurine decreases with decreasing substrate concentrations such that all three enzymes approach nearly identical misincorporation frequencies at the lowest substrate concentration. The increased accuracy of DNA synthesis corresponds to conditions producing a high nuclease/polymerase ratio. The misinsertion frequency for aminopurine is independent of substrate concentrations and enzyme phenotype; therefore, the increased accuracy of DNA synthesis with decreasing substrate concentrations is shown to be a result of increased nuclease activity and not increased polymerase or nuclease specificity. The data are analyzed in terms of a kinetic model of DNA polymerase accuracy which proposes that discrimination in nucleotide insertion and removal is based on the free energy difference between matched and mismatched base pairs. A value of 1.1 kcal/mol free energy difference, delta G, between adenine: thymine and aminopurine:thymine base pairs is predicted by model analysis of the cocentration dependence of aminopurine misincorporation and removal frequencies. An independent estimate of this free energy difference based on the 6-fold higher apparent Km of T4 DNA polymerase for aminopurine compared to adenine also gives a value of 1.1 kcal/mol. It is shown that the aminopurine misinsertion frequency for an enzyme having either extremely low 3'-exonuclease activity, Escherichia coli DNA polymerase I, or no measurable exonuclease activity, calf thymus DNA polymerase alpha, is 12 to 15%, which is similar to that for the T4 polymerases and consistent with delta G approximately 1.1 kcal/mol.


Protein degradation in the mouse blastocyst. The degradation characteristics of 56 individual newly synthesized proteins of the Day 4 mouse blastocyst have been examined employing double isotope labeling of proteins for half-life measurement and two-dimensional electrophoresis for separation of proteins. The half-lives ranged from 1 to approximately 30 h with a mean of 12.4 h. Several proteins appeared to have half-lives greater than 30 h but decay times were insufficient to provide precise information for these proteins. The results suggest there is a tendency for proteins with acidic isoelectric points to be degraded more rapidly than basic proteins, and for high molecular weight proteins to be degraded more rapidly than low molecular weight proteins. Although the regressions of these two parameters on half-life were not significant, the direction and magnitude of the trends were similar to those previously described for liver proteins. Two specific proteins, tubulin and actin, were tentatively identified, and their half-lives determined. Tubulin had a half-life of 9.0 h. The half-lives of the provisionally identified gamma, beta, and alpha forms of actin were 2.2, 8.7, and 5.4 h respectively.


Regulation of hydroxydocosanoic acid sophoroside production in Candida bogoriensis by the levels of glucose and yeast extract in the growth medium. Cells of Candida bogoriensis produce as a major extracellular lipid 13-[(2'-O-beta-D-glucopyranosyl-beta-D-glucopyranosyl)oxy]docosanoic acid 6',6''-diacetate (Ac2Glc2HDA), the diacetylated sophoroside of 13-hydroxydocosanoic acid (HDA), along with mono- and unacetylated derivatives. The HDA glycolipid production is greater than 2 g/liter when cells are grown on a "standard" medium of 3% glucose and 0.15% yeast extract. Either lowering the glucose concentration (0.5 to 2.0% glucose, at 0.2% yeast extract) or raising the yeast extract concentration (2 to 4% yeast extract at 3% glucose) greatly decreased the yield of this glycolipid, as well as its rate of synthesis measured by [14C]acetate incorporation. Total HDA production was also depressed on the low glucose medium, as was the activity of UDP-glucose:HDA glucosyltransferase, the first enzyme involved in the synthesis of Ac2Glc2HDA from HDA. Levels of acetyl-CoA:Glc2HDA acetyltransferase were not decreased by growth on a low glucose medium, however, even under conditions in which glycolipid production was less than 4% of that found in the standard medium. Low levels of the HDA glycolipids were monitored by high pressure liquid chromatography of their p-bromophenacyl esters, formed by the action of alpha,beta-dibromoacetophenone on the sodium salt of the lipid in the presence of a crown reagent catalyst. This regulation of extracellular Ac2Glc2HDA production by the nutrient composition of the growth medium may represent an important property in the adaptation of C. bogoriensis to its natural environment, the phyllosphere.


Optical probe responses on sarcoplasmic reticulum. Merocyanine and oxonol dyes. The fluorescence and absorbance of merocyanine 540 in suspensions of skeletal muscle microsomes is altered by the binding of Ca2+ and other cations to the membrane. The order of effectiveness of various cations in causing this effect is La greater than Ca congruent to Mg greater than K. Competition between Ca2+, Mg2+, and K+ suggests the involvement of low affinity, relatively nonspecific cation binding sites in the process. Changes in the fluorescence and absorbance of merocyanine were also observed during ATP-dependent accumulation of calcium into sarcoplasmic reticulum. These changes are satisfactorily explained by the binding of accumulated calcium to binding sites on the interior of sarcoplasmic reticulum membrane. The small absorbance response of the oxonol dye bis[1,3-dibutylbarbituric acid-(5)]trimethinoxonol to Ca2+ and ATP is qualitatively similar to that of merocyanine 540 and can be readily explained by the same mechanism. We have found no clear evidence that any of the observed dye responses are due to changes in the diffusion potential across the sarcoplasmic reticulum membrane generated by an electrogenic transport mechanism. The possibility is considered that merocyanine and oxonol dyes respond to changes in electrostatic surface potential caused by the binding of cations.


The co-purification and common identity of cholyl CoA:glycine- and cholyl CoA:taurine-N-acyltransferase activities from bovine liver. A procedure for the purification of cholyl CoA:glycine and taurine N-acyltransferase activities from the soluble cell fraction of bovine liver is described. The procedure results is an 900-fold enrichment relative to the soluble cell fraction. The final preparation gives a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Mr = 50,900, and runs as a single peak, Mr = 47,000, on gel filtration. The preparation is approximately 80% pure as judged by isoelectric focusing and focuses at a pH of 6.6. The glycine and taurine conjugating activities co-purified and did not separate to any extent in any of the chromatographic steps employed, including a gradient elution from an affinity column and an isoelectric focusing column. Also, kinetic analysis revealed that glycine and taurine appear to compete for a common active site. The two activities had identical temperature-denaturation curves and were equivalently stabilized against temperature denaturation by taurocholate. This data provides strong evidence for a common enzyme for both glycine and taurine conjugation in bovine liver. A preliminary kinetic characterization of the enzyme revealed non-Michaelis-Menten kinetics.


Further studies on a human lung tumor-associated antigen. Comparison of antigens from different tumors. A human lung tumor-associated antigen was purified to homogeneity from a crude cell-free extract of a human lung adenocarcinoma using standard biochemical procedures. In order to facilitate monitoring the recovery of antigen, trace amounts of previously purified and radioiodinated antigen from another lung tumor were added to the crude extract. The purified antigen was a glycoprotein and contained sialic acid. The antigen had a molecular weight of 76,000 and appeared to contain three subunits, each with a molecular weight of 25,000. The antigen had the following physical properties: Stokes radius, 39.4 A; S20,w, 4.24 S; D20,w, 5.15 x 10(-7) cm2 S-1; and a frictional ratio of 1.40. In addition, the purified, radioiodinated antigen retained complete immune reactivity since it could be quantitatively precipitated with specific immune serum. All of these properties were in close agreement with the properties of another antigen which was purified from a separate human lung tumor. Thus, it appeared from the biochemical and immunochemical criteria presented in this report that a common and identical antigen was isolated from two distinct human lung tumor extracts.


Drug-mediated induction of cytochrome(s) P-450 and drug metabolism in cultured hepatocytes maintained in chemically defined medium. Cytochrome P-450 was induced in monolayer cultures of chick embryo hepatocytes incubated in a chemically defined medium. Phenobarbital induced the de novo synthesis of a microsomal protein of Mr = 51,000, whereas beta-naphthoflavone induced the de novo synthesis of a microsomal protein of Mr = 55,000. The increases in these proteins were associated with differential increases in aminopyrine-N-demethylation and benzo-(a)pyrene hydroxylation.


Relief of patellofemoral contact stress by anterior displacement of the tibial tubercle. Elevation of the patellar tendon by means of displacement of the tibial tubercle has been advocated by Maquet, Ficat and Hungerford, and Murray as an alternative to patellectomy for the treatment of patellofemoral arthralgias. Although tubercle elevation certainly reduces the over-all patellofemoral contact force, its effects on the complex local patterns of contact stress are of more direct significance. In a laboratory series using fresh amputation material, arrays of six miniature contact stress sensors were embedded in the retropatellar cartilage of knees subjected to isometric quadriceps-extension forces. The experimental data revealed that elevation of the patellar tendon generally afforded relief of local contact stress regardless of the joint configuration (zero degrees, 45 degrees, or 90 degrees of flexion), but that its effects were most pronounced at 90 degrees of flexion. Progressive increase in the tendon elevation caused progressive reduction in the contact stress. Most of the contact stress relief was achieved, however, with the first one-half inch of tendon elevation; further elevations to one and one and one-half inches were only marginally useful. In view of the increased superior patellar pole contact associated with distal pole flotation, the results indicate that under most circumstances the optimum amount of elevation of the tibial tubercle is about one-half inch.


The total condylar knee prosthesis. A report of two hundred and twenty cases. The total condylar knee prosthesis is a non-hinged surface replacement which can be used for almost all knee deformities. This report discusses the first consecutive 220 arthroplasties in 183 patients. Follow-up time was three to five years. Before operation eighty-six knees had more than 10 degrees of fixed varus deformity and thirty-one knees had more than 10 degrees of fixed valgus deformity. All patients were assessed using The Hospital for Special Surgery scoring system. Of the total of 220 knees, 137 (62%) were rated excellent; sixty-one (28%), good; ten (4.5%), fair; and twelve (5.5%), poor. Of 139 osteoarthritic knees, 93% were rated excellent or good. Complications included three deep infections and four cases of posterior subluxation. The over-all reoperation rate was 3.6%.


Arthrodesis of the knee following failed total knee arthroplasty. In forty-five patients, who had an arthrodesis because of failed total knee arthroplasty, the cause was infection in forty, instability in two, failure of the prosthesis in two, and loosening in one. The arthrodesis succeeded in twenty-nine (81%) of thirty-six patients who had had a minimally or partially constrained arthroplasty and in five (56%) of nine who had had a hinge-type prosthesis inserted. The reasons for failure were severe bone loss, persistent sepsis, and loss of bone apposition after manipulation. The technique of arthrodesis did not seem to influence the final result. External fixation most commonly had to be used because of the infections and the device was kept in place for an average of ten weeks, after which immobilization in a cast was used until the arthrodesis healed.


Failed lumbar disc surgery and repeat surgery following industrial injuries. One hundred and seventy-nine of the compensation patients in this study who had one low-back operation had to have repeat back surgery. One hundred and three Workmen's Compensation Board patients who were reoperated on by a number of surgeons in the Toronto area were independently reviewed with one to two years of follow-up. Many had residual back pain, limited lumbar movement, presisting nerve-root deficits, and psychological disturbances. Forty per cent of the second operations were successful. Subsequent operations yielded progressively poorer results and made more patients worse than better. Operations were frequently undertaken without clear indications or evidence of correctable organic lesions. The results of repeat operations were better when the preceding operation had given more than six months' relief, when sciatica overshadowed back pain, and when a definite recurrent disc herniation was found. Scarring and neurolysis, previous infection, repair of a pseudarthrosis, and adverse psychological factors precluded a good result. Careful patient selection based on total evaluation of the disability including psychological assessment, accurate localization of the lesion by detailed investigation, and, most important, a logical sequence of decisions based on clear, objective criteria are prerequisites for this complex and demanding surgery. Caution and restraint are required when contemplating repeat back surgery.


Analysis of six hundred and twenty-two intertrochanteric hip fractures. Of 622 intertrochanteric fractures, 57% were stable (Types I and II). Twenty-eight per cent were Type III, and 15% were Type IV (the unstable types). The 150-degree telescoping Massie nail proved superior to the fixed 135-degree Jewett nail (particularly for unstable fractures) because it allowed a controlled impaction of the fracture fragments to a stable position. In about one-third of the fractures, some medial displacement occurred. With anatomical reduction and the use of the Massie or ASIF nails, we achieved a decrease in the morbidity and mortality and 96% satisfactory results. Our prospective study was compared with a retrospective study in which other devices were used. Early ambulation and weight-bearing also was a major contributing factor to the improved results in the prospective study. Intertrochanteric hip fractures that are unstable can be fixed with a collapsible nail, and that treatment appears to give as good or better results than the displacement method of Dimon and Hughston or Sarmiento.


Closed Ender nailing of femoral-shaft fractures. Closed Ender nailing was used in sixty fractures of the femoral shaft. Follow-up examination in fifty-two cases is reported. There were only two failures of the method, both in fractures with supracondylar extension. Simple transverse and short oblique fractures and those with unicortical comminution were shown to be stable after nailing, while long oblique fractures and lesions with bicortical comminution had a tendency to shorten and often required cerclage wiring, cast-bracing, or traction. The average hospital stay was 23.7 days. The operations were simple and quick. There was one case of osteomyelitis, one of clinically apparent thrombophlebitis, and one of fat-embolism syndrome in a patient with multiple fractures and multisystem injuries. There were no cases of breakage of the nail, delayed union, or non-union. In most cases knee motion returned to the preinjury level, although in five knees removal of the nails was necessary. The two failures of nailing which required plate fixation occurred in fractures extending into the supracondylar region. Therefore, these fractures are not suitable for Ender nailing.


The initiation of gait.

The initiation of gait. The initiation of gait was studied utilizing electromyography, force-plate data, measurements of walking velocities, and ranges of motion of joints, all of which demonstrated that gait is initiated by the body becoming unbalanced in such a way as to permit a subject to pick one foot the ground in order to take the first step. The subsequent transition of the body to the steady gait pattern occurs rapidly over a period of appproximately three steps. This transition involves rapid changes in the measured forces and activities of the muscles of the lower extremity and the motion of the joints reflects these changes.


Some vagaries of the capitellum.

Some vagaries of the capitellum. The capitellum is the first epiphyseal center of the elbow to ossify. Its normal position is easily subject to misinterpretation as a separation fracture because: (1) the ossification center for the capitellum is situated anteriorly, (2) it is often tilted downward during development, and (3) its epiphysis tends to be wider posteriorly than anteriorly. The capitellum frequently fuses with the trochlea and lateral epicondyle before uniting with the humerus. We reviewed the normal anatomical configuration of the elbow. Some useful methods for evaluating the position of the normally situated and minimally displaced capitellum and distal end of the humerus are presented.


The pathological anatomy of fatal atlanto-occipital dislocations. Nine atlanto-occipital dislocations were found in postmortem examinations of 112 victims of multiple trauma. Axial traction facilitated roentgenographic identification of the injury. A hyperextension mechanism of injury was suggested by the associated injuries, including submental lacerations and mandibular fractures. Atlanto-occipital dislocations were more frequent in children than in adults. A pure dislocation injury without fracture was identified.


The mechanical bonding of methylmethacrylate to cancellous bone. Effect of a hemostatic agent. The bonding of methylmethacrylate to cancellous bone was tested in shear both in control specimens and in those in which microfibrillar collagen had been previously used as a hemostatic agent. The breaking load and energy to failure were significantly lowered by the microfibrillar collagen.


Subungual exostoses.

Subungual exostoses. From 1910 through 1975, forty-four patients with subungual exostoses were seen at the Mayo Clinic. Thirty-four of them had the exostosis on the great toe. Forty-three of the patients were treated by local excision and one was treated by amputation of the hallux. Five patients had local recurrence. None of the tumors underwent malignant change. Histologically, the tumors consisted of a proliferating fibrocartilaginous gap that merged into mature trabecular bone at its base. The growth in the cap was so active that is sometimes mimicked sarcoma, but no true anaplasia was seen. The subungual exostoses were uniformly benign, and local excision was the treatment of choice.


Fractures at the base of the thumb: treatment with oblique traction. Oblique traction on the thumb metacarpal, originally described for the treatment of Bennett's fracture, has been applied to comminuted intra-articular fractures of the thumb metacarpal to counter the forces that cause shortening and varus angulation. It also has been applied to fractures of the trapezium to restore congruity of the trapeziometacarpal joint. It was successful in achieving and maintaining a satisfactory reduction in eight patients with these three types of fractures.


Recurrent dislocation of the peroneal tendon. Recurrent dislocation of the peroneal tendons is an important, although infrequent, cause of disability of the ankle. We used a simple procedure that corrects the basic deformity of a shallow peroneal groove, without the use of metallic fixation or transfer of a tendon or ligament. The groove for the tendons is deepened by removal of some inner fibular substance, while the smooth tenosynovial channel is maintained as an intact periosteal flap on the fibula. The technique was used successfully in ten cases.


Reduction of neglected congenital dislocation of the hip in children over the age of six years. Thirty-two neglected congenital dislocations of the hip in twenty-two children over the age of six years were treated by traction, open reduction and Chiari osteotomy. In five hips, where prolonged traction failed to bring the femoral heads into the vicinity of the acetabulum, a shortening subtrochanteric osteotomy of the femur was performed. The overall results were good and this regime of treatment is recommended.


Biochemical disturbances associated with total hip replacement. A prospective study on 227 patients undergoing arthroplasty of the hip was carried out with reference to the effects on the cardiovascular and respiratory systems. Investigations revealed that the placing of acrylic bone cement and the prosthesis in the femoral shaft produced clinical and biochemical disturbances which were consistent with pulmonary microembolism. A fall in arterial oxygen tension during the procedure and hypoxaemia extending into the postoperative period with elevation of serum lipase and a fall in triglycerides supported the idea that embolisation with marrow fat occurred. The method of venting (by catheter or proximal hole) did not influence the biochemical disturbances. The implications of these findings are discussed.


Juxta-articular bone cysts (intra-osseous ganglia): a clinicopathological study of eighty-eight cases. The clinical, radiographic and pathological features of eighty-eight cases of histologically verified intra-osseous ganglia in eighty-three patients are described. All were located in the subchondral bone adjacent to a joint and most frequently involved the hip, the ankle (medial malleolus), the knee and the carpal bones. Forty-seven of the eighty-three patients were male and all the patients were between fourteen and seventy-three years of age, with an average age of forty-one years. There are two fundamental types of intra-osseous ganglia, one apparently arising by penetration of juxta-osseous ganglion into the underlying bone, a mechanism proved in fourteen of our eighty-eight cases (16 per cent); in the remaining seventy-four cases, the ganglion cyst was primarily intra-osseous ("idiopathic"). The initial cause of the intramedullary mucoid degeneration is discussed. We believe that mechanical stress and repeated minor trauma near the surface of the bone may lead to intramedullary vascular disturbance with consequent foci of aseptic bone necrosis. The revitalisation of these areas causes fibroblastic proliferation, followed by mucoid degeneration of the connective tissue, possibly due to some unknown local factor. Curettage or excision is usually effective, and recurrence (only four cases) is exceptional.


Hip dysplasia without dislocation in one-year-old boys. Six boys were examined during the second year of life, each with symptoms in one hip. The affected femoral capital epiphysis was smaller or more irregular (or both) than that of the unaffected hip but was neither subluxed nor dislocated. The subsequent radiographs revealed changes similar to those in severe Perthes' disease. Nevertheless, we give reasons for believing that this disorder in boys under two years of age should be distinguished from Perthes' disease.


Rib resection in infantile idiopathic scoliosis. Analysis was made of the results of two methods of treatment of progressive infantile idiopathic scoliosis: a group of forty-eight children treated by rib resection, localiser jackets and Milwaukee brace were compared with a 'control' group of nineteen children treated by localiser jackets and Milwaukee brace alone. This failed to show any significant difference between the two groups. Carefully controlled splintage was effective in slowing the rate of progression of most curves which was not significantly affected by the addition of rib resection.


The management of progressive infantile idiopathic scoliosis. Twenty-seven children with progressive infantile idiopathic scoliosis have been reviewed after long-term management. Twenty-two children had single thoracic curves which were diagnosed at an early age and treated in a modified Milwaukee brace until the age of ten years when the spine was corrected and fused. The mean correction after operation was 40 per cent of the initial degree of curvature seen in early childhood before treatment. Solid spinal fusion led to a further moderate loss in correction due to bending of the fusion mass before the spine became stable several years later. Five children had double structural curves and were treated only in a brace. This provided less satisfactory control of these curves but because of the minimal cosmetic deformity, extensive spinal fusion was avoided.


The association between congenital elevation of the scapula and diastematomyelia: a preliminary report. Of forty-six patients with congenital elevation of the scapula, diastematomyelia was confirmed in four and was probably present in five others. Possible reasons for the previously unsuspected association are presented.


Decompression of the extensor tendons at the wrist in rheumatoid arthritis. Simple decompression of the extensor tendons at the wrist was carried out on fifty-four wrists in a total of forty-one patients with rheumatoid disease. This procedure was combined with excision of the ulnar head in forty-five wrists. Complete resolution of the synovitis occurred in 81.5 per cent of the wrists. In two patients the tendons ruptured soon after operation and in both cases this was due to prolapse of the ulnar stump after an associated Darrach procedure. The clinical results of decompression compare favourably with those of the widely accepted operation of dorsal tenosynovectomy.


Synovectomy of the elbow in rheumatoid arthritis: the place of excision of the head of the radius. The results of thirty synovectomies of the elbow for rheumatoid arthritis are reported. Satisfactory relief of pain was obtained in twenty-seven elbows and the range of movement was improved in twenty-one. The classical operation includes excision of the radial head but in this series approximately half the radial heads were conserved with comparable results. The results of synovectomy do not significantly deteriorate with time up to ten years and the operation can be done with good results, especially in respect of relief of pain, even in elbows with relatively advanced rheumatoid disease. Radiographic assessment is not of much help in evaluating the results of the operation, but is essential in selection of elbows for synovectomy.


The management of late diagnosed congenital dislocation and subluxation of the hip-with special reference to femoral shortening. Thirty hips affected by congenital dislocation or subluxation underwent surgical treatment between the ages of five and fifteen years, and the results are presented. Reduction was aided by concurrent femoral shortening in seventeen. An aggressive approach in older children not only relieves the presenting symptoms in most, but also, by improving the anatomical relationships, increases the prospect of success should total replacement become indicated in the future.


Anomalous insertion of the tibialis posterior tendon in congenital metatarsus varus. Congenital metatarsus varus is a common deformity that usually responds to conservative treatment. In fifteen feet operated on for resistant deformity, an anomalous insertion of the tibialis posterior tendon was found in fourteen. Six cadaveric infant feet were dissected to confirm the normal insertion. The dynamic component of the deformity is stressed.


Tibial defect due to acute haematogenous osteomyelitis: treatment and results in twenty-one children. The management of twenty-one children with a defect of the tibial shaft due to acute haematogenous osteomyelitis is described. Half the defects were due to removal of the sequestrum before the involucrum had formed. Only four patients, all under ten years of age, had spontaneous regeneration of the shaft. Eleven children had a posterior tibiofibular graft and six had a transfer of the ipsilateral fibular diaphysis. The results of operation were superior to those of spontaneous regeneration. All the grafts united and the children returned home to lead normal lives. Shortening was only a problem when growth plates or adjacent joints had been damaged. We now leave the sequestrum for up to one year after the onset of infection. If the involucrum fails to form we reconstruct the tibia as soon as possible after sequestrectomy.


Orthopaedic problems in adult hypophosphatasia: a report of two cases. Hypophosphatasia in adults is rare. Two elderly sisters presenting with pathological fractures of the femur are reported to illustrate the difficulties in orthopaedic management of this disease. All patients with a history of repeated fractures, especially from minor trauma and with generalised radiological bony abnormality, should be screened for this rare disease. A consistently low level of serum alkaline phosphatase with the presence of phosphoethanolamine in the urine is diagnostic.


The ball and socket ankle joint.

The ball and socket ankle joint. This paper reports fifteen cases of ball and socket articulation at the ankle followed up for an average of twelve years. All patients showed inequality of leg length. Ten patients showed coalitions of the bones of the hindfoot and nine patients had a reduction in the number of bony elements of the forefoot. Other associated anomalies are described. The abnormality seems to be part of a congenital short-limb malformation, perhaps modified by adaptive change associated with rigidity of the hindfoot. Surgical intervention was not required in any patient in the series.


Early posterior subtalar fusion in the treatment of fractures of the os calcis. Displaced fractures of the os calcis involving the subtalar joint frequently cause chronic disability due to subsequent osteoarthritis. Early posterior subtalar fusion may prevent this outcome. We have reviewed forty-seven fractures in forty-three patients at an average of seven years after operation. Over 90 per cent of patients had an excellent, good or satisfactory result.


Intracortical haemangioma simulating osteoid osteoma. A case of intracortical haemangioma in the tibial diaphysis is reported. The radiological and macroscopic features were identical with osteoid osteoma. In view of this similarity, haemangiomata, despite their rarity at this site, must be considered in the differential diagnosis of osteoid osteoma.


Fibronectin expression is determined by the genotype of the transformed parental cells in heterokaryons between normal and transformed fibroblasts. The expression of fibronectin, a cell surface-associated transformation-sensitive glycoprotein, was studied in hetero- and homokaryons of normal and SV40-transformed human fibroblasts. In immunofluorescence, fibroblast homokaryons had an intense surface-associated and intracelluar fibronectin fluorescence similar to that of normal fibroblasts. Transformed cells and their homokaryons had a minimal surface-associated and a weak intracellular fibronectin fluorescence. In heterokaryons formed between transformed and normal fibroblasts, the expression of fibronectin fell within 24 h to the level of the transformed cell homokaryons. The change was detectable already at 3 h after fusion and was gene-dose dependent. These results show that the transformed genotype determines fibronectin expression in the heterokaryons.


Inhibition of intercellular adhesion by concanavalin A is associated with concanavalin A-mediated redistribution of surface receptors. The inhibition of adhesion between aggregates and layers of embryonic retinal cells by concanavalin A (Con A) and Con A-mediated rearrangements of Con A receptors on retinal cells were studied. A short incubation of aggregates and layers with 10 micrograms/ml Con A substantially reduced aggregate-to-layer adhesion in a subsequent assay without soluble lectin present. This effect of Con A was dose-dependent, temperature-sensitive, involved events subsequent to Con A binding, and was reduced by cytochalasin B. The inhibition produced by succinylated Con A was substantially increased by incubation with antibody to Con A. Visualization of ConA- receptor complexes by fluorescence microscopy revealed that binding of Con A induced clearing of Con A receptors from filopodia, flattened regions of growth cones, and the edges of axons. This clearing reaction was prevented by the same agents that reduced Con A's inhibition of cell adhesion: low temperature, succinylation of Con A, or cytochalasin B. Aggregate-layer adhesion was restored by releasing Con A at 37 degrees C. Inhibitors of protein and ATP synthesis did not prevent recovery of ability to make adhesions. However, release of Con A at lowered temperatures did not prevent recovery. The results suggest that intercellular adhesion is inhibited by events associated with redistribution of Con A-receptor complexes on retinal cells.


Membrane movements and fluidity during rotational motility of a termite flagellate. A freeze-fracture study. Freeze-fracture electron microscopy was used to examine the structure of a region of plasma membrane that undergoes continual, unidirectional shear. Membrane shear arises from the continual clockwise rotation of one part (head) of a termite flagellate relative to the rest of the cell. Freeze-fracture replicas show that the lipid bilayer is continuous across the shear zone. Thus, the relative movements of adjacent membrane regions are visible evidence of membrane fluidity. The distribution and density of intramembrane particles within the membrane of the shear zone is not different from that in other regions of the cell membrane. Also, an additional membrane shear zone arises when body membrane becomes closely applied to the rotating axostyle as cells change shape in vitro. This suggests that the entire membrane is potentially as fluid as the membrane between head and body but that this fluidity is only expressed at certain locations for geometrical and/or mechanical reasons. Membrane movements may be explained solely by cell shape and proximity to rotating structures, although specific membrane-cytoskeletal connections cannot be ruled out. The membrane of this cell may thus be viewed as a fluid which adheres to the underlying cytoplasm/cytoskeleton and passively follows its movements.


Transfer of potassium. A new measure of cell-cell coupling. Mammalian cells of different species differ in sensitivity to ouabain. This sensitivity is expressed as reduced intracellular K+ content, reduced rates of protein synthesis, and cessation of cell multiplication. Using 86Rb+ as a measure of intracellular K+, we found higher levels of radioactivity in mixtures of ouabain-sensitive and -resistant cells cultured in the presence of ouabain than predicted from pure cultures of the two component cell types. The simplest explanation is that K+ and 86Rb+ are being transferred from ouabain-resistant to ouabain-sensitive cells, enhancing the total intracellular 86Rb+ in the culture. A function, "index of cooperation," expresses this enhancement as a number ranging from 0 to 1, and permits comparisons to be made under various culture conditions and using various cell types. An index of cooperation greater than 0 requires cell contact, since no enhancement occurs when contact between two cell types in the same culture is prevented. The index of cooperation for a number of different cell combinations agrees with other measures of cell-cell interaction associated with gap junctions, such as electrical coupling and metabolic cooperation. Coculture of ouabain-sensitive and ouabain-resistant cells in the presence of ouabain also leads to restoration of the capacity for protein synthesis. Autoradiography shows that this restoration occurs in the sensitive cell type and is dependent upon contact with ouabain-resistant cells. Furthermore, sensitive cells are able to multiply in the presence of ouabain when cocultured with resistant cells. Thus K+, presumably transferred to sensitive cells through gap junctions, is able to counteract the toxic effects of ouabain on intracellular K+ levels and protein synthesis, and to restore growth.


Microtubules in mammalian heart muscle.

Microtubules in mammalian heart muscle. Sections of adult mammalian cardiac muscles fixed at room temperature reveal numerous microtubules (24--28 nm in diameter) both near the nucleus and in the extra-myofibrillar space. Microtubules encircle the nucleus, are associated with the myofibrils in a helical arrangement, and form a network that runs transversely at the level of the I band and axially between the myofibrils. Microtubules are more numerous in muscle cells than previously recognized and may perform more than cytoskeletal function.


Epidermal cells adhere preferentially to type IV (basement membrane) collagen. Epidermal cells from adult guinea pig skin attach and differentiate preferentially on substrates of type IV (basement membrane) collagen, compared to those of types I--III collagen. In contrast, guinea pig dermal fibroblasts attach equally well to all four collagen substrates. Fibronectin mediates the attachment of fibroblasts but not of epidermal cells to collagen.


Membrane biogenesis. In vitro cleavage, core glycosylation, and integration into microsomal membranes of sindbis virus glycoproteins. Sindbis virus 26S RNA has been translated in a cell-free protein-synthesizing system from rabbit reticulocytes. When the system was supplemented with EDTA-stripped dog pancreas microsomal membranes, the following results were obtained: (a) Complete translation of 26S RNA, resulting in the production, by endoproteolytic cleavage, of three polypeptides that are apparently identical to those forms of C, PE2, and E1 that are synthesized in vivo by infected host cells during a 3-min pulse with [35S]methionine. (b) Correct topological deposition of the three viral polypeptides--in vitro-synthesized PE2 and E1 forms are inserted into dog pancreas microsomal membranes in a orientation which, by the criterion of their limited (or total) inaccessibility to proteolytic probes, is indistinguishable from that of their counterparts in the rough endoplasmic recticulum of infected host cells; in vitro-synthesized C is not inserted into membranes and therefore is accessible to proteolytic enzymes, like its in vivo-synthesized counterpart. (c) Core glycosylation of in vitro-synthesized PE2 and E1 forms, as indicated by binding to concanavalin A Sepharose and subsequent elution by alpha-methylmannoside.


The angular distribution of directional changes of guided 3T3 cells. This paper reports that the angular distribution of directional change of 3T3 cells which followed guiding lines on a substrate and left the guidance at various angles shows a peculiar preference for angles between 30 degrees and 60 degrees. Regarding the phenomenon of cellular guidance itself, 3T3 cells faced with a choice between guiding lines toward different directions seem to explore various options before following one. This observation suggests that the outcome of a probing action and not substrate properties alone can explain the guidance behavior of 3T3 cells.


Distribution of cell surface saccharides on pancreatic cells. I. General method for preparation and purification of lectins and lectin-ferritin conjugates. We describe here a simple, general procedure for the purification of a variety of lectins, and for the preparation of lectin-ferritin conjugates of defined molar composition and binding properties to be used as probes for cell surface saccharides. The technique uses a "universal" affinity column for lectins and their conjugates, which consists of hog sulfated gastric mucin glycopeptides covalently coupled to agarose. The procedure involes: (a) purification of lectins by chromatography of aqueous extracts of seeds or other lectin-containing fluids over the affinity column, followed by desorption of the desired lectin with its hapten suge; (b) iodination of the lectin to serve as a marker during subsequent steps; (c) conjugation of lectin to ferritin with glutaraldehyde; (d) collection of active lectin-ferritin conjugates by affinity chromatography; and (e) separation of monomeric lectin-ferritin conjugates from larger aggregates and unconjugated lectin by gel chromatography. Based on radioactivity and absorbancy at 310 nm for lectin and ferritin, respectively, the conjugates consist of one to two molecules of lectin per ferrritin molecule. Binding studies of native lectins and their ferritin conjugates to dispersed pancreatic acinar cells showed that the conjugation procedure does not significantly alter either the affinity constant of the lectin for its receptor on the cell surface or the number of sites detected.


Distribution of cell surface saccharides on pancreatic cells. II. Lectin-labeling patterns on mature guinea pig and rat pancreatic cells. The surface saccharide composition of collagenase-dispersed pancreatic cells from adult guinea pig and rat glands was examined by using eight lectins and their ferritin conjugates: Concanavalin A (ConA); Lens culinaris (LCL); Lotus tetragonolobus (LTL); Ricinus communis agglutinins I and II (RCA I, RCA II); Soybean agglutinin (SBA); Ulex europeus lectin (UEL); and wheat germ agglutinin (WGA). Binding studies of iodinated lectins and lectin-ferritin conjugates both revealed one population of saturable, high-affinity receptor sites on the total cell population (approximately 95% acinar cells). Electron microscopy, however, revealed differences in lectin-ferritin binding to the plasmalemma of acinar, centroacinar, and endocrine cells. Whereas acinar cells bound heavily all lectin conjugates, endocrine and centroacinar cells were densely labeled only by ConA, LCL, WGA, and RCA I, and possessed few receptors for LTL, UEL, and SBA. Endocrine and centroacinar cells could be differentiated from each other by using RCA II, which binds to centroacinar cells but not to endocrine cells. Some RCA II receptors appeared to be glycolipids because they were extracted by ethanol and chloroform-methanol in contrast to WGA receptors which resisted solvent treatment but were partly removed by papain digestion. RCA I receptors were affected by neither treatment. The apparent absence of receptors for SBA on endocrine and centroacinar cells, and for RCA II on endocrine cells, was reversed by neuraminidase digestion, which suggested masking of lectin receptors by sialic acid. The absence of LTL and UEL receptors on endocrine and centroacinar cells was not reversed by neuraminidase. We suggest that the differential lectin-binding patterns observed on acinar, centroacinar, and endocrine cells from the adult pancreas surface-carbohydrate-developmental programs expressed during morphogenesis and cytodifferentiation of the gland.


The surface epithelium of teleostean fish gills. Cellular and junctional adaptations of the chloride cell in relation to salt adaptation. Various species of teleostean fishes were adapted to fresh or salt water and their gill surface epithelium was examined using several techniques of electron microscopy. In both fresh and salt water the branchial epithelium is mostly covered by flat respiratory cells. They are characterized by unusual outer membrane fracture faces containing intramembranous particles and pits in various stages of ordered aggregation. Freeze fracture studies showed that the tight junctions between respiratory cells are made of several interconnecting strands, probably representing high resistance junctions. The organization of intramembranous elements and the morphological characteristics of the junctions do not vary in relation to the external salinity. Towards the base of the secondary gill lamellae, the layer of respiratory cells is interrupted by mitochondria-rich cells ("chloride cells"), also linked to respiratory cells by multistranded junctions. There is a fundamental reorganization of the chloride cells associated with salt water adaptation. In salt water young adjacent chloride cells send interdigitations into preexisting chloride cells. The apex of the seawater chloride cell is therefore part of a mosaic of sister cells linked to surrounding respiratory cells by multistranded junctions. The chloride cells are linked to each other by shallow junctions made of only one strand and permeable to lanthanum. It is therefore suggested that salt water adaptation triggers a cellular reorganization of the epithelium in such a way that leaky junctions (a low resistance pathway) appear at the apex of the chloride cells. Chloride cells are characterized by an extensive tubular reticulum which is an extension of the basolateral plasma membrane. It is made of repeating units and is the site of numerous ion pumps. The presence of shallow junctions in sea water-adapted fish makes it possible for the reticulum to contact the external milieu. In contrast in the freshwater-adapted fish the chloride cell's tubular reticulum is separated by deep apical junctions from the external environment. Based on these observations we discuss how solutes could transfer across the epithelium.


Dexamethasone induces irreversible G1 arrest and death of a human lymphoid cell line. Growth of a human leukemic T-cell line (CEM C7) in 10(-6) M dexamethasone results in inhibition of growth and rapid loss of cell viability after a delay of approximately 18 to 24 hours. Analysis of dexamethasone-treated cells by flow-microfluorometry showed that they were arrested in the G1 phase of the cell cycle. Loss of cell viability began at the same time as G1 accumulation was first detectable, and 20% of all cells were found to be blocked in G1 at this time suggesting that loss of viability and G1 arrest were coincident events. Half-maximal and maximal effects on both viability and G1 arrest after 48 hours in steroid were nearly identical with respect to steroid concentration and corresponded to half-maximal and full occupancy of glucocorticoid specific receptor by hormone, consistent with a glucocorticoid receptor mediated mechanism for both phenomena. Most non-viable cells were arrested in G1, and accumulation of cells in G1 was irreversible; removal of steroid in the presence of colcemid did not result in a decreased fraction of G1 cells. Furthermore, dexamethasone treatment did not protect cells against the effects of 33258 Hoechst-amplified killing of bromodeoxyuridine substituted cells exposed to light. These results show that dexamethasone arrests these leukemic cells in G1 and strongly suggest that dexamethasone-treated cells are killed upon entry into G1.


A transient increase in amino acid transport modulated by insulin in differentiating muscle cells. During synchronous differentiation of embryonic chick muscle cells in cultures, the Na-dependent uptake of an amino acid analog, alpha-amino isobutyric acid (AIB) undergoes in abrupt, transient increase. The increase in AIB uptake is concomitant with the rapid fusion of mononucleated myoblasts, and precedes the accumulation of muscle-specific proteins. Subsequently, Na-dependent AIB transport diminishes markedly during postfusional differentiation of myotubes. The rate of AIB uptake is increased by insulin both before and after myoblast fusion. This stimulation by insulin is restricted to the Na-dependent component of total AIB uptake but is apparently not the result of insulin-mediated increase in the trans-membrane Na gradient.


Frequency difference between nuclear and polysomal sequences of poly(A)-containing RNA from cultured AKR mouse embryo cells. The involvement of post-transcriptional mechanisms in the determination of the frequency distribution of messenger RNA sequences has been studied in cloned mouse embryo cells in culture. Hybridization kinetic experiments between poly(A)-containing RNA and complementary DNA have been used to study the alterations in frequencies which occur in those nuclear poly(A)-containing RNA sequences which are conserved in cytoplasmic poly-ribosomes. Sequences adjacent to nuclear poly(A) tracts are present in a much narrower frequency distribution in the nucleus than in polysomes, with a large proportion of the nuclear sequences present in an average frequency of about one molecule per cell. Few nuclear sequences appear to be present in more than ten copies per cell. A minimum of 70% of these sequences are also found in poly-ribosomal RNA but in greatly altered frequencies. Abundant sequences which comprise a major fraction of the poly(A)-containing polyribosomal RNA are derived from a small fraction of the nuclear poly(A)-adjacent RNA sequences. Very few nuclear poly(A)-adjacent sequences are present in a frequency characteristic of high abundance polysomal sequences. Conversely, poly(A)-containing polyribosomal RNA appears to contain few sequences which are present in as low a frequency as the majority of nuclear poly(A)-adjacent sequences. These observations suggest that post-transcriptional mechanisms play a major role in determining the steady-state frequency of polyribosome-associated messenger RNAs.


The effect of amino acids on the temperature sensitive phenotype of the mammalian leucyl-tRNA synthetase mutant tsHl and its revertants. The temperature sensitive leucyl-tRNA synthetase mutant tsHl and two revertants have been compared to the parental Chinese hamster ovary cells with respect to the effects of amino acid concentrations in the medium on growth. Elevating the leucine concentration 30- or 100-fold allowed tsHl to grow exponentially at 38.5 degrees C, normally the nonpermissive temperature. Partial revertants that had recovered some enzyme activity required smaller supplements for growth. Measurements of the leucine pools indicated that they respond directly to the extracellular leucine concentration and may mediate the effect. Use of combinations of amino acids confirmed that isoleucine has a similar though weaker effect on tsHl and identified an even weaker protection by valine. The triple combination of leucine, isoleucine and valine was a much more efficient medium supplement and three times normal concentrations of these amino acids supported growth of tsHl at 38.5 degrees C. It is postulated that they are acting at their respective aminoacyl-tRNA synthetases to help stabilize a complex which also contains the mutant leucyl-tRNA synthetase. The pool size measurements also showed that the leucine pools of tsHl and a revertant increased 2-fold more in a response to increased temperature than those of WT. It is suggested that this is a regulatory response to low leucyl-tRNA synthetase activity and is important in determining growth phenotypes.


Isolation and characterization of revertants of the mammalian temperature sensitive leucyl-tRNA synthetase mutant tsHl. Nine spontaneous and seven ethyl methanesulfonate induced revertants of the Chinese hamster ovary cell line mutant (tsHl), which possesses a temperature sensitive leucyl-tRNA synthetase, were isolated and characterized with respect to growth rate, leucyl-tRNA synthetase activity and thermolability, intracellular leucine pool size, and rRNA content. Although most revertants had increased leucyl-tRNA synthetase activity, and of those tested, all but one had increased thermostability, each appears to be unique. One revertant may be an intergenic suppressor since it appears to contain an elevated level of tsHl-like synthetase. There was no evidence for any of the revertants having increased rRNA and tRNA contents, however, many showed leucine pools two to three times larger than wild type cells. Since similar increases have been observed in tsHl cells they are believed to result from regulation of leucine pool size by the leucyl-tRNA synthetase and are of a magnitude sufficient to affect significantly the growth of revertants at 38.5 degrees C.


Growth kinetics and collagen synthesis of normal skin, normal scar and keloid fibroblasts in vitro. Fibroblasts were isolated from keloid, normal skin, and normal scar and maintained in tissue culture for four passages. Growth kinetics were the same for all groups on days 2 through 12. However, the rate of collagen synthesis per fibroblast was greater in keloid derived cells than any controls at all growth phases. Keloid fibroblasts have an autonomous capacity to synthesize collagen at a significantly increased level in vitro, which may explain in part why these lesions are characterized by increased collagen deposition.


The nature of cells generating human myeloma colonies in vitro. Freshly explanted human myeloma cells formed colonies of monoclonal plasma cells in soft agar in the presence of medium conditioned by the adherent spleen cells of mineral oil-primed BALB/c mice. The medium showed peak activity at a dilution of 1:4. 2-mercaptoethanol or monothioglycerol was necessary for colony formation. Other thiols tested were ineffective in promoting colony growth. Colony-forming cells adhered to nylon wool, but not glass beads or plastic dishes. The presence of E-rosetting cells was not required for myeloma colony formation. Antibody prepared against a human myeloma cell line, RPMI 8226, reduced colony formation. These studies demonstrate the usefulness of this bioassay for determining functional properties of the myeloma colony-forming cell.


Electrophysiological properties of human oviduct smooth muscle cells in dissociated cell culture. Intracellular recordings were made from human oviduct smooth muscle maintained in cell culture. Solitary cells isolated from one another and cells in contact with one another retained electrical properties of smooth muscle in vivo. Membrane potential of solitary cells and connected cells was -35 mV. Connected cells formed electrotonic junctions which transmitted current from one cell to another. This current spread was responsible for differences in input resistance and time constant in solitary cells, 66 Momega and 96 msec, compared to connected cells, 26 Momega and 56 msec. All cells expressed delayed rectification to depolarizing current pulses. Some cells generated action potentials spontaneously or in response to intracellular current pulses. Action potentials were abolished by cobalt or by EGTA. Slow wave potentials, 5 . 20 mV in amplitude, occurred continuously once every 15 to 45 seconds in connected cells.


Selective changes in tRNA methyltransferase activity in confluent monolayers of WI-38 cells stimulated to proliferate. In quiescent confluent monolayers of WI-38 cells, the specific activity of the tRNA methyltransferases falls to 20% of the level found in log phase cells. When the resting cells are stimulated to proliferate by a change to fresh medium, the enzyme show a rapid rise in specific activity which correlates with early increases in the rate of tRNA synthesis. The specific activity of the enzymes continues to rise throughout the period of DNA synthesis, at the end of which it is somewhat higher than that of log phase cells. The increases in enzyme activity could be blocked by exposure of the stimulated cells to Actinomycin D (2 microgram/ml). The increases in activity were not equivalent for the different base-specific enzymes. The contribution of the N2-methylguanine specific enzyme remained relatively constant, while that of the N2,N2-dimethyl-guanine specific and 1-methyladenine specific enzymes doubled and tripled, respectively, by late S phase. The contributions of the 1-methylguanine and the 7-methylguanine specific enzymes fell to a few percent of the total by late S phase. This indicates non-coordinate variations in the expression of the different base-specific enzymes after stimulation of resting cells and may be related to altered isoaccepting tRNA profiles observed in resting and growing cells.


Colony formation in agar by multipotential hemopoietic cells. Agar cultures of CBA fetal liver, peripheral blood, yolk sac and adult marrow cells were stimulated by pokeweed mitogen-stimulated spleen conditioned medium. Two to ten percent of the colonies developing were mixed colonies, documented by light or electron microscopy to contain erythroid, neutrophil, macrophage, eosinophil and megakaryocytic cells. No lymphoid cells were detected. Mean size for 7-day mixed colonies was 1,800-7,300 cells. When 7-day mixed colonies were recloned in agar, low levels of colony-forming cells were detected in 10% of the colonies but most daughter colonies formed were small neutrophil and/or macrophage colonies. Injection of pooled 7-day mixed colony cells to irradiated CBA mice produced low numbers of spleen colonies, mainly erythroid in composition. Karyotypic analysis using the T6T6 marker chromosome showed that some of these colonies were of donor origin. With an assumed f factor of 0.2, the mean content of spleen colony-forming cells per 7-day mixed colony was calculated to vary from 0.09 to 0.76 according to the type of mixed colony assayed. The fetal and adult multipotential hemopoietic cells forming mixed colonies in agar may be hemopoietic stem cells perhaps of a special or fetal type.


The requirement of DNA synthesis for the induction of alkaline phosphatase by bromodeoxyuridine in a derivative of the HeLa cell line. Non-lethal concentrations of bromodeoxyuridine induce a 2- to 5-fold increase in the specific activity of alkaline phosphatase in a HeLa subclone, S3G. Experiments employing 10-hour pulses of BRdU showed that 48 hours were required before induction commenced, and that maximal induction was attained by 96 hours. Under conditions in which DNA synthesis was prevented with hydroxyurea induction did not occur. Upon removal of hydroxyurea both DNA synthesis and induction were rapidly reestablished. Furthermore, experiments employing radiolabelled BRdU demonstrated that the kinetics of the induction process paralleled the incorporation of the analogue into cellular DNA. These results indicate that DNA synthesis, or some process intimately linked to DNA synthesis, is required for the induction of alkaline phosphatase, and suggest that the mode of the induction may be through the incorporation of the analogue into cellular DNA.


Superoxide dismutase specific activities in cultured human diploid cells of various donor ages. It has been postulated that superoxide dismutase (SOD) protects cells from free radical-induced damage. In these experiments SOD specific activity was measured as established human diploid cell lines from various donor ages progressed through their in vitro lifespan. Significant elevations in activity occurred during the in vitro lifespans of cells from fetal and newborn donors, but no change in activity was detected during the lifespan of cells from an adult donor. In addition, a direct relationship between enzyme activity and donor age was detected with the following relative activities: adult greater than newborn greater than fetal. The possible relationship between these findings and the free radical theory of aging is discussed.