A new amino acid, 3-(2,5-SS-dicysteinyl-3,4-dihydroxyphenyl)alanine, from the tapetum lucidum of the gar (Lepisosteidae) and its enzymic synthesis. The tapetum lucidum of the alligator gar Lepisosteus was shown by t.l.c. to contain a new phenolic amino acid, which is apparently a major constituent of the reflecting material. It was isolated in a yield of 0.5 mg/eye and its physical and chemical characteristics, especially reductive hydrolysis with hydriodic acid giving dopa (3,4-dihydroxyphenylalanine) and cysteine, suggested that it might to SS-dicysteinyldopa. Tyrosinase oxidation of L-dopa in the presence of an excess of L-cysteine yielded, in addition to known 5- and 2-S-cysteinyldopa, the same amino acid as that isolated from the eye of the gar, thus confirming the gross structure. The position of the two cysteine residues was established by the fact that tyrosinase oxidation of catechol and cyteine gave 3-S-cysteinylcatechol and 3,6-SS-dicysteinylcatechol. The natural amino acid is therefore formulated as 3-(2,5-SS-dicysteinyl-3,4-dihydroxyphenyl)alanine (2,5-SS-dicysteinyldopa), which may be formed by two consecutive additions of cysteine, first to dopaquinone and then to 5-S-cysteinyldopaquinone. The enzymic synthesis of 2,5-SS-dicysteinyldopa in vitro suggests that it may also be involved in the biosynthesis of phaeomelanin.


Studies on the purification of rat liver uridine diphosphate glucuronyltransferase. 1. A stable, more highly purified, preparation of UDP-glucuronyltransferase was obtained than previously reported. 2. Enzyme activity towards o-aminophenyl and p-nitrophenyl was increased 43- and 46-fold respectively. 3. The final preparation contains only three staining polypeptide bands visible after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 4. The only known major accompanying protein appears to be epoxide hydratase. 5. The purified enzyme activity towards o-aminophenol can still be activated 3 fold by diethylnitrosamine. 6. On evidence from purification, o-aminophenol and p-nitrophenol appear to be glucuronidated by the same enzyme protein. The possible recognition of the UDP-glucuronyltransferase enzyme is discussed.


Cinnabarinate synthase from baboon (Papio ursinus) liver. Identity with catalase. The enzyme chinnabarinate synthase was purified from the nuclei of baboon liver. Two purified fractions were obtained that exhibited a typical haem protein absorption spectrum; a probable identity with catalase was demonstrated. It was confirmed that catalase in the presence of Mn2+ produces cinnabarinate from 3-hydroxyanthranilate. Doubt is expressed on the existence of a distinct cinnabarinate synthase enzyme.


Enzymic assays for isomers of 2,6-diaminopimelic acid in walls of Bacillus cereus and Bacillus megaterium. An enzymic assay for individual isomers (meso-, LL- and DD-) of 2,6-diaminopimelate was developed. The enzyme 2,6-diaminopimelate decarboxylase specifically attacked meso-diaminopimelate and was used to measure this isomer manometrically. The meso- and LL-isomers were measured together manometrically in a coupled assay with diaminopimelate decarboxylase and diaminopimelate epimerase (which converts LL-diaminopimelate into meso-diaminopimelate). The DD-isomer was not attacked by either enzyme and was measured, as residual diaminopimelate after the coupled assay, by a colorimetric method, which was also used to measure total diaminopimelate before enzymic treatments. The coupled enzymes were also used to prepare pure DD-isomer from chemically synthesized diaminopimelate. A mixture of diaminopimelate isomers was present in walls of four strains of Bacillus megaterium [in each about 75% (w/w) meso-, 18% LL- and 7% DD-] and in walls of two strains of Bacillus cereus (about 85% meso-, 8% LL- and 7% DD-). One strain of B. cereus contained at least 95% meso-diaminopimelate, with only traces of LL- and DD-isomers. Peptidoglycan from Escherichia coli was assayed as containing at least 95% meso-isomer. The proportion of isomers in the wall of a strain of B. megaterium remained constant after growth in a variety of different media.


Azathioprine in early rheumatoid arthritis. Comparison with gold and chloroquine. This study compares the effect of azathioprine with those of gold and chloroquine in early (Class II) rheumatoid arthritis (RA). Thirty-three similar patients with classic or definite RA of less than 5 years duration were randomly entered, 11 into each drug group. Assessment of standard clinical and laboratory measures at 12 and 24 weeks showed significant improvement in all three groups. In general, all three drugs were effective antirheumatic agents with low toxicity. However, because of serious potential toxicity, azathioprine could not be recommended over gold and chloroquine in early RA therapy.


Comparative study of immunologic methods for demonstration of antibodies to soluble nuclear antigens. Immunofluorescence, hemagglutination, complement fixation, and immunodiffusion. Four methods to detect antibodies reactive with soluble nuclear antigens were compared-the fluorescent antibody technique, hemagglutination, complement fixation, and immunodiffusion. Centain advantages and disadvantages of each method are described. The first three methods have greater sensitivity than immunodiffusion, but immunodiffusion discriminates most reliably between different antibodies.


Absorption, distribution and excretion of a dihydroxydibutyl ether labelled with 14C in animals. Kinetic studies were performed in rats, dogs and monkeys after oral administration of 14C-labelled dihydroxy-dibutyl ether (DHBE; Discinil), a known choleretic agent. The data presented indicate that the compound was intirely and readily absorbed and that it was excreted mainly with the urine in all the three species. The complete distribution pattern was investigated in rats. In most tissues high levels of radioactivity were already present after 10 min and the peak concentration was reached within the first 30 min. The decay was rapid and at the fourth hour only traces of radioactivity could be still detected in the tissues. Liver and kidney were the target organs for the radioactive material (which is in accordance with the activity and the excretory route of DHBE). The blood levels paralleled the general tissue distribution. The excretion of radioactivity was completed within 24 h in the three animal species and it took place by the urinary route, mainly witin the first 7--8 h.


Combined T and B lymphocyte marker test in lymphoproliferative disorders. The combined T and B lymphocyte marker test of Mendes et al (1974) has been developed to provide an easy, reproducible test for clinical practice. It has been used to study subpopulations of peripheral lymphocytes in patients with lymphoid malignancies. Significant increases in the numbers of lymphocytes which bind both sheep erythrocytes (E) and the fixed, third component of complement (C3) have been found using this test, in the peripheral blood of all untreated lymphosarcoma (L/SA) patients and T cell acute lymphoblastic leukaemia (ALL) patients. Such increases were not found in blood from patients with chronic lymphatic leukaemia (CLL), other non-lymphoid malignancies, null-cell ALL or normal individuals. These mixed E and C3 receptor bearing cells may be Fc receptor positive and represent a separate subpopulation of lymphocytes.


Immunoglobulins in human aqueous humour. The immunoglobulin concentrations in human aqueous humour from 44 patients aged 35 to 85 years with cataracts were measured by a standard immunodiffusion method. IgG was found in all the samples (mean level 7-0 mg/100 ml. IgD, IgA or IgM could not be detected. There was no significant difference in IgG levels in aqueous humour between the two sexes, in different age groups, and in the different types of cataracts.


Experimental internuclear ophthalmoplegia. A midline experimental lesion separating the medial longitudinal fasciculi at and below the level of the abducens nuclei without damaging either fasciculus at the level of the nuclei has produced defects of ocular motility resembling those of clinical internuclear ophthalmoplegia. Electromyographic recordings during lateral gaze demonstrate: (1) lack of inhibition of the lateral rectus muscle in the adducting eye, (2) delayed inhibition of the medial rectus muscle in the abducting eye, and (3) occasional evidence of excitation of the medial rectus muscle of the abducting eye probably associated with pupillary constriction. The presumed physiologic mechanisms involved in conjugate gaze movements are discussed in the light of the experimental findings.


Optic nerve glioma and phaeochromocytoma associated with von Recklinghausen's disease: A case report. A 32-year-old woman who died suddenly during pregnancy as a result of an adrenal phaeochromocytoma also suffered from von Recklinghausen's disease. 21 years earlier she had an optic nerve glioma successfully removed. The association between von Reckinghausen's disease and optic nerve glioma/phaeochromocytoma is well established. So far as is known, the present case is the first ever published with the triad: von Recklinghausen's disease, optic nerve glioma, and phaeochromocytoma.


Infective keratitis in soft contact lens wearers. Eight cases of infective keratitis are reported in wearers of soft contact lenses. Four of them had normal eyes and were wearing lenses on a continual basis. One was wearing a lens continually for therapeutic reasons. Three others were wearing lenses daily or intermittently. The four latter cases were using contaminated lens solutions. Two of the continual lens wearers lost vision to the point of blindness. A significant factor in their bad outcome was the fact that both lived in areas remote from adequate ophthalmic services. Serratia liquefaciens was implicated in one case. This is thought to be the only reported case of corneal abscess due to this organism in the past 16 years. S. marcescens was grown in another case from the lens solutions and carrying case.


Characterization of N-polyhedrin of two baculovirus strains pathogenic for Orgyia pseudotsugata. N-polyhedrin of inclusion bodies of two nucleopolyhedrosis viruses of Orgyia pseudotsugata was characterized. Alkali-dissolved N-polyhedrin from both virus strains was of similar size and consisted of 12S molecule of 209 000 daltons. Eight subunits of approximately 26 000 daltons were found to form the 12S molecules. N-polyhedrin from both viruses showed two main antigens by immunodiffusion. The subunits appear to possess one antigen and, upon formation of the 12S molecule, a new antigen is created. Both the subunit and 12S antigens from the two virus strains were shown to be antigenically related. The 12S molecule of both viruses also appears to possess a minor antigen unique to each virus.


Restriction endonuclease map of Euglena gracilis chloroplast DNA. A physical map of the Euglena gracilis chloroplast genome has been constructed, based on cleavage sites of Euglena gracilis chloroplast DNA treated with bacterial restriction endonucleases. Covalently close, circular chloroplast DNA is cleaved by restriction endonuclease SalI into three fragments and by restriction endonuclease BamHI into six fragments. These nine cleavage sites have been ordered by fragment molecular weight analysis, double digestions, partial digestions, and by digestion studies of isolated DNA fragments. A fragment pattern of the products of EcoRI restriction endonuclease digestion of Euglena chloroplast DNA is also described. One of these fragments has been located on the cleavage site map.


Kinetics and mechanism of refolding of bovine carbonic anhydrase. A probe study of the formation of the active site. In kinetic studies of the folding of bovine carbonic anhydrase from disorganized to native structure, an azosulfonamide, 2-(4-sulfomylphenylazo)-7-acetamido-1-hydroxynaphthalene-3,6-disulfonate (I), has been used as a probe to follow the dynamics of formation of the active site region. The probe is a specific inhibitor of the native enzyme that binds in the active site crevice. The experiments, with previous data (Yazgan, A., and Henkens, R. W. (1972), Biochemistry 11, 1314), show that a tight binding site for I forms at an intermediate stage in the folding process. A subsequent conformational change perturbs the visible absorption and circular dichroism of bound I and could result in even tighter binding. The subsequent change completes formation of the active site. This is shown by results from separate experiments on the kinetics of recovery of activity (p-nitrophenyl acetate as substrate). Similar probe methods could be used with other proteins and enzymes to study the kinetics and mechanism of regeneration of specific sites--for example, the active site.


Antisera to poly(A)-poly(U)-poly(I) contain antibody subpopulations specific for different aspects of the triple helix. Rabbit antibodies to the triple-helical polynucleotide poly(A)-poly(U)-poly(I) were fractionated into three major antibody populations, each recognizing a different conformational feature of the triple-helical immunogen. Two distinct populations were purified from precipitates made with poly(A)-poly(U)-poly(U) and poly(A)-poly(I)-poly(I). The former reacted with double-stranded poly(A)-poly(U) or poly(I)-poly(C), and similar populations could be purified with either double-stranded form. The second population recognized the poly(A)-poly(I) region of the triple helix, and the third required all three strands for reactivity. These immunochemical studies suggest that the poly(A) and poly(U) have the same orientation in the triple-helicical poly(A)-poly(U)-poly(I) as in the double-helical poly(A)-poly(U), in which they have Watson-Crick base pairing.


Acquisition of the covalent quaternary structure of an immunoglobulin G molecule. Reoxidative assembly in vitro. We recently reported results of an investigation of the reoxidation of a human, monoclonal immunoglobulin G, following selective reduction of its interchain disulfides by dithiothreitol (Sears, D.W., et al. (1975), Proc. Natl. Acad. Sci. U.S.A. 72, 353). In that work, we described the reoxidative behavior of the molecule under nondissociating conditions. In the present paper, results are presented of the reoxidation of heavy (H) and light (L) chains of this protein alone, or mixed in varying proportions after separation, or mixed with the L chains modified prior to recombination and reoxidation. The overall reoxidative asembly patterns in experiments with H and L separated prior to recombination are similar to those observed when the chains remain noncovalently associated throughout. With equimolar mixtures of H and L, the reoxidation rates also are similar to those of unseparated chains. However, when L chains are present in excess, the overall in vitro rates of covalent assembly are generally diminished, probably indicating transient nonproductive interactions. At the highest molar excesses of L (3:1), the assembly pathways may also be modified. In all experiments with excess L chains, covalent L2 dimers form at rates which are comparatively slow relative to the H2L2 assembly rates. Two kinds of reoxidation experiments with modified L chains are described here for the first time. In the first, the free half-cystine of L is irreversibly blocked by reaction with iodoacetamide, and the alkylated L chains are recombined with reduced H chains. This experiment isolates the reactions in which H2 disulfides are formed without the accompanying formation of HL bonds. Although the alkylated L chains do not play a direct role in the reoxidation, their presence is required to inhibit aggregation and precipitation of high-molecular-weight products which otherwise ensue; this suggests a possible biological role for excess L in vivo. In the second kind of experiment, covalent L2 dimers are mixed with reduced H chains. L2 rapidly disappears with the concurrent appearance of HL, H2L, and fully assembled H2L. H2 dimers are also reactive in this process. Special procedures were developed for analyzing the data from these experiments. A complete format is given for the quantitative determination of the concentration of each of the molecular components directly from spectroscopic scans of the gels. The computational methods solve the general analytical problem posed when staining is not proportional to mass and are applicable to a wide variety of systems utilizing gel electrophoresis to study subunit interactions. A theoretical analysis of pathway and kinetic cooperatively in this system is presented in the following paper (Sears, D.W., and Beychok, S. (1977), Biochemistry 16 (following paper in this issue)).


Acquisition of the covalent quaternary structure of an immunoglobulin G molecule. Theoretical reoxidation models. A theoretical format suitable for analyzing diverse complex kinetic systems where reaction pathways may exhibit cooperativity, is developed to account for the in vitro kinetics of air reoxidation of a human IgGlkappa immunoglobulin, in which the four interchain disulfide bonds have been reduced (Sears, D.W., et al. (1977), Biochemistry 16 (preceding paper in this issue)). The equations relate experimentally determined concentrations of the reactants, product, and macroscopic intermediates to the probability of occurrence of any of 12 distinct microscopic states. The concentrations of the macroscopic species--light chains (L), heavy chains (H), covalently associated intermediates HL, H2, H2L, and product H2L2--are also explicitly related to an observable function of those concentrations, the sulfhydryl titer, r. Since values of r can be calculated for any microscopic state of the system, the theory is in principle capable of a complete description of the experimental reaction course. In practice, limited experimental information precludes a unique solution of the equations at present, and it was not judged worthwhile to attempt curve fitting or approximation of probability terms with adjustable parameters of unknown physical significance. Certain special cases of the theory are, however, readily and exactly solved. These include models in which reoxidation is a random process and others in which the probabilities for inter-HL and inter-HH bond formation are different but independent of one another. The experimental results in the case of the IgGlkappa studied here clearly depart form the predicted behavior in either of these models. The initial probability for formation of a bond between a heavy and light chain is 1.5 to 2 times greater than for a bond between heavy chains. From the fact that this ratio changes as the reaction proceeds, and from the pattern of variation in concentration of intermediates during the reaction, it is concluded that the reoxidation process is not random, and that the bonds do not form independently, exhibiting instead kinetic cooperativity. The results are discussed in terms of assembly pathways in this and related systems. A novel feature of the theory is that it eliminates time as an explicit variable in the treatment of the kinetic process. This makes it especially useful for discerning whether the formation of one bond influences the reaction probability of another in a system where several similar or intrinsically identical reactions occur, and where kinetic order is difficult to establish. While the theory here is formulated in terms of the reoxidation reactions, it is, for example, equally applicable to the reduction process described (Sears, D.W., et al. (1977b), Biochemistry 16 (following paper in this issue)).


Relative susceptibilities of the interchain disulfides of an immunoglobulin G molecule to reduction by dithiothreitol. The reduction by dithiothreitol (DTT) of the four interchain disulfides of a human IgGlkappa immunoglobulin has been studied by two methods: variation of the concentration of DTT relative to the protein concentration (incremental reduction); and variation of the time of reduction at fixed levels of DTT and protein (kinetic reduction). In both cases, the results depend on whether the reduction is carried out aerobically or anaerobically. Under aerobic conditions, the relative levels of intermediates (HL, H2, and H2L) which are generated as native molecules (H2L2) are converted to reduced heavy (H) and light (L) chains depend on the concentrations of protein and DTT as well as on the exposure time to DTT; no stable equilibrium is reached between reduced and oxidized states and conditions gradually revert from those favoring reduction to those favoring reoxidation. By contrast, anaerobic reduction is independent of protein concentration or time of exposure to DTT, beyond about 30 min, indicating that an equilibrium between partially reduced and oxidized states is achieved. The distribution of intermediates observed under anaerobic conditions has been analyzed according to theoretical models (Sears, D.W., and Beychok, S. (1977), Biochemistry 16 (second in a series of three articles in this issue)). Within experimental error, both kinds of anaerobic experiments resemble a random reduction process wherein the four disulfides are equivalent and independent of each other with respect to rate and extent of reduction by D. It is concluded that there are no readily detected pathways in the process, as would occur if the intrinsic reactivities of the bonds were distinct, and no marked cooperatively between the four reaction sites, as would be observed if reduction of one bond materially facilitated or hampered reactivity at another site. Both of these characteristics of the reduction are in direct contrast to those of the reoxidative process, which is marked by the initial preference for formation of a bond between heavy and light chains, and by kinetic cooperativity in bond formation during the course of the reaction (Sears, D.W., et al. (1977), Biochemistry 16 (first in a series of three articles in this issue); Sears, D.W., and Beychok, S. (1977), Biochemistry 16 (second in this series)).


Evidence for two types of P-700 in membrane fragments from a blue-green alga. The mathematical analysis described in the preceding paper (Biochim. Biophys. Acta (1977) 460, 65-75), in which the steady-state photooxidation of P-700 was compared with overall electron flux in Photosystem I chloroplast fragments, was applied to membrane fragments from the blue-gree alga Nostoc muscorum (Strain 7119) noted for their high activity of both Photosystem I and Photosystem II. The same analysis, which gave good agreement between the photooxidation of P-700 and the overall light-induced electron flux (measured as NADP+ reduction) in Photosystem I chloroplast fragments, revealed in the algal membrane fragments two P-700 components: one responding to high light intensity (P-700 HI), the photooxidation of which was in good agreement with the overall electron flux (measured as NADP+ reduction by reduced 2,6-dichlorophenolindophenol), and the other component responding to low light intensity (P-700 LI), the photooxidation of which was not correlated with the reduction of NADP+ by reduced 2,6-dichlorophenolindophenol.


Two plant-type ferredoxins from a blue-green alga, Nostoc verrucosum. Two plant-type ferredoxins were isolated and purified from a blue-green alga, Nostoc verrucosum. They were separable by chromatography on a DEAE-cellulose column. The slow-moving band was designated ferredoxin I (Fd I) and the fast-moving band was ferredoxin II (Fd II). The ratio of the yield of ferredoxins I and II was about 1 : 0.84. Both ferredoxins had absorption spectra similar to those of plant-type ferredoxins. Two atoms of non-heme iron and two of labile sulfur were found per mol of both ferredoxin I and ferredoxin II. Their molecular weights were identical and estimated to be about 18 000 by a gel filtration method. The biochemical activities of these Nostoc ferredoxins were studied: the NADP photoreduction activity on one hand and the NADP-cytochrome c reductase activity on the other.


Involvement of iron in the biogenesis of the cyanide-insensitive respiration in the yeast Saccharomycopsis lipolytica. The involvement of iron in the biogenesis of the cyanide-insensitive respiration in the yeast Saccharomycopsis lipolytica has been established on the following basis: (1) endogenous metal chelation by either benzyl- or salicylhydroxamic acid, EDTA or nitrilotriacetate prevented the biogenesis of the cyanide-insensitive respiratory pathway in S. lipolytica. (2) Addition of Fe(III) during the biogenesis increased both the rate of the appearance of the alternative respiratory pathway and its extent. Neither Fe(II), nor Co(II), Cu(II), Al(III), La(III), Mn(II) or Mg(II) could substitute for Fe(III). (3) The biogenesis of the alternative respiratory pathway could be dissociated into two steps: (a) a first one, slow, cycloheximide-sensitive, temperature-dependent, iron-independent, leading to cells still fully cyanide-sensitive, presumably involving the de novo biosynthesis of an inactive protein moiety and (b) a second step, fast, iron-dependent, temperature-independent, cycloheximide-insensitive, leading to cells with a cyanide-insensitive respiration, presumably the activation by iron of the inactive precursor.


Temperature dependence on the delayed fluorescence of chlorophyll a in blue-green algae. 1. The delayed fluorescence of chlorophyll a was measured with a phosphoroscope by changing the temperature in a range of room temperatures in intact cells of blue-green algae, Anacystis nidulans, two strains of Anabaena variabilis and Plectonema boryanum, and other kinds of algae, Cyanidium caldarium and Chlorella pyrenoidosa. The induction of delayed fluorescence remarkably depended on the temperature of measurment. Nevertheless, the induction pattern was characterized by three levels of intensity; the initial rise level at the onset of excitation light, the maximum level after a period of excitation and the steady-state level after 10 min of excitation. 2. In A. nidulans and a strain of A. variabilis grown at various temperatures, close relationship was found between the phase transition of membrane lipids and the initial rise and the steady-state levels of delayed fluorescence. The initial rise level showed the maximum at the temperature of phase transition between the liquid crystalline and the mixed solid-liquid crystalline states, The steady-state levels showed a remarkable change from a high in the liquid crystalline state to a low level in the mixed solid-liquid crystalline state. 3. The millisecond decay kinetics of the delayed fluorescence measured at the steady-state level in A. nidulans grown at 38 degrees C consisted of two components with different decay rates. The half-decay time of the fast component was about 0.17 ms and was constant throughout the temperature range of measurement. The half decay time of slow component ranged from 0.6 to 1.5 ms, depending on the temperature of measurment.


Evidence for monomeric bacteriochlorophyll in P800 of the photoreaction center from Rhodospirillum rubrum. To find out whether weak or strong coupling exists between the bacteriochlorophyll molecules of the photoreaction center, the relative efficiency of energy transfer to P870 was measured at 795 nm and at 808 nm, at room temperature and at 77 degrees K. At room temperature, both relative efficiencies are close to 100%. However, at 77 degrees K, 795 nm light has a quantum efficiency of 76% and 808 nm light has an efficiency of 87%. These results confirm the fact that P800 is formed of at least one short wavelength component and one long wavelength component. Moreover, the short wavelength component is weakly coupled to both P870 and to the long wavelength component of P800. The conclusion is that the short wavelength component is due to monomeric bacteriochlorophyll. By comparison with other data, all four bacteriochlorophyll molecules of the photoreaction center are inferred to be monomeric.


Localisation of the subunits of the photosynthetic reaction centers in the chromatophore membrane of Rhodospirillum rubrum. Reaction centers were isolated with the detergent lauryl dimethyl amine oxide from chromatophore membranes of Rhodospirillum rubrum. The subunit composition of these reaction centers is similar to the one obtained from Rhodopseudomonas spheroides: three subunits with the molecular weights of 21 000, 24 000 and 29 000. Reaction centers prepared from chromatophores labeled with 131I were heavely labeled in their large subunit (H). The smaller subunits (L and M) contained only little label. Sonication during labeling yielded a slightly higher incorporation of 131I in subunit H compared to the smaller ones. It is concluded that the H protein is largely exposed at the cytoplasmic side of the membrane but might also be accessible for iodination on the inside of the membrane while the L and M proteins are almost completely embedded in the membrane. Iodination of spheroplasts results in only a slight binding of 131I to chromatophores and reaction centers.


Mitochondrial respiratory chain of Tetrahymena pyriformis. The thermodynamic and spectral properties. The mitochondria isolated from the ciliate protozoon Tetrahymena pyriformis carry an oxidative phosphorylation with P/O ratio of 2 for succinate oxidation and P/O ratio of 3 for the oxidation of the NAD-linked substrates. The respiration is more than 90% inhibited with 1 mM cyanide while antimycin A and rotenone inhibit at concentrations of 1000-fold higher than those effective in mammalian mitochondria. Using a combination of spectral studies and potentiometric titrations, the components of the respiratory chain were identified and characterized with respect to the values of their half-reduction potentials. In the cytochrome bc1 region of the chain a cytochrome c was present with an Em7.2 of 0.225 V and two components with absorption maxima at 560 nm and the half-reduction potential values of -0.065 and -0.15 V at pH 7.2. The cytochrome with the more positive half-reduction potential was identified as the analogue of the cytochrome(s) b present in mitochondria of higher organisms, while the cytochrome with the more negative half-reduction potential was tentatively identified as cytochrome o. In addition ubiquinone was present at a concentration of approx. 4 nmol per mg mitochondrial protein. In the spectral region where cytochromes a absorb at least three cytochromes were found. A cytochrome with an absorption maximum at 593 nm and a midpoint potential of -0.085 V at pH 7.2 was identified as cytochrome a1. The absorption change at 615-640 nm, attributed usually to cytochrome a2, was resolved into two components with Em7,2 values of 0,245 and 0.345 V. It is concluded that the terminal oxidase in Tetrahymena pyriformis mitochondria is cytochrome a2 which in its two component structure resembles cytochrome aa3.


Spin label studies on the human erythrocyte membrane. Two sites and two phases for fatty acid spin labels. Human erythrocytes, untreated and glutaraldehyde-treated, were spin labeled with three kinds of fatty acid labels, and their electron spin resonance (ESR) spectra were studied in detail at various temperatures. 1. The better spectral resolution could be obtained by packing the erythrocytes in a hematocrit capillary tube, because of the preferential parallel orientation of the cylindrical axes of erythrocyte-disc to the centrifugal axis. 2. It was demonstrated by the incorporation and the release of the labels that the membrane possessed two kinds of the fatty acid "sites": the tightly and weakly binding "sites" at the approximate molar ratio of 1:1. The rough estimates of the binding constants were obtained, which reproducibly varied with the blood donors over a period of a year. 3. The temperature dependency of the ESR spectra revealed the presence of two distinct phases, perhaps the solid and fluid phases. With lowering of the temperature, the fluid phase became more solid but the solid phase unchanged. The pretreatment of the erythrocytes with glutaraldehyde increased the amount of the frozen phase, corresponding to the decrease of the membrane flexibility.


Architecture of the outer membrane of Escherichia coli K12. II. Freeze fracture morphology of wild type and mutant strains. Freeze fracturing electron microscopy of Escherichia coli K12 cells showed that the outer fracture face of the outer membrane is densily occupied with particles. On the inner fracture face of the outer membrane, pits are visible, which are probably complementary to the particles at opposite fracture face. This observation suggests that the particles are micelle-like. In some mutants which lack one or more major outer membrane proteins the density of particles is reduced. The loss of protein d appeared to a prerequisite for this phenomenon. However, mutants which lack all glucose and heptose-bound phosphate in their lipopolysaccharide also have a reduction in particle density whereas, the amount of protein d is normal. Moreover, loss of lipopolysaccharide by EDTA treatment also caused a reduction in the density of particles. From these results it is hypothesized that the particles consist of lipopolysaccharide aggregates stabilized by divalent cations and probably complexed with protein and/or phospholipid.


Interaction of ethidium bromide with ribosomes. Absorption, fluorescence, circular dichroism and sedimentation studies. It is shown in this work that the binding of ethidium bromide to yeast ribosomes occurs through intercalation in the double-stranded rRNA regions and produces changes in the ribosomes structure, yielding unfolded subparticles, and even partial separation of proteins from rRNA at very high binding ratios. The addition of Mg2+ prevents these structural changes, probably by partial inhibition of the dye binding.


Initiation of protein synthesis in bacillus subtilis in the presence of trimethoprim or aminopterin. Initiation of protein synthesis has been studied in the presence of the tetrahydrofolic acid analogues trimethoprim or aminopterin in Bacillus subtilis. This bacterium can grow in the presence of the inhibitors, when the medium is supplemented with the low molecular weight products of tetrahydrofolate-dependent pathways. In an attempt to show whether formylation of initiator tRNA is a prerequisite for the iniation of protein synthesis in procaryotic cells, the amount of N-formylmethionine in tRNA and in protein has been determined. The level of formylation of methionyl-tRNA was found to be 70% in control cells and approximately 2% in inhibitor-treated cells. The content of formyl groups in protein has also been found to be drastically reduced. Trimethoprim or aminopterin did not alter the amount of tRNAMet nor the degree of aminoacylation of tRNAMet in vivo. These results indicate that in B. subtilis inititation of protein synthesis is possible without prior formylation of initiator tRNA.


Rat renal and erythrocyte carbonic anhydrases. Purification and properties. Rat renal and erythrocyte carbonic anhydrases (carbonate hydro-lyase, EC 4.2.1.1) were isolated by affinity chromatography. The erythrocytes contain two major forms of the enzyme. One of the forms has a specific activity (towards CO2) 30 times higher than the other and constitutes the major part of the total cellular carbonic anhydrase. The amino acid compositions of this high-activity type and of the low-activity type are similar to the compositions reported for these types in other species. The kidney appears to have only one high-activity form of carbonic anhydrase which is very similar to and probably identical with the erythrocyte high-activity form.


Involvement of NADPH-cytochrome c reductase in the rat liver squalene epoxidase system. Microsomal squalene epoxidase has previously been solubilized with Triton X-100 and resolved into fractions, FA and FB, by DEAE-cellulose chromatography (Ono T. and Bloch K (1975) J biol. Chem. 250, 1571-1579). It has now been found that FB is identical with NADPH-cytochrome c reductase (denoted FPT, EC 1.6.2.3). Although both NADPH and NADH served as electron donors, the former was preferred for squalene epoxidase activity in the reconstituted system of FA and FB. FB is characterized by its ability to reduce cytochrome c by NADPH. In place of FB, partially purified FPT was tested for its ability to support squalene epoxidation in the presence of FA. A stepwise purification of the deoxycholate-solubilized FPT yielded an increase in specific FPT activity with a parallel increase in squalene epoxidase activity. Bromelain-solubilized FPT was less effective. Rabbit antisera preparations to the purified FPT solubilized with trypsin were shown to inhibit concomitantly FPT activity and squalene epoxidase activity. These observations support the concept that squalene epoxidation is primarily mediated via a flavoprotein, NADPH-cytochrome c reductase, and a terminal oxidase, squalene epoxidase, which is distinct from cytochrome P-450.


Stimulation of prostaglandin biosynthesis by lipoic acid. Enzyme preparations from sheep seminal vesicles display an enhanced ability to synthesize prostaglandins, particularly prostaglandin F from polyunsaturated fatty acids if alpha-lipoic acid is present in the incubation mixture prior to the addition of fatty acid. The stimulation by lipoate is reversible, time dependent, and involves modifications of V and Km for oxygenase activity. Product studies, structure vs. activity studies, and purification data indicate that lipoate exerts it effect by a mechanism distinct from a glutathione-like metabolism of the endoperoxide linkage in prostaglandin G and prostaglandin H. In addition, product studies suggest that lipoate is not a cofactor for the endoperoxide isomerase component of prostaglandin synthetase. Purification of the endoperoxide synthesizing activity by ion-exchange chromatography and isoelectric focusing yields preparations which are more responsive to lipoate than microsomal preparations.


The influence of ethylenediaminetetraacetate on white skeletal muscle myosin. Myosin from rabbit white skeletal muscle was treated with 10 mM EDTA in 150 mM phosphate buffer. After precipitation of myosin by dialysis against a 14-fold volume of water, EDTA-treated myosin, myosin before treatment and the supernatant from the treatment of myosin with EDTA were examined on sodium dodecyl sulphate-polyacrylamide gels by electrophoresis. It has been found that the quantity of LC2 light chains diminished after treatment with EDTA, and the supernatant contained the LC2 light chains. Treatment of myosin with EDTA in the presence of Mg2+ does not change the stoichiometry of the LC2 light chain and the supernatant is free from LC2 light chains. The treatment of myosin with p-chloromercuri-benzoate leads to dissociation of the same amount of LC2 light chains. It is suggested that divalent cations and thiol groups are engaged in the attachment of LC2 light chain to the myosin molecule.


The binding of calcium to glycerinated muscle fibers in rigor. The effect of filament overlap. The binding of Ca2+ to glycerinated rabbit psoas fibers of varying sarcomere length was measured with a double isotope technique and ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid buffers. Experiments were carried out under rigor conditions with fiber bundles pre-set at different lengths prior to extraction with detergent and glycerol. These experiments were designed to test whether rigor complex formation, determined by the degree of filament overlap, enhances Ca2+-receptor affinity in the intact filament lattice, as it does in reconstituted actomyosin systems. The Ca2+-receptor affinity, as indicated by the free Ca2+ concentration at half-saturation and by the slopes of Scatchard plots, was found to be relatively unaffected by variations in filament overlap. However, the maximum bound Ca2+ was significantly reduced in stretched fibers. With maximum filament overlap the bound Ca2+ was equivalent to 4 mol per mol troponin. When stretched to zero overlap the fibers bound a maximum of 3 mol Ca2+ per mol troponin. When fibers with maximum overlap were incubated in the presence of 5 mM MgATP there was a reduction in the number of Ca2+-binding sites equivalent to that caused by stretching the fibers. These findings, taken together with other data in the literature, suggest that in the intact filament lattice at least one of the Ca2+-binding sites is present only when cross-bridge attachments are formed.


The modification of tryptophan in bovine thrombin. 2-Hydroxy-5-nitrobenzyl bromide, at a 100-fold molar excess, was observed to react withthrombin at pH 4.0 to give a modified enzyme which possessed 20% of the fibrinogen clotting activity and 80% of the esterase activity compared to a control preparation. Spectrophotometric analysis of the modified protein indicated that this effect on catalytic activity was associated with the incorporation of 1 mol of reagent per mol of thrombin. Amino acid analysis showed no loss of amino acids other than tryptophan. The reaction of N-bromosuccinimide with thrombin at 2-fold molar excess resulted in the modification of one tryptophan per mol of enzyme with the loss of 80% of the fibrinogen clotting activity with, as above, a considerably smaller loss of esterase activity. Oxidation of thrombin with N-bromosuccinimide decreased the extent of subsequent tryptophan modification with 2-hydroxy-5-nitrobenzyl bromide. Thrombin modified with 2-hydroxy-5-nitrobenzyl bromide showed a 3-4 fold increase in Km and a decrease in V for the ester substrate. The reaction of thrombin with 2-acetoxy-5-nitrobenzyl bromide, a substrate analogue, also resulted in the inactivation of the enzyme. The data are interpreted to show the presence of a tryptophan residue at or near the enzyme's substrate binding site.


Some properties of a glycoprotein with calcium binding ability found in human biological fluids in macroglobulinaemia IgM. An acidic glycoprotein with calcium-binding properties was isolated from the urine of patients with severe macroglobulinaemia IgM. The molecular weight of this protein determined by Sephadex gel filtration was found to be 62 000 +/- 2800 in Tris - HCl buffer and 21 000 +/- 1000 in 6 M guanidine - HCl. The amino acid and carbohydrate composition of the isolated glycoprotein is presented. Electrophoretic migration of this protein was observed to be greatly affected by calcium ions present in the buffer in a concentration of 10(-3) M. At least two sets of binding sites seem to participate in binding calcium. The values 2.2 - 10(6) M-1 for the apparent association constant and 4.4 - 10(-4) mol of Ca2+ bound per g of protein for high affinity bindings sites were estimated, on the basis of data from the equilibrium dialysis. The origin and possible biological role of this protein is discussed.


Plant glycoprotein biosynthesis. Uridine diphosphate N-acetyl-glucosaminyltransferase from horseradish root. A particulate enzyme preparation from horseradish root tissue was shown to catalyze the transfer of 2-acetamido-2-deoxy-D-[14C1]glucose from uridine diphosphate 2-acetamido-2-deoxy-D-[14C1]glucose to an exogenous acceptor molecule derived from horseradish peroxidase. The acceptor was produced from purified peroxidase by the action of a mixture of glycoside hydrolases covalently bound to Sepharose. The membrane preparation containing the transferase was purified approximately 12-fold by aqueous two phase distribution and by discontinuous sucrose density gradient centrifugation. Hydrolysis of the reaction product yielded glucosamine as the only radio-labeled substance. Precipitation of the reaction product by antiserum against peroxidase showed that the label was incorporated into peroxidase. The transferase utilized the acceptor most efficiently when only 12% of the 2-acetamido-2-deoxy-D-glucose was removed from the acceptor. The acceptor lost no accepting capabilities when heated to 100 degrees C for 3 min prior to assay. Trypsin treatment caused a 14% decrease in label incorporated while pronase treatment caused a 93% decrease,


Purification and specificity of antibodies to adenosine. Antibodies to adenosine were elicited in rabbits by immunization with bovine serum albumin-adenosine conjugate. The antibodies were purified and fractionated on two affinity columns (Sepharose-oligo(A) and Sepharose-AMP). Two families of antibodies have been obtained. The antibodies purified on the Sepharose-oligo(A) column react with poly(A) while those purified on the Sepharose-AMP column do not, as shown by gel diffusion. The association constants for the binding of Fab fragments or IgG purified on the Sepharose-oligo(A) column and several haptens were deduced from dialysis equilibrium, fluorescence quenching and displacement of AMP-fluorescein conjugate. The antibodies mainly recognize adenine, and the ribose or the phosphate group of (or AMP derivatives) do not play a critical role in the interaction. Thermodynamic parameters for adenosine-Fab fragments complexes have been determined deltaH degrees = 16 kcal/mole and deltaS degrees = - 15 cal. degree-1 mole-1. Circular dichroism studies indicate that about three nucleotide residues penetrate the binding site of Fab fragments.


[Quaternary structure of oligomeric immunoglobulin A forms from human blood serum]. A character of forces stabilyzing quaternary structure of dimer and more high molecular human immunoglobulin A oligomers is found to be different. Quaternary structure of IgA dimer is formed when joining subunits with disulfide bonds and is stabilized by non-covalent interactions between them. Disulfide bonds play a main part in the formation of trimers and tetramers. Dimer IgA reconstructs by 40% from subunits with intact interchain S--S bonds. The addition of exogenous J-chain does not significantly affect the process of dimer self-assembling from subunits with recovered and intact interchain disulfide bonds.


[Metabolism of phenolic compounds in plants]. A review is given on mechanisms of hydroxylation (the introduction of the first hydroxyl group into C-2 and C-4 positions of cinnamic and benzoic acids; the introduction of the second and the third hydroxyl groups into molecules of phenolcarboxylic acids and flavonoids), dehydrogenation (with participation of phenol oxydase and peroxydase) and deep oxidative degradation of phenolic compounds in plant tissues and cell cultures. Data on compartmentation of respective enzyme systems are also considered.


[Multiple forms of Bacillus subtilis subtilisin and effects of mutations on the distribution of its molecular forms]. Using polyacrylamide-gel electrophoresis, isoelectric focusing and gel-filtration it was demonstrated that the auxotrophic mutant strains of Bac. subtilis A-50 and their prototrophic revertant strains produce multiple molecular forms of subtilisin. Three of them are the same as the corresponding molecular forms of subtilisin from the wild strain A-50. In different mutant strains the relative amounts of the main three forms varies considerably resulting in the absence of certain forms in several strains. There is the additional minor form of subtilisin possessing high electrophoretic mobility in four prototrophic revertant strains and one Arg--auxotrophic strain of Bac. subtilis A-50. It would be reasonable to suppose that different molecular forms of subtilisin derive from the product of its single structural gene as a result of post-translational modifications (limited proteolysis). This enzyme and probably most, if not all secretory proteins may be synthesised as larger precursors and then specifically modified in the bacterial cell membranes. Thus, certain mutations, without affecting the structural gene of this secretory protein -- subtilisin -- have pronounced effects on this structural gene expression, varying the degree of its product modification and the amount of resulting secretory molecular forms of subtilisin.


[Effect of metalloproteins on the photochemical activity of chloroplasts treated with polyene antibiotics]. The effects of various metall-containing proteins (plastocyanin, plantacyanin, azurine and cytochromes of the f type) on the activity of photosystem I of chloroplasts, treated with polyene antibiotics, were studied. The inhibiting effect of the polyenes, surgumycin and philipin, was completely removed by an addition of copper-containing protein plastocyanin. No similar effect was exerted by other Cu-containing proteins--azurine and plantacyanin. The cytochromes of the f type isolated from the green algae chlorella, blue-green algae spiruline and aphanezomenone, having different electrophoretic properties, restored the activity of photosystem I of chloroplasts incubated with antibiotics in a different degree. Acid cytochrome f of chlorella restored the activity by 80--100%; less acid cytochrome f from spiruline-only by 50%. The least restoring effect was exerted by aphanezomenone cytochrome, which possesses some basic properties. The chloroplasts treatment with surgumycin did not affect the isolation of the terminal enzyme of the chloroplast electron-transporting chain of ferredoxin--NADP--reductase. Possible environment of plastocyanin in the chloroplast membrane and the mechanism of photosystem I restoration are discussed.


Considerations of species-related hematological differences on the evaluation of biomaterials. Species-related hematological differences of experimental animals are important in the proper assessment of biomaterials intended for human use. This area, which has received less than adequate attention in the past, requires the development of new test methodologies with blood of primates and humans, under conditions that better simulate the physiological environment.


Preparation of specific immune sera against rabbit skeletal, pig and human cardiac heavy meromyosins. Antibodies were produced in guinea-pigs against heavy-meromyosin (HMM) and tested by immunodiffusion and microcomplement fixation (MCF). A similar response was obtained for HMM extracted either from human and pig left ventricles, or from rabbit white skeletal muscles. For each muscle, the antisera cross-react quantitatively with the native intact myosin and are highly specific. Thus, this procedure permits the preparation of muscle type-specific anti myosin antibodies, by a simpler means than those previously published.


[Antigens common to Mycobacterium bovis (BTsZh) and several malignant and normal tissues of laboratory animals]. Agglutination, gel precipitation and immunoelectrophoresis tests demonstrated the presence of antigens common for the Mycobacterium bovis (BCG) and some malignant and normal rat tissue cells. The method of absorption of immune sera in the agglutination test showed that this antigen was specific for the BCG microorganisms and cells of primary induced tumours of rat muscle tissue. This antigen had the electrophoretic mobility in the beta-globulin zone.


[Changes in the immunologis status of the body during carcinogenesis detected by the physico-chemical characteristics of cells of the immunocompetent system]. The state of cell population of the lymph nodes and the thymus was studied on a model of rat sarcoma of the hip(induced by dimethlbenzanthracene) by the method of fluorescent sounds. Statistically significant changes of incorporation of the negatively charged sound 1-anilino-8-naphthalinosulfonate (ANS) into cell suspension, depending on the stage of sarcoma development, were detected. The appearance of cell forms with a lesser hydrophobic character of the surface and with less binding points for the ANS were noted at the early carcinogenesis periods. It is supposed that these cells were referred to immature ones. A conclusion was drawn on the applicability of the method of fluorescent sound for recording changes in the immunological state of the organism in carcinogenesis.


[Change in the distribution of rat liver and myocardial carbonic anhydras activity in acute dichloroethane pisoning (histophotometric study)]. A study was made of the histochemical changes in the distribution of the carboanhydrase activity in the myocardium and the liver of rats in acute oral dichlorethane poisoning. Kurate's method modified by Hausler was used. The histophotometric method was applied for the semiquantitative assessment of the changes in the enzyme activity. The majority of the experimental animals displayed a distinct increase in the activity of carboanhydrase in the myocardium and the liver, the correlation of these changes remained the same as in the intact rats.


[Immunochemical analysis of glial and neuronal fractions of rat brain]. A method of obtaining antineuronal and antiglial immune sera is described. Data of the quantitative immunochemical analysis of the antigens of the neuronal and glial fractions of the rat brain are presented. The neurons proved to contain 4, and the glia--3 brain-specific proteins; one or two of them are common for these cell fractions.


[Opsonizing activity of the serum of gnotobiotic guinea pigs contaminated with individual representatives of intestinal microflora]. The influence of contamination of germfree guinea pigs with individual representatives of the intestinal microflora (Bac. mesentericus, Bac. subtilis, S. albus, and S. faecalis) on the formation of the serum opsonic activity was studied. An increase of the opsonic activity to all the microorganisms on the 11th day after a corresponding monocontamination and a stimulating influence of the serum on the intracellular digestion of Bac. mesentericus and Bac. subtilis microbes was noted. As to the pathogenic microorganisms (E. coli 055), S. Faecalis only were capable of stimulating the serum opsonic activity. The results indicated the presence of an association between the microflora composition and the opsonic activity of the animal blood serum. The value of this index also depended on the properties of the phagocytosis object.


[Effect of Sonne shigella endotoxin on cholinergic regulation of intestinal circulation]. A method of extracorporeal autoperfusion and resistography of the intestinal vessels and synchronous multichannal recording the systemic blood circulation were applied to the study of the influence of the Sh. sonnei endotoxin on the cholinergic regulation of the cardiovascular system in Macacus rhesus. It was shown that development of intoxication was accompanied by the changes in the cholinergic regulation of the intestinal and systemic circulation leading to its disturbance. Possibly affection of the cholinergic control structures and disturbances of the circulation determined the development and the course of the infectious process.


[Protective effect of chloramphenicol and dextramycin following the adrenocorticolytic action of 7, 12-dimethylbenz(a)anthracene]. The influence of chloramphenicol (CAP) and dextramycine (DMC) on the adrenocorticolytic action of 7,12-dimethylbenz(a)anthracene (DMBA) was studied in rats. It appeared that CAP in doses of 1.0-0.1 mg/g and DMC in doses of 1.0-0.05 mg/g completely prevented the development of necroses of the adrenal glands in administration 1.5 hours before the carcinogen. With lower doses of CAP and DMC and also when they were administered 48 hours before or 1.5 hours after the carcinogen the protective effect was retained only partially. The toxic action of the carcinogen remained unchanged when CAP and DMC were administered 3, 6 or 24 hours after DMBA.


[Changes in the hypothalamus, reticular formation and pituitary of rats during development of subcutaneous tumors]. Dynamics of structural changes was studied in the hypothalamus, reticular formation and the hypophysis of rats, depending on the phases of development in them of 9m10-dimethyl-1,2-benzanthracene-induced tumours. The phase of subcutaneous tumours appearance proved to be accompanied by structural and functional changes in the hypothalamus, reticular formation and the hypophysis.


[Micro-quantitative determination of alpha1-antitrypsin by unidimensional capillary counter-immunodiffusion]. The immunoprecipitation reaction for the determination of microamounts of alpha1-antitrypsin was conducted in the capillaries filled with a microvolume of a 1% agarose gel (the gel length was 7--8 mm, and the diameter--0.6 mm). Microamounts of the antigen and antiserum were applied to the surface of the opposite ends of the gel. Precision control of these manipulations was carried out by means of an ocular micrometer of a binocular lens. The amount of the formed immunoprecipitate in gel was calculated by the summated optic density after staining with amido black recorded by the method of double-wave microspectrophotometry. Sensitivity of the method was 0.3--1.0 ng of alpha1-antitrypsin. The linear plot between the amount of alpha1-antitrypsin and the amount of the precipitate formed was found within the range of 1--25 ng of protein. The error was about 10%.


[Use of a highly-sensitive method of electrophoresis-precipitation for detecting hepatitis B virus antigen in human serum]. A modification of the highly-sensitive method of electrophoresis-precipitation in polyacrylamide gel used for detection of the virus hepatitis B antigen (HBsAg) and the anti-HBsAg is described. The method completely retains the specificity of the gel immunodiffusion test, but is 100--2000 times more sensitive in HBsAg detection. The sera of patients with different pathology were checked by this method. In comparison with the methods used before the HBsAg determination by the mentioned method proved to be more effective.


Heterogeneity of factor VIII antibodies: further immunochemical and biologic studies. Previous studies using immunoneutralization techniques have shown that many factor VIII inhibitors are IgG antibodies of a single light chain type. We have investigated this apparent homogeneity by immunoneutralization assay and liquid isoelectric focusing of inhibitor fractions from five hemophiliacs and two nonhemophiliacs. By immunoneutralization assay, inhibitors from four hemophiliacs and one nonhemophiliac were exclusively k light chain type: the fifth hemophilic inhibitor was predominantly k1 and the second nonhemophilic inhibitor was a mixture of k and gamma. However, heavy chain subtyping of the six predominantly or exclusively k inhibitors showed all to be mixtures of IgG4 and IgG1. By isoelectric focusing, two inhibitors showed multiple peaks of activity between pH 5 and 9. The remaining five showed predominant peaks of activity, which were solely IgGk1 between pH 5.8 and 7, with smaller peaks between pH 7 and 9. The most acidic major peak, focusing at pH 6, was IgG4 in the three cases tested. Two of these acidic peaks neutralized factor VIII more efficiently than other peaks in the same focusing profiles, suggesting greater affinity for factor VIII. These studies demonstrate that factor VIII inhibitors are composed of heterogenous subpopulations of immunoglobulins which can be separated by isoelectric focusing.


Immunochemical and ultrastructural investigations in a patient with cold-agglutinine-active monoclonal IgMK-gammopathy. A patient with "chronic cold-agglutinin disease" is described. Initially, the process was a benign monoclonal gammopathy. After an intervall of 6 years clinical, cytomorphological and immunochemical studies showed progress towards malignancy. In the bone marrow intranuclear lipid-like inclusions appeared in the plasma cells.


The prolonged exposure of Japanese quail to carbaryl and its effects on growth and reproductive parameters. Carbaryl fed to Japanese quail over a 14 week period resulted in decreased body weight, increased relative brain, liver and kidney weights in one or both sexes at levels of 900 ppm or higher. Significant differences were not found in the F1 birds at the same levels after 6 weeks. It appears that the time of exposure is critical and that parental diet is not. There were no significant differences in egg production, hatchability or fertility although trends toward reduction of these parameters were developing at the 900 and 1200 ppm levels.


The influence of gastric emptying on plasma concentrations of the analgesic, meptazinol. 1. The influence of gastric emptying on the rate of absorption and peak plasma concentrations of meptazinol was studied in monkeys by comparison of the plasma concentrations of the drug achieved after intragastric and intraduodenal administration of the compound. Absorption was much more rapid and plasma concentrations were very much higher after intraduodenal than after intragastric dosage. 2. Determination of the 'normal' gastric emptying time in rats and monkeys showed that this was much longer in the monkey than in the rat. In addition, meptazinol was shown to have a pronounced retarding action on gastric emptying in the monkey, an effect which was not apparent in the rat. 3. The significance of these observations in relation to the species difference in oral potency of the drug is discussed.


Comparative physical and immunological aspects of the chimpanzee and guinea-pig subcutaneous chamber models of Neisseria gonorrhoeae infection. Physical and immunological characteristics of the chimpanzee and guinea-pig subcutaneous chamber models for Neisseria gonorrhoeae infection were compared to evaluate their usefulness for gonococcal research. Urethral infection in chimpanzees anatomically resembled the human infection; however, individual variation in response, limited availability, and the presence of interfering micro-organisms in the urethra were found to limit the usefulness of the chimpanzee in immunological research. Although the guinea-pig subcutaneous chamber model may not be suitable for studying the attachment of gonococci to host cells or for the local production of IgA, it does have the immunological advantages of being more sensitive to infection, less variable in response, free of interfering micro-organisms, and is readily available to investigators. Except for differences in sensitivity and variability, results with the guinea-pig model paralleled results obtained in experiments with chimpanzees. Unlike chimpanzees, guinea-pigs are a comparatively inexpensive, rapidly replenishable animal, which after subcutaneous implantation with small porous chambers provide a convenient model for studying most immunological aspects of gonococcal infections.


Isolation of Neisseria meningitidis from the urethra, cervix, and anal canal: further observations. Neisseria meningitidis was recovered from the urethra of nine and from the anal canal of seven men, and from the cervix of two women. Twelve of the 16 men were admitted homosexuals. Seven men had no symptoms, while the remainder had mild to moderate symptoms. One woman was asymptomatic and the other was in hospital with acute salpingitis. Reports of previous isolations of meningococci from the urethra, cervix, or anal canal are reviewed. It is concluded that in men, these infections are usually mild and self-limited, but in women, meningococcal genital infections frequently proceed to severe disease.


Triple tetracycline (Deteclo) in the treatment of chlamydial infection of the female genital tract. The efficacy of treating genital Chlamydia in women using Deteclo 300 mg twice daily for seven and 21 days has been studied. Forty-four patients were treated for seven days and 20 for 21 days. Seven days of treatment was as effective in eliminating Chlamydia trachomatis from the female genital tract as 21 days. The reproducibility of the technique of isolation of C. trachomatis was assessed by delaying treatment in 10 patients for up to 156 days and find C. trachomatis still present.


Preparation and properties of the brain specific protein 14-3-2. A brain specific protein, 14-3-2, has been isolated from bovine brain by the use of ammonium sulfate precipitation, gel filtration on Sephadex G-150, and ion exchange chromatography on DEAE-cellulose and DEAE-Sephadex. It is an acidic protein, in agreement with the high content of glutamic and aspartic acid, that is composed of a single polypeptide chain of mol. wt. 50,000. Immunochemical tests using antiserum to purified 14-3-2 showed that the protein is present in at least 100-fold greater amounts in brain than in any other rat tissue. Furthermore, 14-3-2 was found in brains of a number of vertebrate species, although the antigen is apparently not entirely identical in all species tested. The protein 14-3-2 can be considered to be a species non-specific protein which is neuronal in origin.


Anatomic localization of topically applied [14C]penicillin during experimental focal epilepsy in cat neocortex. 14C-labeled penicillin was topically applied to the suprasylvian gyri of adult cats in order to study the distribution of the convulsant agent at the onset of focal epileptogenesis. Using radioassay and autoradiographic techniques, a steep gradient of penicillin was found. At the time interictal EEG spike discharges appeared, 95% of the labeled drug was in the uppermost cortical layers (laminae I-III). Analysis of the concentration profiles obtained by scintillation counting showed that penetration of penicillin into brain occurs primarily by passive diffusion. An apparent diffusion coefficient for penicillin in neocortex of 1.5 sq. mm/h was calculated using modifications of standard diffusion equations. It is apparent that with a rapidly acting topical convulsant such as penicillin, the dimensions of the neuronal pool actually in contact with the drug will change significantly over time. The changing boundaries of the epileptic neuronal aggregate must be taken into account when interpreting observations made within and around such experimentally produced epileptic foci.


Identification by microinjection of TRH-sensitive sites in the cat's brain stem that mediate respiratory, temperature and other autonomic changes. Cats were prepared with an array of stereotaxically implanted guide tubes, the tips of which rested just above selected structures in the brain stem. Thyrotropin-releasing hormone (TRH) was microinjected in a volume of 0.5 micronl at 347 individual sites scattered throughout the hypothalamus and mesencephalon Polypnea, hypothermia, vocalization, salivation, defecation and vasodilation were evoked by 10-20 ng of TRH injected only at loci in the mesencephalon, principally in the reticular substance. TRH failed to lower body temperature when it was infused at the same sites in the anterior hypothalamus at which norepinephrine produced its characteristic hypothermia. These results suggest that the TRH-induced hypothermia is a secondary effect of tachypnea which results from the action of the tripeptide on the mesencephalic respiratory-autonomic mechanism.


Purification and characterization of human muscle pyruvate kinase. The M1 isozyme of pyruvate kinase has been purified from human psoas muscle in a seven-step procedure. Fractionation by ammonium sulfate precipitation, heat treatment, acetone precipitation, diethylaminoethyl cellulose batchwise treatment followed by chromatography on carboxymethyl cellulose and Sephadex G-200 gave a product with a specific activity of 383 U/mg representing a 294-fold purification with a yield of 11%. The product formed orthorhombic crystals and was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate, sedimentation velocity, sedimentation equilibrium, and immunodiffusion. The purified enzyme has a molecular weight of 240700 and has a sedimentation coefficient (S20,W) of 10.04S. It contains four subunits with identical molecular weights of 61000. No free N-terminal amino acids could be detected. Antibody prepared against the purified human M1 isozyme does not cross-react by immunodiffusion or enzyme inactivation with the human erythrocyte isozyme and in the reverse experiment antibody prepared against human erythrocyte pyruvate kinase does not cross-react with the purified M1 isozyme. The amino acid composition of the M1 isozyme is presented.


The attachment of bacterial spinae.

The attachment of bacterial spinae. Spinae are attached to protease-sensitive structural proteins in the external surface of the outer membrane. Agents and (or) treatments affecting ionic, hydrophobic, or hydorgen bonds are ineffective in releasing spinae from bacteria. As judged by thin-sectioning and freeze-fracturing techniques, the outer membrane is not modified at the attachment site to a detectable extent, and the other surface layers are not involved. The attachment of spinae is thus differentiated from that of flagella.


Outer-membrane damage in sublethally heated Escherichia coli K-12. Exponentially grown cells of Escherichia coli K-12 heated at 48 degrees C in potassium phosphate buffer at pH 7.0 were structrually injured before death. During heating for 60 min about 20% of the cellular lipopolysaccharide (LPS) was released from the outer membrane into the heating medium. Removal of 30% of the cellular LPS, by washing the cells in buffer containing ethylenediaminetetraacetic acid (EDTA), caused no significant increase in the rate of death and structural injury produced by heating. The addition of EDTA to the heating medium produced only a slight increase in the rate of thermal death but  a large increase in the rate of structural injury. By a combination of heating at 48 degrees C and washing with EDTA, a maximum of 50% of the LPS was released from cells. These results taken together suggest that structural injury and loss of LPS are not the direct causes of death. The addition of 5 m M Mg2+ to the heating medium protected the cells from death and structural injury caused by heating at 48 degrees C.


The effect of hyperimmunization with Neisseria gonorrhoeae on the presence of gonococcal antibody in serum, tissues, and secretions of the rabbit. Antibody responses in sera, tissues, and secretions of the urogenital tract and lower respiratory tract of rabbits hyperimmunized with Neisseria gonorrhoeae were examined. Antibody was detected by passive hemagglutination, whole-cell agglutination, bentonite flocculation, and in some cases immunodiffusion-in-gel. Immunization of rabbits either intravenously or intramuscularly resulted in the presence of gonococcal antibodies in the sera, spleens, and tissue of the urogenital tract (vagina, cervix, uterus, and fallopian tubes). Gonococcal antibody was also found in secretions bathing the mucosa of the urogenital tract and lower respiratory tract. Antibodies were not detected in sera, tissues, and secretions of non-immunized rabbits. The spleen was shown to synthesize gonococcal antibody in vitro in response to hyperimmunization. Tissues of the urogenital tract did not appear to synthesize gonococcal antibody thus suggesting and antibodies present in secretions of the urogenital tract were derived mainly from serum.


Distribution of anticonvulsant drugs in gray and white matter of human brain. Gray and white matter were obtained during neurosurgical therapy of focal epilepsy from 17 patients. In 10 patients, receiving only phenobarbital, the drug was uniformly distributed between gray and white matter. Phenytoin concentrations averaged 1.4-fold greater in white matter than in gray matter when expressed per gram wet weight of tissue. The gray matter/plasma ratio of phenytoin was approximately 2-fold greater than that of phenobarbital. Carbamazepine levels were also slightly greater in white matter. The data revealed wide differences between drugs in the relative concentrations in gray and white matter, which must be taken into account in any quantitative studies of anticonvulsant drug levels in the brain.


Effects of antihypertensive drugs on blood velocity: implications for prevention of cerebral vascular disease. The treatment of high blood pressure prevents death from congestive heart failure, hypertensive nephropathy, and encephalopathy, and strokes from cerebral arteriolar disease (lacunes, hemorrhage from microaneurysms). However, atherosclerosis, manifested as coronary artery disease is just as frequent a cause of death in well-controlled hypertensives as in poorly-controlled patients. Increasing evidence suggests that increased blood velocity, by causing turbulence and high shear rates at the endothelial surface of arteries, may be important in the pathogenesis of atherosclerosis. Turbulence has been observed in cerebral berry aneurysms. In order to measure the effects of antihypertensive agents on blood velocity, a new method of analysing Doppler ultrasound velocity recordings has been developed. Studies in Rhesus monkeys show the following: In doses which reduce diastolic pressure by 13-28%, propranolol decreased mean blood velocity (MV) by 17%, clonidine decreased MV by 14%, while methyldopa increased MV 12%, and hydralazine increased MV by 52%. (p less than .00001). It is hypothesized that enlargement of berry aneurysms, the progression of cerebral atherosclerosis, and embolism from carotid lesions might all be decreased by the selection of antihypertensive agents which decrease blood velocity.


Lactic acidosis and ketoacidosis: biochemical and clinical implications. A case of lactic acidosis presented the opportunity for review of the association between lactic acidosis and ketoacidosis. The diagnosis of lactic acidosis or the combination of lactic acidosis and ketoacidosis is established clinically by the detection of a metabolic acidosis of the "unmeasured anion gap" type in the absence of significant renal failure, poison intake or a strongly positive clinical test for ketones. Before treatment can be planned the biochemical basis of lactic acidosis and ketoacidosis must be understood -- especially the fact that lactic acidosis is not a single disease entity but has many possible causes. Among important considerations is the relation between the blood concentrations of bicarbonate and organic acid anions. After recovery from metabolic acidosis of the unmeasured anion gap type, metabolic alkalosis is common. Decreased bicarbonate excretion plays an important role in the pathogenesis of the latter and may be the result of potassium or chloride loss, or both. The deficits, if present, should be corrected with appropriate therapy.


Total body irradiation with 8 MeV X-rays in generalized malignant disease. The authors describe their experience with total body irradiation (TBI) with 8 MeV-x rays in the treatment of disseminated tumors. With a dose of 125 rad, given three times with 2-week intervals, some objective remissions (6/26) were obtained, especially in patients with lymphomas. For solid tumors only subjective improvement could be achieved. The side-effects were acceptable. The authors came to the conclusion that total body irradiation may be an alternative to chemotherapy for certain cases of generalized cancer.


Ultrastructure of myeloma cells in a case with crystalcryoglobulinemia. The bone marrow of a patient with multiple myeloma of the IgG2 Kappa type with spontaneously crystallizing cryoglobulin was studied by electron microscopy. The ultrastructure of the myeloma cells disclosed the presence of a crystalline material in the cytoplasm within the rough endoplasmic reticulum (RER) as well as in extracisternal sites. The crystalline material was also seen extracellulary with a distinctly unique subunit structure. The tubular units measured 200 +/- A (SEM) externally with an internal diameter of 100 +/- A (SEM). The intracellular distribution did not indicate a characteristic organelle association usually observed in protein synthesizing cells. It is suggested, based on the present observations and the findings of others, that the crystalline material may represent polymerized protein synthesized by free ribosomes mostly in extracisternal locations, a pattern often seen in neoplastic plasma cells. Diffusion to extracisternal sites of precrystalline material through the membranes of the RER is a possible alternative mechanism.


Familial cancer syndromes: a survey.

Familial cancer syndromes: a survey. Several pre-malignant diseases are known to have a genetic etiology. This study focuses attention upon precancerous disorders wherein the mode of inheritance is either well established or wherein it remains unclear even though familial aggregation of the particular diseases have been amply documented. These conditions will be discussed as useful models for systematic investigations of the host etiologic component in carcinogenesis. Our survey of hereditary precancerous syndromes includes multiple polyposis of the coli, the multiple mucosal neuroma syndrome, the Cancer Family Syndrome, Sipple's syndrome, Von Recklinghausen's neurofibromatosus, the multiple nevoid basal cell carcinoma syndrome, tuberous sclerosis, familial cutaneous malignant melanoma, and carcinoma of the breast. We have emphasized the heterogeneous character of many forms of familial cancer. Familial breast cancer associations clearly show such heterogeneity, as do colon cancer syndromes. Certain of these precancerous states are characterized by phenotypes which are clinically apparent, polyposis coli being the classic example. Others, such as Sipple's syndrome are amenable to routine screening for biochemical markers. The bulk of putative genetic cancer-predisposing problems require further basic investigation of modes of inheritance. Cancer control may be enhanced through communication of useful genetic and diagnostic information to primary care physicians. Referral of cancer clusters of possible genetic etiology from clinicians to human geneticists facilitates the necessary basic research.


Leukemia and other cancers following radiation treatment of pelvic disease. Follow-up studies of patients treated for cancer of the cervix with radiotherapy have shown such women to be at little or no increased risk of leukemia subsequent to the radiation exposure. However, women exposed to lower doses of radiation in the pelvic area, in the induction of an artificial menopause, appear to show increased risks of both leukemia and cancers of those sites directly in the radiation field. The studies of these two types of radiation exposure are reviewed. The findings may possibly be reconciled with each other on the basis of the distribution of radiation dose to the bone marrow. Irradiation for cancer of the cervix delivers radiation doses to a small portion of the marrow which are probably lethal for most marrow cells. The mean dose to cells distant from the cervix may be too small to produce a detectable increase in leukaemia incidence. The lower and more uniformly distributed radiation dose used to induce an artificial menopause will be less lethal for marrow cells and may consequently deliver a higher "effective" marrow dose to surviving--cells, resulting in an increased leukemia risk.


Long bone ossifying fibromas.

Long bone ossifying fibromas. Ossifying fibromas involving the tibia were seen in two patients. In both patients, the radiographic appearance of the lesions suggested fibrous dysplasia, but histopathologic evaluation demonstrated findings similar to ossifying fibroma of the mandible and facial bones. Radiologic and pathologic recognition of this entity is necessary for proper treatment.


Crystalloids of prostatic carcinoma: relationship to Bence-Jones crystals. Previously undemonstrated protein crystalloid structures were found to occur in the malignant acini of prostatic carcinomas in 23% of cases systematically reviewed. Histochemically and immunohistochemically, they appear closely related, if not identical, to Bence-Jones crystals. Thus, the prostatic carcinoma cell is the first neoplastic cell besides those of the lymphocytic-plasmacytic series shown to produce such crystalline structures. Like the proteolytic splitting of amyloid from Bence-Jones proteins, it is postulated that prostatic epithelium may enzymatically split corpora amylacea from a Bence-Jones-like protein which it synthesizes. It is further postulated that malignant prostatic cells may lack the necessary enzymatic machinery to accomplish this with resultant accumulation of crystalloids within their acini. Prostatic crystalloids were never found in benign glands with rare exceptions and then were limited to benign appearing glands adjacent to carcinoma. The presence of such crystalloids in microscopic sections of prostate may be of diagnostic aid in determining the presence of malignancy or of a premalignant state.


Pathological study of alpha-chain disease, with special emphasis on evolution. The pathology of six cases of alpha-chain disease (alpha-CD), four of which were followed until complete remission or death, was studied by histologic, immunofluorescence and ultrastructural techniques. The lesions could be classified in three histopographical stages. The late stage C is an immunoblastic sarcoma probably deriving from the same clone as the initial plasmacytic stage A, stage B being a transitional one between A and C. The asynchronism of the lesions in different organs in the same patient requires a laparotomy for an accurate staging which determines the prognosis and the treatment. Complete and prolonged remissions have been observed at stage A only, sometimes with oral antibiotic treatment alone. At all stages, alpha-CD and the "Mediterranean lymphoma" share identical aetiological, clinical and pathological features. Accurate immunological studies will determine the precise-frequency of alpha-CD protein synthesis in the latter syndrome.


The therapeutic implications of splenic involvement in stage IIIA Hodgkin's disease. In an effort to determine the most appropriate initial therapy for patients with stage IIIA Hodgkin's disease, a comparative analysis was undertaken of 13 pathologically staged IIIA Hodgkin's disease patients whose abdominal disease was localized to the spleen (IIIsA); 17 IIIA patients with spleen and abdominal lymph node involvement (IIIs+n+A) and 44 stage IIA patients. The three groups were treated concurrently with either extended field irradiation alone or limited field irradiation followed by MOPP chemotherapy. Relapse rates after irradiation alone were 17% for the IIIsA patients; 63% for the IIIs+n+A patients and 25% for the stage IIA patients. Following therapy with irradiation and chemotherapy no relapses occurred among the IIIsA and IIIs+n+A patients while 2/16 (13%) stage IIA patients relapsed. After irradiation alone stage IIIs+n+A patients had a significantly shorter remission duration and survival than the stage IIA patients (p = 0.03 and 0.002, respectively) but remission duration and survival were similar for the IIIsA and IIA patients. When therapy was irradiation and chemotherapy no significant differences in remission duration or survival were noted for the three groups. The most common sites of relapse for the IIIs+n+A patients were extralymphatic (60%) while no extralymphatic relapse have occurred among the IIIA patients (p less than 0.02). The only relapse among the IIIsA patients was at an extralymphatic site. These data have shown that patients with IIIsA Hodgkin's disease have a similar prognosis to stage IIIA disease but after therapy with irradiation along stage IIIs+n+A patients have a poorer prognosis. Combined irradiation and chemotherapy should, therefore, be considered for stage IIIs+n+A Hodgkin's disease when abdominal nodal disease cannot be included in the initial radiation port.


New assessment of the prognostic significance of histopathology in Hodgkin's disease for laparotomy-negative stage I and stage II patients. This paper describes preliminary radiotherapy results in 90 patients with Stage I and II Hodgkin's disease who were evaluated by laparotomy, including splenectomy, and liver and bone marrow biopsies. As a result of selection by laparotomy, the estimated five-year survival rate for these patients was 96%. No statistically significant differences were detected in the disease-free survival for patients with mixed cellularity, nodular sclerosis, and lymphocytic predominance disease. Since only one patient with lymphocytic depletion was in this series, no statement can be made regarding this rare histopathology. Patterns of new disease differed for Stage I and II patients. The major difference was that patients with nodular sclerosing Stage II presentations involving the mediastinum were at considerable risk of developing subsequent disease in the pulmonary parenchyma or the pleura. This finding, together with the demonstration that a histologic diagnosis of mixed cellularity did not carry an inferior prognosis, indicates the need for reassessment of the appropriateness of applying treatment programs based on results of lymphangiographically staged patients to Stage I and II patients evaluated by laparotomy.


Asynchronous development of two monoclonal proteins (IgM lambda and delta 1 chains) in a patient with abdominal lymphoma. An IgM paraprotein was detected in a patient with abdominal lymphoma. Due to some doubts about the malignant nature of the situation, no cytostatic drugs were administered. When seen two years later the patient had considerably deteriorated in clinical status, with evidence of extra-abdominal involvement. At this time he presented a second paraprotein of low of molecular weight (74,000 to 80,000), consisting of two covalently linked delta1-chain fragments.


Prolactin dynamics following transphenoidal hypophysectomy for metastatic carcinoma of the breast. In nine patients who had undergone trans-sphenoidal hypophysectomy, prolactin dynamics were studied with intravenous thyrotropin releasing hormone (TRH). Residual prolactin secretory reserve was demonstrated in seven. Five patients were TRH tested both before and after trans-sphenoidal hypophysectomy. Hypophysectomy did not alter base line prolactin concentration but did decrease prolactin response to TRH from 55 ng/ml to 21 ng/ml (p less than 0.001). Post-hypophysectomy L-Dopa suppressed baseline prolactin concentrations to undetectable levels. There was no correlation between alterations in prolactin dynamics and tumor response to hypophysectomy. Trans-sphenoidal hypophysectomy is not effective in ablating prolactin secretion and serious doubts are raised about the role of altered prolactin dynamics in inducing breast cancer remission.


Quantitative exposure of grafted rat tracheas to 7, 12-dimethylbenz(a)anthracene. A method was developed for continuously exposing tracheal epithelium to measured amounts of carcinogen. Beeswax was the vehicle for sustained release of carcinogen, and tracheas transplanted to s.c. sites were target tissues. In the experiment reported here, transplanted rat tracheas were exposed to a potent carcinogen, 7,12-di-methyl benz(a)anthracene (DMBA). The rate of release of DMBA from the beeswax carrier within the tracheal lumen approached first order when the initial concentration of carcinogen was high (3200 to 325 microng in a 24.45-mg pellet). With lower concentrations, where the carcinogen was dissolved in the beeswax, initial release was rapid, and most of the carcinogen was delivered within 4 weeks. At high DMBA dose levels, the entire tracheal epithelium was uniformly replaced by keratinizing squamous metaplasia after 1 week of exposure, and after 2 months, when from 280 to 910 microng DMBA had been delivered, all transplants had developed invasive squamous carcinomas. Sarcomas also developed in 19% of the transplants. At lower dose levels the epithelial reactions were more varied, and tumor development was more protracted. The lowest DMBA dose presently known to diduce carcinomas in this experimental model is 40 microng, which is in the dose range used for tumor initiation in skin carcinogenesis studies in mice.


Antagonism of development and growth of 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors by the antiestrogen U 23,469 and effects on estrogen and progesterone receptors. This study analyzed the effectiveness of a nonsteroidal antiestrogen, cis-(3-[p-(1,2,3,4-tetrahydro-6-methoxy-2-phenyl-1-naphthyl)phenoxy]-1,2-propanediol (U 23,469) previously shown to be potent in antagonizing estrogen-induced uterine growth, in preventing the development of 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumors and in eliciting the regression of established tumors; the study also attempts to elucidate the mechanisms of the tumor antagonism of U 23,469. Virgin female Sprague-Dawley rats that receive DMBA at 47 to 50 days of age and then receive U 23,469 (250 micrograms s.c. in 0.15 M NaCl daily) have a greatly reduced number of mammary tumors and a markedly decreased tumor area. Treatment with U 23,469 for increasing time periods (3, 6, or 12 weeks) beginning 2 weeks after DMBA results in a progressive delay in onset of tumor appearance. U 23,469 treatment beginning 1 week after DMBA or given prior to DMBA is even more effective. The time course of tumor regression (3 months after DMBA) by U 23,469 or ovariectomy is similar, with 50% regression in ca. 2 weeks, and both elicit regression of almost all tumors (greater than 90%). After ovariectomy, cytosol progesterone receptor levels are greatly diminished in tumors and uteri, while cytosol estrogen receptor (ER) levels are high; in both tissues little (ca. one-third) of ER is in the nucleus. During U 23,469 treatment, cytosol ER content is very low in regressing tumor and uterus and over 90% of ER is in the nucleus; cytosol progesterone receptor is slightly depressed in the uterus but is at the untreated level in mammary tumor. These receptor studies suggest that the effectiveness of this antiestrogen in antagonizing mammary tumor development and growth may reside in its ability markedly to perturb the distribution of ER, maintaining over 90% of ER in the nucleus with concomitant low levels of cytoplasmic ER, a situation that may render the mammary tissue insensitive to the animal's own endogenous estrogens.


The effects of antioxidants on skin tumor initiation and aryl hydrocarbon hydroxylase. Butylated hydroxytoluene, butylated hydroxyanisole, and vitamins C and E are effective inhibitors of 7,12-dimethylbenz(a)anthracene tumor initiation in a two-stage system of tumorigenesis. These antioxidants did not significantly induce epidermal aryl hydrocarbon [benzo(a)pyrene]hydroxylase, nor did they have any effect when added directly to the in vitro aryl hydrocarbon [benzo(a)pyrene]hydroxylase assay. However, butylated hydroxytolene and butylated hydroxyanisole, when topically to mice, inhibited the in vitro, epidermally mediated, covalent binding of radioactive benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene to DNA. When butylated hydroxytolene and butylated hydroxyanisole were added in vitro, they did not inhibit the epidermally mediated covalent binding of the hydrocarbons to DNA. The inhibition of polycyclic hydrocarbon tumorigenesis by antioxidants may be related to the ability of antioxidants to prevent the in vivo activation of hydrocarbons to carcinogenic epoxides and/or other electrophilic intermediates or may be related to their ability to increase detoxification of the reactive intermediate that requires intact cells to be operational. In any event, the results suggest that the antioxidants have an indirect effect on the epidermal metabolizing system which leads to a decrease in covalent binding to DNA.


Species comparison of in vitro metabolism of aflatoxin B1. The metabolism of [14C]aflatoxin B1 by 9000 X g supernatant fraction of livers of duck, rat, mouse, monkey, and humans was compared by incubating the compound and liver fractions in the presence of cofactors and air for 30 min. The incubation medium was extracted with chloroform, and the soluble metabolites were separated on thinlayer plates and quantified; radioactivity remaining in the aqueous phase was determined in order to quantify metabolism to water-soluble derivatives. Duck, monkey, and human livers were most active in total conversion, each metabolizing approximately 80% of available substrate in 30 min. Rat and mouse livers had lower activites, metabolizing 15 to 20%. Duck liver produced mainly (60%) chloroform-insoluble derivatives, but all other species produced larger quantities of chloroform-soluble than insoluble metabolites. Aflatoxin Q1 was the principle chloroform-soluble metabolite produced by monkey, human, and rat liver, whereas duck liver produced mainly aflatoxicol in that fraction. Aflatoxin P1 was produced by monkey, human, and mouse liver, but not by duck and rat. The chromatographic region containing M1 and B2alpha contained low levels of radioactivity in all species except human. No consistent pattern of metabolism emerged which could be correlated with species differences in response to aflatoxin B1 toxicity or carcinogenicity.


Enumeration and identification of human leukemic lymphocytes by their natural binding of bacteria. The recently described property of bacteria to bind to human lymphocytes was used to distinguish between normal and chronic leukemic lymphocyte (CLL) populations. Strains of the following bacteria were used in this study: Arizona hinshawii, Escherichia coli strains 1 and 2, Bacillus globigii, Brucella melitensis, Corynebacterium diphtheriae strains 1 and 2, Corynebacterium xerosis, Sarcina lutea, Staphylococcus aureus, and Staphylococcus epidermidis. For identification of immunoglobulin-bearing lymphocytes, a strain of E. coli that did not bind to human lymphocytes was coated with anti-human light-chain antibody. Labeling of lymphocytes with bacteria was promoted by centrifugation. In the eight CLL patients studied, in which greater than 90% of the lymphocytes were leukemic cells, 52 to 77% were labeled by anti-human light-chain antibody-E. coli, 80 to 93% were labeled by Br. melitensis, and 78 to 95% were labeled by E. coli 1 compared to 11 to 24, 11 to 22, and 30 to 44%, respectively, in normal individuals, Thus, Br. melitensis, E. coli 1, and the anti-human light-chain antibody-E. coli may have diagnostic value for CLL. The percentage of the lymphocyte population that bound each of the other bacteria varied from patient to patient. Preliminary results obtained by studying the pattern of binding of E. coli 2, B. globigii, Sa. lutea, or S. aureus by leukemic lymphocytes suggest that categories of CLL patients may be distinguished by this method.


Increased in vitro growth capacity of tracheal epithelium exposed in vivo to 7, 12-dimethylbenz(a)anthracene. Heterotopic tracheal transplants of rats were exposed in vivo to 150 or 640 microng of 7, 12-dimethylbenz(a)anthracene (DMBA) delivered over 2 weeks, and the epithelium was studied in vitro in an attempt to identify changes in growth behavior during early phases of neoplastic development. Explants were made from the exposed tracheas, as well as from a series of controls, and the rate of epithelial outgrowth from the explants was measured. Secondly, the capacity of the outgrowths to survive as primary cultures and their ability to survive multiple in vitro passages were studied. During the initial planting, the rate of outgrowth was by far the greatest from the explants preexposed to 150 microng DMBA. Outgrowth from explants preexposed to 640 microng DMBA was sparse during the first planting but increased markedly during repeated planting when insulin- and hydrocortisone-supplemented medium was used. Establishment of outgrowth during repeated planting of carcinogen-exposed tracheal pieces was clearly hormone dependent. Control explants did not exhibit this hormone dependency. Primary cultures could be established from only three of six explants preexposed to 150 microng DMBA. These explants had been initiated in insulin- and hydrocortisone-supplemented medium. The primary cultures were successfully subcultured. Primary cultures were also established from five of five explants preexposed to 640 microng DMBA and cultured in hormone-supplemented medium. At least one cell line was obtained from each of the explants. To establish and maintain primary cultures from control tracheas required an enriched medium containing amino acid supplements, sodium pyruvate, and putrescine, as well as the hormone supplements. However, such cultures could not be subcultured. The primary cultures from the carcinogen-exposed explants and the subsequently developed cell lines all exhibited morphological characteristics of keratinizing squamous epithelium. These characteristics include: epithelioid cell morphology, multilayering and sloughing of orangeophilic squamous cells, and the presence of keratohyalin granules. These experiments demonstrate a markedly increased in vitro growth capacity of tracheal epithelium after a short in vivo exposure to carcinogen.


Detection of chemical carcinogens by unscheduled DNA synthesis in rat liver primary cell cultures. Unscheduled DNA synthesis was observed in primary rat liver cell cultures treated with members of five different classes of chemical procarcinogens requiring enzymatic activation as well as with a direct-acting carcinogen. In total, ten carcinogens and one related analog not commonly accepted as carcinogenic were active, while one weak carcinogen and four noncarcinogens were inactive. The production of unscheduled DNA synthesis by this spectrum of chemical carcinogens indicates that these cultures have substantially retained the metabolic capability of liver for activating diverse procarcinogens. Thus, such cultures may be useful for detecting the ability of chemicals to interact with DNA and, thereby, assigning them priority for consideration as potential cancer-causing agents.


Chemical modification of neamine.

Chemical modification of neamine. The aminocyclitol antibiotic neamine has been modified chemically by removing one or two hydroxyl groups from the 2-deoxystreptamine moiety to give 5- and 6- deoxyneamines (5 and 10), as well as 5,6-dideoxyneamine (15). Their antimicrobial activities were determined against several microorganisms, including kanamycin-resistant strains.