Metabolic aspects of the secretion of stored compounds from blood platelets. V. Effect of ionophore A23187 on washed platelets. 1. The A23187-induced secretion of preabsorbed serotonin from human blood platelets at 37 degrees C is studied. Preincubation at the same temperature before the addition of ionophore is necessary for maximal release induction. When total incubation time is kept constant, longer time with ionophore results in a smaller decrease in the level of metabolic ATP and increase in metabolic ATP and increase in metabolic IMP. This coincides with the reduction in secretion, but statistical treatment of the results suggests that the reduced secretion only partially explains the reduced drop in metabolic ATP, and that therefore a resynthesis of metabolic ATP from IMP may have taken place. 2. In some experiments induction of secretion takes place over a very narrow range of ionophore concentration. 3. When K+ substitutes for Na+ in the extracellular medium, the need for preincubation for maximal secretion becomes less evident, and at times is abolished, while there is still a significant increase in the metabolic ATP level by prolonged incubation with ionophore. 4. A reduction in secretion is observed with metabolic blockers when the ionophore is added after preincubation, but to a much less degree than when secretion is induced by thrombin, in spite of a great  reduction in the level of metabolic ATP. This may partly be explained by the increase in secretion induction by A23187 in the presence of inhibitors when the ionophore is added in the cold, suggesting that the inhibitor may cause "weakening" of the platelets' "resistance" to induction of secretion by ionophore. 5. When the effect of Ca2+ and of Mg2+ on the level of intermediates of the TP leads to hypoxanthine conversion is studied, it is evident that the addition of Ca2+ causes enhanced IMP accumulation and a reduction in the level of inosine plus hypoxanthine, while Mg2+ has the opposite effect. This suggests that the two metals affect the enzymes of the IMP leads to hypoxanthine conversion differently. 6. Indomethacin inhibits secretion induced by A23187, suggesting that prostaglandin intermediates may amplify the ionophore-induced release. The adenine nucleotide metabolism is not affected. 7. The results indicate that there is an indirect, rather than direct, link between the major metabolic changes and the secretion induced by A23187, but that the ionophore may cause intracellular changes which are not connected to its effect as release inducer.


Effects of dextran-linked chloromercuribenzoic acid on insulin release from microdissected pancreatic islets. Insulin release in response to dextran-linked p-chloromercuribenzoic acid was studied in microdissected pancreatic islets of non-inbred ob/ob-mice. No contamination of the dextran-linked mercurial with free chloromercuribenzoic acid was detected before or after the incubation with islets. In comparison with free mercurial, of the same thiol-blocking activity, the dextran-linked compound had a weak insulin-releasing action with a different dose vs. response relationship. The dextran-linked mercurial had no demonstrable effect on the islet content of cyclic AMP. The results support the hypothesis that free organic mercurials mainly stimulate insulin release by blocking thiol ground that are embedded within the beta-cell plasma membranes beneath their surfaces.


Spectroscopic studies of pepsin and its complex with Streptomyces pepsin inhibitor. Binding of Streptomyces pepsin (EC 3.4.23.1) inhibitor to the active site of pepsin causes a characteristic ultraviolet difference spectrum having a trough around 298 nm which suggests that tryptophan residue(s) are involved in a decreased refractive index or different charge density. The fluoreschat of the pepsin-inhibitor complex. Relatively large circular dichroism (CD) spectrum change at 280--310 nm was observed upon binding of the inhibitor. Solvent perturbation difference spectra of pepsin alone and the pepsin-inhibitor. Solvent perturbation difference spectra of pepsin alone and the pepsin-inhibitor complex obtained with 20% ethylene glycol as perturbant showed that the exposed 2.5 tryptophan residues were not buried upon binding of the inhibitor, whereas 1.5 tyrosine residues were buried. It is speculated that the microenvironmental change around tryptophan residue(s) which are not located at the inhibitor binding site is induced upon binding of the inhibitor.


Phosphoenolpyruvate carboxylase of Escherichia coli. Studies on multiple conformational states elicited by allosteric effectors with a fluorescent probe, 1-anilinonaphthalene-8-sulfonate. Conformational change of phosphoenolpyruvate carboxylase (orthophosphate: oxaloacetate carboxy-lyase (phosphorylating), EC 4.1.1.31) induced by allosteric effectors was investigated using a hydrophobic probe, 1-anilinonaphthalene-8-sulfonate (ANS). Kinetic experiments suggested that ANS binds with the enzyme at the sites which are not involved in the catalytic and regulatory functions, though it partially inhibits the enzyme activity with half-saturation concentration (S0.5) of 38.5 muM. Binding experiments showed that a maximum of 2 mol of ANS are able to bind with 1 mol of the enzyme subunit presumably with an equal dissociation constant to each other (34.5 muM). Flourescence emission of ANS was markedly increased by binding with the enzyme. L-Aspartate, the allosteric inhibitor, and CoASAc and fructose 1,6-bisphosphate (Fru-1,6-P2) the allosteric activators, produced various degrees of change in fluorescence, when added singly or in combinations. The changes were shown to be attributable to the allosteric interactions between the enzyme and effectors from some criteria such as structural specificity, half-saturation concentrations, and heterotropic-homotropic interactions of the ligands. It was concluded from these analyses that the enzyme can be in at least four conformational states which are distinct from each other. Especially noteworthy is the finding that the enzyme, upon simultaneous binding of CoASAc and Fru-1,6-P2, takes a new conformation which is enterely different from those induced by sole binding of each effector. In addition, the heterotropic interaction between the activator and the inhibitor was observed through conformational change by the ANS method, as observed in the kinetic studies.


The incorporation of tritium from tritium-enriched water into UDP-N-acetylglucosamine and UDP-N-acetylmannosamine catalyzed by UDP-N-adetylglucosamine 2-epimerase from Escherichia coli. Uridine diphosphate N-acetylglucosamine 2-epimerase from Escherichia coli 014 K7 H- catalyzes the reversible epimerization of uridine diphosphate N-acetylglucosamine to uridine diphosphate N-acetylmannosamine. During epimerization, tritium from tritium-enriched water is incorporated into both uridine diphosphate N-acetylglucosamine and uridine diphosphate N-acetylmannosamine. The position of incorporation is C-2 of the N-acetylhexosamine moieties.


Effect of genetic and physiological manipulations onthe kinetic and binding parameters of the adenine nucleotide translocator in Saccharomyces cervisiae and Candida utilis. 1. Ghe kinetic and binding parameters of adenine-nucleotide transport have been studied in mitochondria isolated from yeast cells in which the mitochondrial protein-synthetizing system had been inhibited by growth in the presence of erythromycin. These parameters have also been studied in promitochondria isolated from yeast grown in anaerobiosis aesence of ethidium bromide results in a loss of cytochromes b, alpha and alpha 3, but it does not affect the rate constant of ADP transport in isolated mitochondria, nor the number of binding sites for atractyloside, bongkrekic acid and ADP. 3. Promitochondria from S. cerevisiae grown in anaerobiosis, mitochondria from a qo mutant (qo mitochondria) and mitochondria from S. cerevisiae grown in the presence of erythromycin (ERY-mitochondria) are able to transport ADP by the same exchange-diffusion mechanism, sensitive to carboxy-atractyloside, and with the same rate constant as the wild type mitochondria. Promitochondria, qo mitochondria and ERY-mitochondria bind atractyloside, bongkrekic acid and ADP with the same high affinity as the wild type mitochondria. They only differ from the wild type mitochondria by a lower number of binding sites for ADP and for specific inhibitors of ADP transport. 4. Mitochondria isolated from the nuclear mutant p9 of S. cerevisae, called also op1, are characterized by a much lower affinity for bongkrekic acid than mitochondria from the wild type (20 times less). 5. Manipulation of the fatty acid composition of the mitochondrial membranes in the desaturase auxotroph mutant KD115 does not modify the number of sites, no their affinity of bongkrekic acid. 6. The above results are interpreted to mean that the structure and function of the mitochondrial adN translocator are not affected by any change in the mitochondrial protein synthetizing system.


[New approach to the interpretation of vibrational spectra of microbial cells]. IR-technique was applied to study baker's yeast cell and its envelope fragments. The quantitative evaluation of spectra was made using the polysaccharide band at 1070 cm-1 as the bench one. The comparison of the spectra of the whole cells and of the envelope fragments suggested that the spectrum of the yeast cell was governed by the composition of its envelope only. ATR-spectra of the whole cells and of envelope debris were obtained and the comparison of these with the "absorption" ones made it possible to conclude that the yeast cells vibration spectrum was for the most part the reflection spectrum governed by the composition of the cell envelope rather than the absorption one.


Heart rate, muscle tension, and alpha production of transcendental meditators and relaxation controls. A group of transcendental meditators, and a group of control subjects instructed to relax, were compared with respect to degree of relaxation reached as measured by changes in heart rate, tension of the frontalis muscle, and occipital alpha production. The only significant changes were decreases in these measures over time in the control subjects. The changes in the controls were probably the consequence of sleep or the onset of sleep.


[Use of rare earth elements as markers for the study of biologically active compounds. V. Luminescence spectra and binding centers of europium in proteins]. Luminescence spectra of europium ions bound to some proteins are investigated. Europium is shown to coordinate no more than two charged groups, first of all carboxyles. The possibility of more specific coordination of rare earths in proteins is discussed.


[Physical state of crystalline proteins. IV. Melting of trypsin and pepsin crystals]. The nature of heat transitions in trypsin and pepsin crystals during their heating at various rates is studied. Heating is shown to bring about only the denaturation of protein molecules in crystals.


[Effect of yeast cell membrane components on the IR spectrum of the cell]. IR-technique was used to study the effect of yeast cells treatment by water-alcohols mixtures on the quantitative relationship between the intensities of different bands in the spectra. The analysis of spectra made it possible to suggest that the reflection of the IR-irradiation from the cell is governed by the nucleoprotein layer of its envelope. It is shown that the treatment of yeast cell with ribonuclease raises the "IR-permeability" of the cell envelope.


[Spectroscopic study of conformational changes in proteins in aqueous solutions and mixed solvents]. The character of temperature changes of the absorption spectrum of water in protein solutions is the evidence of conformational changes of protein macromolecules. Along with the denaturation processes changes are also clearly seen at predenaturation stage. These changes involve a break of hydrophobic bonds in protein molecules and subsequent exponing of non-polar radicals into water. Thermodynamic characteristics of conformational changes of some proteins in aqueous solutions and water-organic mixtures are determined.


Allograft nephrectomy.

Allograft nephrectomy. The indications, complications and results of 54 allograft nephrectomies undertaken in 44 patients have been reviewed. There were 7 deaths following 39 nephrectomies done within 6 months of transplantation and complications occurred in a further 14 instances. Many of these resulted from pre-existing infection. When nephrectomy was carried out 6 months after transplantation there were no deaths and morbidity was less. If graft failure is diagnosed within 6 months of transplantation immediate nephrectomy is recommended to prevent future complications. When graft failure occurs later it is suggested that nephrectomy be undertaken only when definite indications for this are present.


The value of prophylactic antibiotics in transurethral prostatic resection: a controlled trial, with observations on the origin of postoperative infection. Prophylactic antibiotics significantly decrease the incidence of urinary tract infection following endoscopic prostate resection. They are ineffective in reducing the incidence of postoperative fevers, or in reducing the frequency of positive blood cultures during or after operation. A majority of prostate glands harbour colonies of potentially pathogenic organisms, and these are the commonest source of infection of the urinary tract following release by prostatic surgery.


Limited intertransverse fusion using iliac grafts. Fusion of the lower lumbar spine or lumbosacral joint can be achieved by the use of limited intertransverse iliac grats inserted by a lateral approach. The method offers certain special advantages and is no less successful than other techniques in common use.


Prophylactic lincomycin in the prevention of wound infection following appendicectomy: a double blind study. The prophylactic use of a single dose of lincomycin in 100 patients undergoing appendicectomy reduced the incidence of postoperative wound infection from 17 to 6 per cent compared with a similar group of control patients. There was no correlation with the histological finding in the appendix, but patients with perforated appendicitis were excluded from the study. Bacteriological examination of the appendix area before surgery showed that Bacteroides fragilis and Escherichia coli were the organisms most frequently isolated. The majority of wound infections were due to B. fragilis, either alone or in association with aerobic organisms, but infection due to E. coli and Staphylococcus aureus also occurred. Systemic antibiotic therapy can successfully reduce the complications following appendicectomy but it is necessary to use a compound with activity against anaerobic organisms.


Combined dexamethasone and low-molecular-weight dextran in acute brain infarction: double-blind study. Intramuscular dexamethasone combined with intravenous low-molecular-weight dextran (dextran 40) was compared with placebo in 40 patients with acute ischaemic cerebral infarction. A double-blind procedure was used. Dexamethasone was given for up to 14 days and dextran 40 for up to three days after the infarction. A weighted scoring system was used to evaluate neurological state and mobility. There were no differences in mortality or in improvement of the neurological or mobility scores between the two groups.


Therapeutic fibreoptic bronchoscopy in intensive care. Experience with therapeutic bronchoscopy using the fibreoptic bronchoscope in intensive care has shown it to be a useful procedure. The Nosworthy connection has also been modified to allow intermittent positive-pressure ventilation to be maintained during bronchoscopy. This procedure is valuable in those cases where sputum or blood are retained in the airways despite adequate physiotherapy and endotracheal suction. Fibreoptic bronchoscopy should be available as a routine service in intensive therapy units.


Endotoxaemia and renal failure in cirrhosis and obstructive jaundice. An investigation into the possible role of endotoxins in the pathogenesis of renal failure in cirrhosis and obstructive jaundice showed the two to be closely related. None of the patients with cirrhosis who had endotoxaemia had other evidence of Gram-negative infection at the time of the study, and the endotoxaemia was therefore probably due to impaired hepatic clearance of toxins normally absorbed from the gastrointestinal tract. In contrast, bacteriological evidence of Gram-negative infection was found in most of the patients with obstructive jaundice and endotoxaemia.


Differential localization of N-acetylated indolealkylamines in CNS and the Harderian gland using immunohistology. Using two different antisera, one binding only melatonin (M) and the other binding both M and N-acetylserotonin (NAS), brain tissue and the Harderian gland were investigated using fluorescence and peroxidase labeled double antibody immunohistology. M has been identified in the outer nuclear layer of the retina, in the optic nerves, chiasma and the tracts, suprachiasmatic nucleus and the Harderian gland. NAS, but not M, was found in the granule layer of the cerebellum, spinal tract of the trigeminal roots and the pontal and spinal reticular formation. The possible relationship of cerebellar and brain stem NAS to brain excitability as well as localization of M in the visual pathway, suprachiasmatic nucleus and the Harderian gland in the relation to the regulation of light dependent circadian periodicity is discussed.


Differentiation of pathogenic, gram-negative, bacterial genera in the presence of beta-2-thienylalanine. Described in a fast screening assay for aid in the differentiation of bacterial genera. Salmonella is separated from Escherichia and Shigella, as a generic group on the basis of an inhibition assay incorporating beta-2-thienylalanine, and using a defined medium. The assay is also usable for confirmation of taxonomic differences between genera. The inhibition reaction, readable as an 'all-or-none' phenomenon, follows a dose-response relationship for cultures affected by beta-2-thienylalanine, and is unaffected by the growth phase of the culture.


Identification of carbon dioxide as a dermatophyte inhibitory factor produced by Candida albicans. A factor produced by Candida albicans, which inhibits dermatophyte growth and induces arthrospore formation is characterized and identified. Candida dermatophyte inhibitory factor (CDIF) is volatile and fungistatic. Analysis of volatile materials produced by C. albicans was subsequently identified as carbon dioxide. The involvement of carbon dioxide in the inhibition of dermatophytes was demonstrated by: (1) utilization of commercial carbon dioxide to produce dermatophyte inhibition as well as arthrospore formation, and (2) prevention of dermatophyte inhibition by C. albicans through incoporation of soda lime into the incubation atmosphere. The ability of carbon dioxide to inhibit dermatophyte growth was shared with other gases (methane and helium), but arthrospore formation was observed only with carbon dioxide. The possible significance of carbon dioxide's induction of arthrospores, a form occasionally observed in active dermatophyte lesions, is discussed.


A miniaturized counting technique for anaerobic bacteria. A miniaturized counting technique gave results as good as the pour-plate and Most Probable Number (MPN) techniques for enumeration of clostridia spp. and anaerobic isolates from the gut. Highest counts were obtained when ascorbic acid (1%) and dithiothreitol (0.015%) were added to the reinforced clostridial medium used for counting. This minimized the effect of exposure to air before incubation. The miniature technique allowed up to 40 samples to be plated and incubated in one McIntosh-Filde's-type anaerobic jar, compared with 3 or 4 by the normal pour plate.


Isolation of Klebsiella pneumoniae from lake water. The question of the importance of aquatic borne Klebsiella pneumoniae to public health has been argued as the organism has appeared often in both polluted effluents and oligotrophic systems. Using a selective medium, double violet agar, which also differentiates K. pneumoniae from other organisms found in aquatic systems, we sampled water from three ponds and a large man-made lake, over a 3-month period. Results indicate that K. pneumoniae can be isolated consistently and in high numbers from eutrophic waters even when environmental stress reduces total numbers, but that isolations from cleaner waters are erratic. We conclude that mere isolation of the organism as an occasional contaminant probably does not present a hazard to public health.


Survival value of chemotaxis in mixed cultures. A motile, chemotactic strain of Proteus mirabilis outgrew a motile, non-chemotactic mutant in a semisolid, amino acid medium, although the two strains grew equally well in broth.


Presumptive identification of Klebsiella pneumoniae on m-FC medium. Klebsiella pneumoniae is a human and animal pathogen frequently encountered in the environment. The membrane-filtration method utilizing m-FC medium for the detection of fecal coliforms yields a colony type easily distinguishable as K. pneumoniae. These colonies appear as atypical light blue, nucleated, mucoid colonies differing considerably from the typical dark blue fecal coliform colonies.


Studies on adriamycin using a weekly regimen demonstrating its clinical effectiveness and lack of cardiac toxicity. Adriamycin has been given to 442 patients using the weekly regimen as initially described by Bonadonna et al. Hematologic toxicity and clinical effectiveness have been similar to those described with regimens in which the drug is given every 3 weeks. Stomatitis is more frequent with the weekly regimen than with the usual triweekly regimen. The incidence of electrocardiographic changes and arrhythmias was similar to that reported in other studies. Nine patients received between 500 and 550 mg/m2, nine between 550 and 600 mg/m2, 28 between 600 and 1000 mg/m2, and 22 between 1000 and 2500 mg/m2. None of the patients developed definite evidence of cardiomyopathy although six showed some disturbance of myocardial function. In each of the six patients, factors other than adriamycin cardiotoxicity were believed to play a major role in the myocardial abnormality. The difference between the incidence of cardiomyopathy seen in this series and that previously reported is statistically significant. The reasons for the difference are not clear but are probably related to the schedule that was used.


Phase I trial of dianhydrogalactitol administered Iv in a weekly schedule. Dianhydrogalactitol was given to 28 patients with a variety of advanced solid tumors on a weekly schedule in iv doses ranging from 2 to 80 mg/m2. No significant toxicity was encountered at doses up to 40 mg/m2/week for 4 weeks. At higher doses mild-to-moderate nausea and vomiting and hematologic toxicity were noted. Thrombocytopenia was more common than granulocytopenia and frequently resolved more slowly. No adverse drug-realted effects on liver, renal, coagulation, or cardiac function were seen. Although no patient had significant antitumor response (as strictly defined), objective improvement was noted in two patients, one with hypernephroma and the other with malignant melanoma. for phase II studies, a weekly dose of 70 mg/m2 is recommended for patients with normal hematopoiesis, with reduction by 25% (55 mg/m2) in patients with extensive prior radiation therapy, prior chemotherapy, and/or widespread metastasis to the bone.


Porfiromycin in the management of epidermoid and transitional cell cancer: a phase II study. Porifiromycin has been tested in many groups of patients over the past several years (1-7). This report is an analysis of greater than 100 patients with epidermoid and urinary tract transitional cell carcinomas treated with this agent. A review of these data and the available literature on this agent shows that porfiromycin offers definite usefulness in disseminated squamous cell carcinomas of the cervix, and definite but lesser effectiveness in epidermoid carcinomas of the lung, the head and neck region, and other sites. Responsiveness was also demonstrated in transitional cell carcinomas of the urinary tract. Toxicity was tolerable and consisted primarily of myelosuppression and local skin necrosis in sites where extravasation had occurred.


Identification and location of the histone H2A and H3 genes by sequence analysis of sea urchin (S. purpuratus) DNA cloned in E. coli. A 2000 base pair (bp) DNA fragment can be excised from sea urchin (S. purpuratus) histone gene repeat units with restriction endonuclease Eco R1. This DNA, which has been cloned in a bacterial plasmid, is known to encompass two of the five histone genes. The fragment has a single endonuclease Hind III cleavage site in one of the genes and a Hae III cleavage site in the other gene. We now report the nucleotide sequences of 62 bp adjacent to the Hind III site and 42 bp adjacent to the Hae III cleavage site. The results identify the cloned DNA as histone genes, show that it codes for histone proteins H2A and H3, and locate and orient H2A and H3 genes with respect to restriction endonuclease sites in the repeat unit.


Radioallergosorbent test (RAST) studies with insolubilized polysaccharides. Polysaccharide preparations from Aspergillus fumigatus and Candida albicans when used with some human sera were found to fix complement and precipitate with specific antibodies present in the sera. Precipitins consisted predominantly of IgG with traces of IgM and IgA. Following insolubilization of the fungal polysaccharide preparations with the N-carboxyanhydride of L-leucine, reactivity with serum precipitins was retained and reaction with IgE antibodies was demonstrated using the radioallergosorbent test (RAST). Results showed that for A. fumigatus and C. albicans at least, polysaccharide-specific IgE antibodies probably occur in sera from some hypersensitive individuals. We suggest that chemical methods of the type described which induce polymerization or cross-linking will be useful for detecting IgE antibodies in the RAST. Such an approach could be used with a variety of polysaccharide preparations which cannot be coupled to polysaccharide solid supports by the usual methods.


A double blind cross over trial of gonadotrophin releasing hormone (LHRH) in sexually impotent men. A double blind cross over trial of 500 mug of gonadotrophin releasing hormone or placebo subcutaneously every 8 h for 4 weeks in ten men with secondary sexual impotence is reported. No obvious clinical improvement occurred but statistical analysis of a libido score showed some overall improvement, especially the spontaneous occurrence of erections during the treatment period (P LESS THAN 0-05).


Antibody-mediated immunity in CFW mice infected with Mycobacterium lepraemurium. Humoral immune response in murine leprosy. A depression in antibody-mediated immunity (AMI) measured both in terms of circulating antibody and plaque-forming cells in the spleen was observed in CFW mice infected with M. lepraemurium when sheep red blood cells (SRBC) and human gammaglobulin (HGG) were used as antigens. The impairment in AMI was evident only after 75 days of infection thereafter the antibody response to SRBC antigen progressively decreased until the last day of experimentation (135 days). Within the first 60 days of infection no alteration in AMI was observed with the HGG antigen while the response to the SRBC antigen was significantly higher in the infected animals than in uninfected controls.


Determination of anti-DNA antibodies by a modified 125I-labelled DNA-binding test. Elimination of non-specific binding of DNA to non-immunoglobulin basic proteins by using an anionic detergent. A modified 125I-labelled DNA-binding test was developed. By using an anionic detergent, 0-025% sodium dodecyl sulphate, the non-specific binding of DNA by basic proteins was completely eliminated, while the specific binding of DNA by anti-DNA antibodies was not interfered with. The level of DNA-binding activity in normal human sera was decreased in the modified DNA-binding test, while its sensitivity was similar to that of the Farr assay in presence of sera from SLE patients.


Leukocyte migration inhibitory factor (LMIF) profile in primary and secondary immunodeficiency disease. Lymphocytes from patients with primary and secondary immunodeficiency disease were tested for capacity to produce LMIF after mitogen and antigen stimulation as well as for ability to stimulate and respond in unidirectional MLC-LMIF assay. Different patterns of immune abnormality in vitro were detectable when Con A and Candida albicans antigen were used. In addition, significant abnormalities in LMIF responding and stimulatory capacity were demonstrated in patients with Hodgkin's disease. LMIF production after stimulation with different agents allows for a better characterization of cellular defects in immunodeficiency disease.


Immune function in sarcoidosis. Studies on delayed hypersensitivity, B and T lymphocytes, serum immunoglobulins and serum complement components. An assessment of immune function was performed in twenty-four patients with recently diagnosed active sarcoidosis. Four patients manifested skin anergy to four antigens. All subjects except one were capable of generating a positive skin response to a croton oil patch test. The incorporation of [3H]thymidine by lymphocytes in vitro in response to the nonspecific mitogens--phytohaemagglutinin, pokeweed mitogen and Con A did not differ between anergic and non-anergic thymidine incorporation in vitro when stimulated by the specific antigens, streptokinase/streptodornase or Candida albicans. Lymphocytes obtained from nine of eleven patients having positive delayed hypersensitivity skin reactions demonstrated MIF production in vitro upon specific antigen challenge. Quantities of circulating B and T lymphocytes did not differ between anergic and absolute numbers of circulating B and T lymphocytes, as well as hypercomplementaemia and hypergammaglobulinaemia when compared to the control group.


Leucocyte aggregation and lymphocyte transformation induced by mercuric chloride. Leuco-aggregation tests carried out on normal subjects using the non-specific mitogen mercuric chloride produced micro-aggregates in the concentration range 2-100 mug/ml with a peaking effect at about 10 mug/ml. These results showed good correlation with lymphocyte transformation test thymidine uptake ratios which were maximal at 2-10 mug/ml.


In vitro effect of thymosin on T-lymphocyte rosette formation in rheumatic diseases. The in vitro effect of calf thymosin fraction 5 on T-rosette forming cells (E-RFC) was studied in Sjögren's syndrome (SS), rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE). The baseline percent E-RFC in sixteen normal controls was67-2 +/- 6-9. E-RFC was significantly decreased in SLE (42-6 +/- 17-0, P less than 0-0001) and SS (51-8 +/- 16-9, P less than 0-002) but not in RA (59-7 +/- 14-1). Ten of twenty-five SS patients and two of eleven RA patients had less than 50% E-RFC, and all showed a significant increase after incubation with thymosin (+ 16-5 +/- 6-5%, P less than 0-0001, and + 11 +/- 4-9%, P less than 0-001, respectively). Eleven of sixteen SLE patients had less than 50% E-RFC. Their response to thymosin was less dramatic but still statistically significant (+ 5-3 +/- 6-0%, P = 0-03). There was no response to thymosin in control subjects or in patients with baseline E-RFC greater than 50%. No increase in E-RFC was seen after incubation with calf spleen fraction 5 or known stimulators of cyclic-AMP. Sera from four active SLE patients, as well as the supernatant obtained from overnight culture of the lymphocytes from one SLE patients, were able to block T-rosette formation by normal lymphocytes, even after exposure to thymosin. Two 'blocking' sera were fractionated by sucrose density gradient ultracentrifucation. In one, the blocking capacity was found to reside in the 19S region containing IgM. In the second, the blocking capacity was in the 7S region containing IgG. Four 'blocking' lupus sera were depleted of IgG or IgM by immunoabsorption with goat anti-human IgG or goat anti-human IgM sepharose 4B. The blocking ability in three sera was partially decreased by depletion of either IgG or IgM, and in a fourth, only by removing IgG. The percent of lymphocytes staining with fluorescein labelled goat anti-human immunoglobulin antisera was increased in SLE patients (35-9 +/- 20-2 vs 21-7 +/- 5-9 in controls, P = 0-02). After overnight culture, the percent of staining cells decreased to normal values. These results suggest that thymosin can stimulate the differentiation of T-lymphocytes in patients with SS, SLE, and RA when the baseline E-RFC is decreased. Furthermore, the decreased percent E-RFC in SLE is probably due to cell-bound anti-lymphocyte antibodies that block sheep erythrocyte receptors on the T-cell and, possibly, thymosin receptors on undifferentiated lymphocytes.


Identification of mouse T and B lymphocytes from cytocentrifuged cell smears. A method enabling direct enumeration of mouse T and B cells from cytocentrifuged cell smears is described. The acid alpha-naphthyl acetate esterase (ANAE) staining was used as marker for T cells. The distribution of the ANAE marker-carrying lymphocytes closely followed the percentual distribution of T cells in spleen and lymph node. Electrophoresis analysis demonstrated that while more than 95% of resting spleen and lymph node T cells carried the ANAE marker, only less than 5% of the B cells were ANAE positive. The B cells (surface-bound Ig-carrying small lymphocytes) were identified by anti-Ig serum followed by rosetting of the B cells with Staphylococcus aureus strain Cowan 1 (StaCw). The ANAE marker-carrying cells did not overlap with the StaCw rosette-forming lymphocytes. Thus we conclude that the combination of StaCw rosetting and ANAE staining enables accurate identification of resting T and B cells from a single microscope slide. Ninety to 100% of Con A-activated blasts expressed the ANAE marker but only 60-85% of PHA and MLC-activated blasts were positive. Twenty to 33% of the blast cells stimulated by LPS expressed the ANAE marker. Thus the ANAE marker is not a reliable criterion for T cells in actibated state.


Immune and phagocytic function in patients on maintenance dialysis and post-transplantation. Immune, phagocytic and opsonic function was assessed in stabilized hemodialysis patients and compared with patients on maintenance immunosuppression following successful renal transplantation. Dialysis patients had a lymphopenia and a qualitative defect in lymphocyte function but opsonic and neutrophil metabolic function was normal. Post-transplant patients had normal intrinsic lymphocyte function but overall immunosuppression due to lymphopenia, selective T cell depletion, and drug inhibition. Neutrophil metabolic function was normal though there was a defect in opsonization.


A micromethod for stimulation of chicken lymphocytes in vitro using whole blood. A simple, rapid and reproducible micromethod for determination of in vitro mitogenic responses of chicken peripheral blood lymphocytes is described. The test utilizes 5 mul volumes of heparinized whole blood, 125I-labelled 5-iodo-2'-deoxyuridine with 5-fluoro-2'-deoxyuridine in place of [3H]thymidine and a multiple cell-culture harvester. The method permits a close follow-up of the mitogenic responses in the chicken.


The carriage of Escherichia coli on the peri-urethral area and in the feces in patients on long term low dose cotrimoxazole therapy. Peri-urethral and fecal carriage of E. coli has been studied in 30 patients maintained on long term low dose cotrimoxazole and in abacteriuric subjects not receiving any antibiotic. Cotrimoxazole treatment is associated with a dramatic reduction in E. coli carriage both in the feces and especially on the peri-urethral area which persists over months or years. Recovery of trimethoprim-resistant E. coli from the feces was uncommon but when observed antedated subsequent relapse due to resistant organisms. Possible mechanisms for low peri-urethral carriage rates include reduction in fecal carriage, vaginal secretion of trimethoprim or low urinary concentrations of that drug.


Restriction endonucleases.

Restriction endonucleases. This review provides a comprehensive account of the current status of the biology and biochemistry of restriction endonucleases. Both Class I and Class II restriction endonucleases will be considered. However, emphasis will be placed on the Class II group, which recognizes and cleaves a specific duplex DNA sequence. Their occurrence, purification, and characterization is discussed in detail. The characterization includes physical mapping information and determination of recognition sequences. In addition to detailed discussions of the biochemical properties of the enzymes, considerable attention is paid to the uses of these enzymes as tools for research in molecular biology. These uses include physical mapping of genomes and their transcripts, genetic analysis (marker rescue, etc.), DNA sequence analysis, analysis of complex genomes, and genetic engineering. Specific examples of each use are outlined. Practical aspects of both the isolation and use of the restriction endonucleases form the major theme of this review.


Once daily administration of a fluphenazine/nortriptyline preparation in the treatment of mixed anxiety/depressive states. A study of 223 patients diagnosed as suffering from mixed anxiety/depressive states was carried out in general practice to compare the effectiveness of treatment with a once-daily tablet preparation containing 1.5 mg fluphenazine plus 30 mg nortriptyline, taken either at night or in the morning, with the same total daily dose taken as 1 tablet t.d.s. Patients were randomly assigned to 4-week's treatment with one of the three drug schemes, and patients' self-ratings as well as physicians's ratings were used to assess symptoms. Both rating assessments showed that there were highly significant improvements in each of the three treatment groups over the 4-week period. Although there were no clinically important differences between improvements in the night-time dose and t.d.s. groups, there was a higher incidence of drowsiness and tablet defaulting among patients taking the single morning dose.


Local treatment of psoriasis of the scalp with clobetasol propionate and betamethasone-17,21-dipropionate: a double-blind comparison. A double-blind evaluation was carried out in 40 patients with moderate to severe psoriasis of the scalp to assess the effectiveness of a 0.05% alcoholic solution of clobetasol propionate as compared with a 0.05% alcoholic solution of betamethasone-17,21-dipropionate. Patients were treated on a non-selective basis with a twice a day application for 2 weeks. When comparing the effects on the parameters scaling, induration, erythema and itching it was concluded that clobetasol propionate was superior to betamethasone-17,21-dipropionate as an antipsoriatic compound. Clobetasol propionate did not cause any side-effects, while 2 of the patients using betamethasone-17,21-dipropionate developed a forehead folliculitis during the second week of treatment.


A double-blind, multi-centre comparison of naproxen and indomethacin in acute musculo-skeletal disorders. A double-blind, multi-centre trial was carried out in 191 patients with acute musculoskeletal conditions to compare the efficacy of naproxen and indomethacin. Patients were randomly allocated to receive either 250 mg naproxen or 50 mg indomethacin twice daily. Treatment was for 7 or 14 days. The results, assessed by the patients' subjective responses, showed that statistically significant improvements in pain, mobility and general condition were produced by both drugs from the first day of treatment. Improvement continued throughout the treatment period but there was no significant difference in the degree or rate between the two drug groups. Twenty-seven patients (13 on naproxen; 14 on indomethacin) withdrew from the study because of side-effects.


Electron microscopy of membrane-free folded chromosomes from Escherichia coli. Membrane-free folded chromosomes were purified from log-phase cultures of Escherichia coli and prepared for electron microscopy by aqueous (Kleinschmidt and Zahn) spreading. The appearance of the chromosomes depended on the salt concentrations in spreading. At certain salt concentration, the chromosomes resembled rosettes, with supercoiled loops of DNA radiating from a central core containing RNA. The rosettes support previous models deduced from physical studies of folded chromosomes. Apparently, cores contain must of the visible RNA, and the organization of the core is linked to the organization of the DNA loops.


Post-coital contraceptive effects of an agonistic analogue of luteinizing hormone releasing hormone. An analogue of synthetic hypothalamic LRH, D-[ALA]6-DES-[GLY]10-PRO9-ethylamide-LRH (Wy-18,481) was evaluated for agonistic (in vivo LH-releasing and ovulation-inducing), post-coital contraceptive and reproductive target organ effects. Both LRH and the analogue terminated pregnancy; there appeared to be a direct relationship between agonistic and post-coital contraceptive potency and activity. The analogue proved to be a potent agonist and both a pre-and post-implantational post-coital anti-fertility agent. In contrast to LRH, the congener did not produce a uterotrophic effect in the hypophysectomized rat. The data suggest that agonist analogue(s) of LRH can terminate pregnancy via hyperstimulation of the pituitary-ovarian-reproductive complex and the use of members of this neurohormonal class as potential clinical pro-fertility agents should be weighted with caution.


The effect of hydrocortisone treatment and adrenalectomy on insulin and glucagon secretion from the perfused rat pancreas. Hydrocortisone treatment increased blood glucose and plasma insulin levels in adrenalectomized rats. Insulin secretion during the immediate secretory response as well as the late phase of glucose-induced insulin release from the perfused pancreas from adrenalectomized rats was three times higher after hydrocortisone treatment. Glucose inhibited glucagon secretion from the perfused pancreas of adrenalectomized rats after hydrocortisone treatment but not from the pancreas of untreated animals. The immediate insulin secretory response to tolbutamide was increased by hydrocortisone treatment whereas tolbutamide did not affect glucagon release from the perfused pancreas of adrenalectomized hydrocortisone treated rats or control animals.


Glucose-induced insulin secretion and insulin sensitivity of peripheric organs of Egyptian sand rats before manifestation of diabetes. In Egyptian sand rats (Psammomys obesus) fed with native food and a low caloric vegetable diet after capture it was possible to study endocrinologic and metabolic changes of the early stages during the progression to diabetes. According to body weight gain and fasting blood glucose the animals were differentiated into two groups classified as basic and protodiabetic group, respectively. Isolated pancreatic islets as well as the perfused pancreases of sand rats responded to low concentrations of glucose with high insulin release during early stages of the development to diabetes. Changes in the insulin content of the islets could not be detected at this time although in the B-cells of the protodiabetic animals a degranulation was visible. During these early stages of the development to diabetes the in vitro insulin action on glucose utilization in soleus muscle and especially in epididymal fat pads as well as the basal glucose metabolism in adipose tissue were low. This strikingly reduced utilization of glucose by adipose and muscle tissues may be a factor which challenges the B-cell.


CNS-endocrine pancreas system. I. The hypothalamus response to insulin deficiency. The anterior, medial and posterior hypothalamic nuclei in alloxan-diabetic male rats were studied by karyometry. Selective responsiveness of separate nuclei of the medial basal hypothalamus (MBH) and the anterior hypothalamus was revealed. In the MBH cell nuclear-size changes were most prominent in the ventromedial nucleus and less pronounced, but significant, in the arcuate nucleus. In the anterior hypothalamus a significant response was produced by the supraoptic nucleus. The authors do not exclude the possibility that the reaction of the neurosecretory cells of the supraotic nucleus is indirect and reflects disturbed vasopressin balance in alloxan-diabetic rats in response to impairment of water-salt metabolism. The responsiveness of the MBH nuclei is considered as evidence for their involvement, the bentromedial nucleus in particular, in hypothalamic control of the endocrine pancreas. Various means by which insulin-sensitive hypothalamic areas may be implicated in this control are discussed.


CNS-endocrine pancreas system. II. Response of dorsal nucleus of the vagus nerves to insulin deficiency. Sensitivity of the vagal dorsal nucleus nerve cells to insulin deficiency was revealed in alloxan-diabetic rats by karyometry. Control measurements showed absence of similar changes in cell nuclear sizes of the adjoining area, the Nucltractus solitarii. The results provide evidence in favour of a selective response of the dorsal nucleus of the vagus nerves to insulin deficiency and allow assumption that not only the peripheral vagus but the central component of this cholinergic system contribute to endocrine pancreas control. It is suggested that the medial basal hypothalamus and the system of the vagus are links of a single control mechanism regulating endocrine pancreas and represented at different levels of the central and peripheral nervous system.


The effect of a single injection of testosterone propionate and estradiol benzoate on the LH-RH induced release of FSH and LH in the male rat. Male adult, intact rats were treated with 1000 or 2000 mug/kg testosterone propionate (TP), 500 or 1000 mug/kg estradiol benzoate (EB) or ol. arachidis only. 30 min, 24h, 48h and 7 days later they were injected with 5 mug/kg LH-RH or saline solution.  30 min after this FSH, LH and testosterone were determined in plasma by radioimmunoassay. It is shown that pretreatment with 500 mug/kg TP enhances significantly the FSH release after LH-RH even on day 7, while the greater dose of TP does not significantly affect the release of FSH. The LH-RH induced release of LH is also increased by the pretreatment with 500 mug/kg TP, while 2000 mug/kg TP do not alter the reaction to LH-RH. EB in both doses does not influence the LH-RH induced release of FSH and LH.


Immunohistological identification of neurophysin and neurophysin-like substances in different vertebrates. Using rabbit antisera to porcine neurophysin, the immunohistological unlabelled-antibody-peroxidase technique was applied to brains of man, rat, quail, pigeon, tortoise, frog, two cyprinid fishes, sturgeon and lamprey as well as to the urophysis of carp and the brain of the cockroach. In each of the investigated vertebrate speices the magnocellular neurosecretory system is immunoreactive, known to give a specific reaction for neurosecretory material by means of appropriate histological and histochemical methods, too. Moreover, in the rat brain we found immunoreactive material in neurons of the suprachiasmatic nucleus, in the parvocellular part of the paraventricular nucleus, in fibres of the external region of the median eminence and in neurosecretory exohypothalamic fibres. The subcommissural organ and Reissner's fibre as well as the urophysis of the fish spinal cord and neurosecretory cells of the insect brain are devoid of neurophysin or neurophysin-like substances.


The mechanism of insulin secretion after oral glucose administration. VIII. Pancreatic juice insulin excretion after glucose loading and meal ingestion in normal and vagotomized dogs. On pancreatic fistula dogs, in the pancreatic juice, immunoreactive insulin has been identified. Insulin excretion is shown to be inhibited by an intravenous glucose load and to be stimulated in a biphasic manner by an oral glucose load or by ingestion of mashed meat. Insulin excretion is reduced 2 months after bilateral truncular vagotomy and the reaction to both oral stimuli is abolished. -- There is no statistical correlation between pancreatic juice insulin and blood glucose, plasma IRI, or plasma amino nitrogen concentrations. Single extrainsular B-cells shown by histologic examination are suggested to be the source of pancreatic juice insulin. Compared to insulin secreted into the portal blood flow, insulin in the pancreatic juice amounts to only 0.1--0.2% of the daily secretory rate of the islets of Langerhans.


Pancreatic B-cell behaviour after changing the natural environment of sand rats (Psammomys obesus. On the basis of the blood glucose increase during the capitivity sand rats born in the desert were classified as normals, protodiabetics and diabetics, indicating a different adaptation to the new environment within a definite period. Isolated islets of animals, which did not develop a hyperglycemia, enhanced their insulin content during the adaptation period. The absolute insulin secretion rates in response to 16.5 mM glucose were rather similar between the three investigated groups and not modified by the insulin as well as glucagon content of pancreatic islets. But, since islets of hyperglycemic sand rats could not increase the insulin content, a significantly enhanced fractional secretion (as % of the content) could be observed. The results let us assume that the B-cell reaction during the adaptation period can be modified by further factors additionally to the changed environment.


Arginyl-tRNA synthetase from Escherichia coli. Influence of arginine biosynthetic precursors on the charging of arginine-acceptor tRNA with [14C]arginine. The behaviour of arginyl-tRNA synthetase (EC 6.1.1.19) in the presence of the arginine biosynthetic precursors, argininosuccinate, ornithine and citrulline, was studied in several Escherichia coli K12 strains and in E. coli W. The results of kinetic measurements with partially purified extracts indicate that the arginyl-tRNA synthetase of E. coli is not inhibited by the arginine precursors. The apparent affinity constant Km for arginine of the K12 enzyme is about 3.4 muM in the absence and in the presence of these precursors, whereas the W enzyme an apparently slightly lowered Km and a decreased [14C]arginyl-tRNA equilibrium level in the presence of argininosuccinate. This however was shown to be due to isotopic dilution of [14C]arginine by non-radioactive amino acid formed from argininosuccinate by argininosuccinate lyase (EC 4.3.2.1) contaminating the synthetase preparation. This finding emphasizes the necessity of using pure arginyl-tRNA synthetase in order to study the possible regulatory involvement of this enzyme in the control of the arginine regulon in vitro.


Interpretation of tRNA-mischarging kinetics. Incorrect tRNA aminoacylation reactions are characterized by very slow reaction rates and by the fact that in most cases they are incomplete. In a previous study some of us explained the incompleteness of the correct aminoacylation reactions of tRNA, which can be encountered under certain experimental conditions (for instance low enzyme concentration or high ionic strength) by an equilibrium between the aminoacylation and the deacylation reactions [J. Bonnet and J.P. Ebel (1972) Eur. J.Biochem. 31, 335-344]. In the present report we bring evidence that the incorrect valylation of yeast tRNAfMet by yeast valyl-tRNA synthetase studied under standard experimental conditions, can also be described by a kinetic rate law including the rate equations of the aminoacylation and of the various deacylation reactions. In particular we show that the incomplete mischarging plateaus reflect the existence of an equilibrium between the valylation reaction on the one hand and the spontaneous and enzymic deacylation of valyl-tRNAfMet and the reverse of the valylation reaction on the other hand. However, when the valyl-tRNA synthetase concentration is not very high the reverse reaction of the amino-acylation does not play a predominant part in the establishment of the plateau. These interpretations have been extended to other mischarging systems: valylation of yeast tRNAPhE by yeast valyl-tRNA synthetase and mischarging of tRNAfMet and tRNA2Val from yeast by yeast phenylalanyl-tRNA synthetase. Unusual mischarging kinetics have been discussed. Furthermore, and as in correct systems, we found that during the mischarging of tRNAfMet one ATP is hydrolyzed per tRNA charged with valine. We conclude that the correct and the incorrect amino-acylation of tRNA behave kinetically in a similar way.


The cell-wall lipopolysaccharide of Escherichia coli K-12. Structure and acceptor site for O-antigen and other substituents. The lipopolysaccharides of two wild-type Escherichia coli K-12 strains, two core-deficient mutants and one SR recombinant with Salmonella typhimurium specificity were analyzed. The respective oligosaccharides were dephosphorylated and methylated. Chemical analysis of the oligosaccharides and mass spectrometric analysis of their methylated derivatives indicated the presence of core structures with different degrees of completion. In different strains of E. coli K-12 the complete core is substituted at the non-reducing end with N-acetylglucosamine or with another substituent. There are indications that the latter may be N-acetylmannosaminuronic acid. In the SR recombinant the complete (N-acetylglucosamine-free) K-12 core is substituted with one S-specific oligosaccharide of S. typhimurium. The attachment site for all these substituents is the 6-position of the non-reducing core-terminal glucose. The heterogeneity of the K-12 core preparations and mode and nature of their substitution are discussed.


Heterogeneity of sites in isolated catalytic subunits of aspartate transcarbamoylase. Carbamoyl phosphate, a substrate of aspartate transcarbamoylase from Escherichia coli, binds with different modes of association in 3 sites in the unmodified catalytic subunits. Over a narrow pH range (6.6--8.0), positive, negative or no interactions are observed. Several substrate analogues also bind to 3 sites in the catalytic trimer. The association of pyridoxal phosphate, CTP and ATP, all competitive inhibitors of carbamoyl phosphate, exhibit negative interactions. Binding of succinate, an analogue of the second substrate, aspartate, is also characterized by heterogeneity. Dissociation constants to high and low-affinity sites differ by factors of 10-100. These observations clearly indicate that, although not observed kinetically, the active sites in the catalytic subunits of aspartate transcarbamoylase are heterogenous.


Calorimetric investigations on thermal stability of tRNAIle (yeast) and tRNASer (yeast). Variation with temperature of the partial heat capacities of tRNAIle (yeast) and tRNASer (yeast) has been determined in two buffers at various salt conditions by scanning microcalorimetry. The overall molar transition enthalpy, deltaHt = 320 +/- 20 kcal mol-1 (1339 +/- 84 kJ mol-1) is identical for the two tRNA species within the limits of experimental error. deltaHt does not show any dependence on the nature of the buffer, nor does it vary on addition of 1 mM MgCl2 or 150 mM NaCl. Thermal unfolding of the native structure to the random coil cannot adequately be described by a two-state, concerted transition under the experimental conditions applied in this study, but exhibits a multistep mechanism characterized by sequential unfolding of separable cooperative domains.


Interaction of substrate analogues with Escherichia coli DNA-dependent RNA polymerase. The inhibition of RNA polymerase with ATP and UTP analogues modified in the phosphate and ribose moieties has been investigated. 1. Modification of the terminal phosphate with a loss of the negative charge [adenosine 5'-(3-O-methyl)triphosphate, Ki = 1.75 mM] substantially weakens the binding ability of these analogues to the enzyme whereas modification with retention of the charge is not so detrimental [adenosine tetraphosphate, Ki = 0.17 mM]. 2. 2'-Modified analogues are only weak competitive inhibitors [2'-amino-2'-deoxyadenosine 5'-triphosphate, Ki = 2.3 mM] of their corresponding substrates [ATP, Km = 0.07 mM] whereas 3'-modified analogues are extremely potent in their inhibition [3'-amino-3'-deoxyadenosine 5'-triphosphate, Ki = 2.3 muM]. 3. A difference was observed in the inhibition of the elongation step of RNA polymerase by ATP and UTP analogues. Thus ATP analogues showed a strong binding to the CT form of the poly[d(A-T)] ternary complex and only a weak binding to the CA form. UTP analogues, on the other hand, showed a similar binding to both forms of the complex.


The mode of action of thiostrepton in the initiation of protein synthesis. The inhibition by thiostrepton of the initiation of protein synthesis is exerted at a different level from the inhibition of reactions mediated by EF-Tu and EF-G in the elongation of protein synthesis. The presence of thiostrepton on the 50-S subunit completely prevents the binding of the EF-Tu - GTP - aa-tRNA complex and EF-G - GTP complex to the 70-S ribosome, resulting in cessation of protein synthesis at a concentration of 1 muM thiostrepton. On the other hand, during initiation thiostrepton impairs the coupling of the 50-S subunit with the 30-S initiation complex, indirectly causing inhibition of IF-2-dependent reactions. Impairment of the coupling is strongly influenced by the conditions of incubation. Since formation of formylmethionylpuromycin and the IF-2-dependent GTP hydrolysis are inhibited to the same extent and recycling of IF-2 can take place in the presence of thiostrepton, we conclude that the basic mechanism of inhibition of initiation differs from that of inhibition of elongation.


Interaction of concanavalin A with external mannan-proteins of Saccharomyces cerevisiae. Glycoprotein nature of beta-glucanases. beta-Glucanases secreted into culture fluid by protoplasts or intact cells of the yeast Saccharomyces cerevisiae were investigated for the presence of covalently linked carbohydrates. Gel filtration of the enzymes on Biogel A-1.5m showed that endo-beta-1,3-glucanase is a polydisperse enzyme of high-molecular weight which elutes in about the same volume as external yeast invertase. Exo-beta-glucanase was eluted from the gel as a much lighter enzyme. Endo-beta-1,3-glucanase added to a mixture of extracellular mannoproteins was precipitated by concanavalin A to a similar extent to mannan, invertase and acid phosphatase. Under the same conditions exo-beta-glucanase did not interact with the lectin, but was partially precipitated from the solution in the absence of foreign mannan or mannan-proteins. The results show that endo-beta-1,3-glucanase of S. cerevisiae is a mannoprotein of a similar nature to external invertase and acid phosphatase. However, exo-beta-glucanase appears to be a glycoprotein which does not contain the highly branched mannan polymer in its molecule.


Derivatives of guanosine triphosphate with ribose 2'-hydroxyl substituents. Interactions with the protein synthetic enzymes of Escherichia coli. This report describes the preparation of four methylated and phosphorylated derivatives of GTP, 2'-O-methylguanosine 5'-triphosphate (PPP-Me2' Guo), and guanosine 2'-monophosphate 5'-triphosphate (PPP-Guo-2'P), 3'-O-methylguanosine 5'-triphosphate (PPP-Me3'Guo), and guanosine 3'-monophosphate 5'-triphosphate (PPP-Guo-3'P). These compounds were compared to GTP in their ability to support reactions catalyzed by Escherichia coli initiation factor 2(IF-2), elongation factor Tu (EF-Tu), and elongation factor G )EF-G). As with previously studied GTP analogues, the nucleotide specificities of IF-2-dependent N-formylmethionylpuromycin formation and EF-Tu-dependent Ac-Phe2-tRNA formation were similar. There was little difference between the reactions supported by GTP, PPP-Me2' Guo, PPP-Me3' Guo, and PPP-Guo-3'P, but PPP-Guo-2'P was a poor substrate with both enzymes. A spectrum of activity was observed in EF-G-dependent formation of N-acetylphenylalanylphenylalanylpuromycin. While PPP-Me2' Guo was almost as effective as GTP in supporting translocation, PPP-Guo-2'P was a very poor substrate, having even less activity than guanosine 3'-diphosphate 5'-triphosphate. Intermediate activities were observed with PPP-Me3' Guo and PPP-Guo-3'P, the former nucleotide being more active than the latter.


Replication of M-13 DNA in plamolysed Escherichia coli cells. Formation of fully synthetic duplex DNA. The replication of the double-stranded replicative-form DNA of bacteriophage M-13 was studied in a cellular system in vitro prepared by plasmolysis of M-13-am5-infected Escherichia coli cells. Newly synthesized DNA was density-labelled with bromodeoxyuridine triphosphate and analysed by equilibrium centrifugation in neutral CSCL. After a 60-min incubation at 30 degrees C 15-20% of the radioactive label in corporated from (32P)DGTP was found in fully synthetic duplex DNA, corresponding to 7-9 replicative form molecules/cell. The plasmolysed cell system is therefore capable of re-initiating new rounds of replicative form replication in vitro. The kinetics of labelling indicate that molecules are selected for replication at random from an intracellular pool of approximately 150 replicative form molecules. Rifampicin and nalidixic acid, which interfere with the semiconservative replication of replicative form DNA, completely prevent the formation of fully synthetic duplex DNA.


Mapping of promoter sites on the genome of bacteriophage M13. With the aid of transcription studies on restriction fragments of bacteriophage M13 DNA it has been demonstrated that at least eight promoter sites are located on the M13 genome. Five of these promoters initiate the synthesis of RNA chains which contain at their 5'-terminal end pppG (G promoters), while the other three promoters initiate RNA chains which start exclusively with pppA (A promoters). The positions of these promoter sites on the physical map are: 0.82 (G0.82), 0.88 (G0.88), 0.94 (G0.94), 0.01 (G0.01), 0.08 (G0.08), 0.36 (A0.36), 0.51 (A0.51) and 0.56 (A0.56). The G promoters were found to be clustered within a distance of one-third of the genome length from the central termination site for transcription (map position 0.77). The A promoters, however, were found at greater distances from this termination signal. Based upon the incorporation of [gamma-32P]ATP or [gamma-32P]GTP, the capacity of these promoters to initiate the synthesis of RNA chains varies. The strongest G promoters are G0.82, G0.94 and G0.08 and the strongest A promoter is A0.36. As judged from their position on the genetic map, it is postulated that two promoters, namely G0.94 and G0.01, are located within gene II. The other promoters are most probably located immediately in front of the gene VIII/VII boundary (G0.82), and immediately in front of gene V (G0.88), gene II (G0.08), gene IV (A0.36), gene I (A0.51) and gene VI (A0.56). No evidence has been obtained so far for the existence of a promoter immediately in front of gene III.


Conformational studies on murein-lipoprotein from the outer membrane of Escherichia coli. Conformational studies on an isolated integral membrane protein are reported. Lipoprotein of Escherichia coli outer membrane was released from murein by treatment with either lysozyme or trypsin. The isolated lysozyme-released lipoprotein (lipoprotein I) contained 2 or 3 muropeptides covalently linked at the C-terminal end, while the trypsin-released lipoprotein (lipoprotein II) was free of muropeptides and lacked the C-terminal peptide Tyr-Arg-Lys. Circular dichroism spectra of the two preparations were essentially identical, and they show an alpha-helix content of about 80%. According to calculations based on the Chou-Fasman rules for proteins of known sequence, lipoprotein is 64% alpha-helix and 15% beta-structure. Infrared spectroscopy qualitatively supports these values. The conformation was stable in the pH range of 5 - 12. Danaturation of lipoprotein by heat, 8 M urea, or sodium dodecylsulphate was a fully reversible, cooperative process. The thermal denaturation of lipoprotein occurs in two steps with transition points at 79.4 degrees C for lipoprotein I and at 85.1 degrees C for lipoprotein II. Lioprotein markedly changes conformation at dodecylsulphate concentrations where micelle formation sets in. The unusual behaviour of the lipoprotein convormation in sodium dodecylsulphate is discussed in relation to the lipoprotein conformation and aggregation within the membrane.


Sequence studies on D-serine dehydratase of Escherichia coli. Primary structure of the tryptic phosphopyridoxyl peptide and of the N-terminus. An improved procedure for large-scale production of crystalline D-serine dehydratase (EC 4.2.1.14) from Escherichia coli is described. The N-terminal sequence of the enzyme (Mr 45500) was determined in a solid-phase sequencer as Met-Glu-Asn-Ala-Lys-Met-Asn-Ser-Leu-Ile-Ala-Gln-Tyr-Pro-Leu-Val-Lys-Asp-Leu-Val-Ala-LEU-Lys. Four of the first five N-terminal residues are homologeous with tryptophanase, another pyridoxal-phosphate (P-Pxy) enzyme that catalyzes alpha,beta-elimination reactions. After borohydride reduction and tryptic digestion of the enzyme, a peptide was isolated showing the sequence Lys-Asp-Ser-His-Leu-Pro-Ile-Ser-Gly-Ser-Ile-Lys(P-Pxy)-Ala-Arg. No clear homology of this portion of the enzyme with tryptophanase or another pyridoxal-phosphate enzyme was observed.


Free 3'-OH group of the terminal adenosine of the tRNA molecule is essential for the synthesis in vitro of guanosine tetraphosphate and pentaphosphate in a ribosomal system from Escherichia coli. tRNAPhe from yeast modified at the 3'-terminal adenosine residue or missing part of its CpCpA end was investigated for its ability to participate in the synthesis of guanosine tetraphosphate and pentaphosphate using Escherichia coli ribosomes. Only tRNA containing complete CpCpA end and an intact ribose residue in the terminal adenosine was active in this process. The absence of the 2'-hydroxyl group of the terminal adenosine does not influence the reaction, but the 3'-hydroxyl group is essential and cannot be replaced by an amino group or a hydrogen atom. tRNA in which the 3'-terminal adenosine is replaced by formycin is active in the induction of guanosine tetraphosphate and pentaphosphate synthesis, but the reaction proceeds relatively slowly.


Degradation of abnormal proteins in Escherichia coli. Differential proteolysis in vitro of E. coli alkaline phosphatase cyanogen-bromide-cleavage products. Escherichia coli alkaline phosphatase has been purified and modified by either carboxymethylation or treatment with cyanogen bromide (CNBr). Only the CNBr-treated protein was degradable in an E. coli cell extract. Separation of the CNBr cleavage products by gel filtration in non-denaturing conditions gave rise to a number of oligomeric complexes, of which only those of molecular weight less than approximately 29000 were degradable in E. coli cell-free extracts. Carboxymethylation of the non-degradable complexes (greater than 29000 molecular weight) resulted in the formation of some complexes of less than 29000 molecular weight: such newly formed complexes were degradable by E. coli cell-free extracts. It is suggested that E. coli cell-free extracts may contain a protease/peptidase system which is active against peptide complexes below 29000 molecular weight, but inactive against peptide oligomers of greater molecular weight.


DNA blockade by rifampicin-inactivated Escherichia coli RNA polymerase, and its amelioration by a specific mutation. 1. Partially diploid strains of Escherichia coli containing both rifampicin-sensitive and rifampicin-resistant RNA polymerase are, in general, sensitive to the drug: of the two copies of the rpoB gene present in such strains, that which codes for sensitive enzyme is dominant. 2. RNA polymerase purified from a normal sensitive strain of E. coli, and inactivated by rifampicin, can "blockade" bacteriophage T7 DNA in vitro, inhibiting its transcription by drug-resistant enzyme molecules. 3. A mutation, rcs-40, reverses the normal dominance relationship in vivo, without detectably affecting the concentrations of resistant and sensitive RNA polymerase in the diploid cell. I show that rcs-40 is closely linked to the rpoB gene which codes for the rifampicin-sensitive enzyme. 4. Rifampicin-sensitive RNA polymerase purified from E. coli rcs-40, although indistinguishable from the normal enzyme by many criteria, is significantly less efficient in the production of drug-dependent DNA blockade.


Isolation and properties of an inhibitor of Escherichia coli RNA polymerase. An inhibitor of the Escherichia coli RNA polymerase has been isolated from E. coli and has been partially characterized. The inhibitor, a polypeptide of molecular weight 70000, acts to shut off RNA synthesis at about the time that the first round of RNA synthesis is over, preventing any further RNA synthesis. The inhibitor apparently does not recognize specific termination sequences in the DNA template, since it works equally well with double-stranded DNA, single-stranded DNA and poly[d(A-T)] as templates for RNA synthesis, and because the RNA molecules synthesized from T7 DNA appear to be terminated at the same site either in the presence or absence of the inhibitor. Several experiments indirectly indicate that the inhibitor may reversibly bind to the RNA polymerase at the termination step, in a ratio of approximately one inhibitor molecule per polymerase molecule.


Chorismate mutase/prephenate dehydratase from Escherichia coli K12. 1. The effect of NaCl and its use in a new purification involving affinity chromatography on sepharosyl-phenylalanine. A new simplified procedure for the purification of chorismate mutase/prephenate dehydratase, based on affinity chromatography on Sepharosyl-phenylalanine, has been developed. The method utilizes the effect of NaCl on the binding properties of the enzyme. NaCl inhibits both the mutase and dehydratase activities of the enzyme. In each case this inhibition is cooperative indicating homotropic interactions between NaCl binding sites on the enzyme. In addition NaCl induces homotropic cooperative effects between chorismate binding sites and between prephenate binding sites. NaCl also increases the sensitivity of the enzyme to inhibition by phenylalanine.


Chorismate mutase/prephenate dehydratase from Escherichia coli K12. 2. Evidence for identical subunits catalysing the two activities. On the basis of amino acid composition, tryptic fingerprints and the determination of amino acid sequences around the four cysteine residues, it can be concluded that chorismate mutase/prephenate dehydratase from Escherichia coli K12 consists of identical, or closely similar subunits. It follows from this that the mutase and dehydratase activities of the enzyme are probably catalysed on the one subunit.


Catalase biosynthesis in yeast: formation of catalase A and catalase T during oxygen adaptation of Saccharomyces cerevisiae. Catalase A from Saccharomyces cerevisiae and its biosynthetic precursors can specifically be immunoprecipitated from extracts obtained from yeast cells grown in the presence of L-[3H]leucine or 59FeCl3. The enzyme and its precursors recognized by a specific antiserum are absent from anaerobic cells. During oxygen adaptation of yeast pre-grown on 0.3% glucose under anaerobic conditions catalase A is formed via a heme-less precursor, probably the apomonomer of the protein, and a heme-containing intermediate. When cells are grown in the presence of Tween 80 the amount of catalase A, but not of catalase T, increases 4-fold. Comparison of the mode of synthesis of catalase T and A shows that no precursor-product relationship exists between the two proteins.


Stopped-flow studies of changes in fluorescence of 8-anilino-1-naphthalene sulfonic acid caused by magnesium and salt binding to yeast enolase. Stopped-flow studies of magnesium and salt (potassium chloride and acetate) effects on yeast enolase were carried out by following 8-anilino-1-naphthalenesulfonic acid fluorescence changes. The fluorescence changes appear to be largely caused by subunit association and dissociation, though there is evidence in some reactions for large changes in fluorescence occurring within the dead time of the stopped-flow measurements. These data are combined with measurements of initial enzyme activity after incubation in various solvents with or without magnesium to obtain subunit association and dissociation rates. From these, it is concluded that magnesium and the salts act by directly changing the affinities of the subunits for each other, apparently by producing a rapid change in protein conformation.


Thermodynamic studies on the specificity of L-isoleucine-tRNA ligase of Escherichia coli MRE 600. Calorimetric investigations on binding of amino acids and isoleucinol to the enzyme. The association enthalpies, delta Ha, involved in the reactions between L-isoleucine:tRNA ligase (AMP-forming) from Escherichia coli MRE 600 (EC 6.1.1.5) and various amino acids have been determined calorimetrically in 50 mM potassium phosphate buffer, at pH 7.5, in the presence of 1 mM dithioerythritol. The delta Ha values for binding of L-isoleucine, L-leucine, L-valine, L-norvaline and L-2-amino-3S, 4-dimethyl pentanoic acid agree within the limits of experimental error in magnitude (3.7 +/- 0.9 kcal mol-1 or 15.5 +/- 3.8 kJ mol-1 at 25 degrees C) and variation with temperature (delta cp = -430 +/- 20 cal mol-1 K-1 or 1799 +/- 84 J mol-1 K-1). In view of the large differences in the equilibrium constants for the corresponding binding equilibria, the identical association enthalpies suggest that the enthalpic contribution to the Gibbs free energy of binding, delta Ga, cannot be responsible for the specificity of the interaction of the enzyme with the amino acids. It has rather to be inferred that the entropic term, delta Sa, is decisive in discriminating the correct amino acid. Analogous calorimetric binding studies on the reaction between L-isoleucinol and the enzyme suggest that the absence of the carboxyl group renders the association enthalpy more positive (by 4-5 kcal mol-1 or 16.7-20.9 kJ mol-1) with respect to that of the amino acids. The variation with temperature of the delta Ha values, however, practically parallels that of the amino acids.


Enzyme-linked sandwich immunoassay of macromolecular antigens using the rabbit antibody-coupled glass rod as a solid phase. A highly sensitive sandwich immunoassay of macromolecular antigens using the rabbit antibody Fab' - beta-D-galactosidase complex and the rabbit antibody immunoglobulin-G-coupled glass rod as a solid phase is described. The Fab' fragments of rabbit antibody IgG are conjugated with beta-D-galactosidase from Escherichia coli using N,N'-o-phenylenedimaleimide. Rabbit antibody IgG is coupled to the aminoalkylsilyl glass rods (3 mm in diameter and 5 mm in length) using glutaraldehyde. A wide range of the concentrations of rabbit IgG fraction (20-2000 mug/ml) is effective for coupling, and the amount of rabbit immunoglobulin G coupled can be controlled. The smallest amounts of ornithine delta-aminotransferase from rat liver, human immunoglobulin G and 2,4-dinitrophenyl human immunoglobulin G that can be determined are 0.03, 0.3 and 0.04 fmol, respectively. The sensitivity of the assay for these antigens is affected mainly by the non-specific binding of the complexes to the solid phase and the ability of antigen molecules, adsorbed on the solid phase, to bind specifically the complexes. The assay with the rabbit antibody immunoglobulin-G-coupled glass rods is simpler and more sensitive than that with the rabbit antibody immunoglobulin-G-coupled Sepharose 4B.


Characterization and site of action of a soluble protein that stimulates peptide-bond synthesis. A recently identified soluble protein, named EF-P, stimulates peptide bond synthesis from ribosomal-bound N-formylmethionyl-tRNA and the aminoacyl-tRNA analog, puromycin. Using this model of peptide bond formation we have purified this activity approximately 100-fold from ribosome-free extracts of Escherichia coli. In order to study the mechanism by which the EF-P factor stimulates peptide bond formation, we examined and compared the requirements and site of action of the spontaneous and the EF-P-mediated synthesis of peptide bonds. We find that "enzymic" peptide bond synthesis (+EF-P) is characterized by relatively broad temperature and NH4Cl optima, a sharp Mg2+ optimum at 12 mM, and an apparent pKa of approximately 8.5. The characteristics of enzymic peptide bond synthesis closely resemble those reported for native peptidyl-puromycin formation rather than other models of peptide synthesis. Factor EF-P requires both 30-S and 50-S subunits for activity. The 30-S particle is inactive by itself and may function in the reaction merely to bind the fMet-tRNA substrate. Both the peptidyl transferase and the EF-P binding site may be part of the 50-S subunit. Unlike all other propagation factors, EF-P does not require the 50-S ribosomal proteins L7 and L12 and may therefore occupy a different ribosomal site.


Symmetry and asymmetry of the pyruvate dehydrogenase complexes from Azotobacter vinelandii and Escherichia coli as reflected by fluorescence and spin-label studies. Fluorescence-lifetime measurements of FAD bound to lipoamide dehydrogenase from Azotobacter vinelandii and Escherichia coli were performed. It is shown from these results that the two FAD groups in the isolated dimeric enzyme, as well as in the enzyme in the intact complex of E. coli, are in non-equivalent surroundings. This contrasts with the near equivalence of the FAD groups of both the enzyme and complex isolated from A. vinelandii. Reduction of the complex with Mg2+, thiamine pyrophosphate and pyruvate or with NADH enables the attachment of a maleimide analogue specifically to the lipoyl moieties of the transacetylase(s). Spin label [N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)maleimide] introduced in such a way proves the existence of at least two different micro-environments around the lipoyl moieties in complex isolated from A. vinelandii. Electron paramagnetic resonance spectra of the specifically spin-labelled complexes from E. coli and A. vinelandii, when dissolved in tricine [N-tris(hydroxymethyl)-methylglycine] buffer, show interactions of at least two electron spins with each other, which indicate that the lipoyl moieties are rather close together. Fluorescent label [N-(1-anilinonaphthyl-4)maleimide] is specifically attached to the lipoyl moiety of the high-Mr transacetylase of the freshly isolated complex from A. vinelandii. From the large differences in the apparent lifetimes tau p and tau m, as detected by phase fluorimetry, it is shown that this fluorscent label is distributed in different micro-environments. The differences observed in energy transfer between fluorescent label, attached to the lipoyl moiety of the high-Mr transacetylase, indicate different conformations of the complex from A. vinelandii. Upon introduction of the label after reduction with NADH a much larger energy transfer, thus a shorter distance, is observed between the label and FAD than when reduction is performed with Mg2+, thiamine pyrophosphate and pyruvate. A similar conformation dependence upon reduction is found for the pyruvate dehydrogenase complex from E. coli. It is thus proposed that the transacetylase of E. coli and the high-Mr transacetylase of A. vinelandii are both non-symmetrically distributed within the complex.


Purification of the membrane-bound DD-carboxypeptidase of the unstable spheroplast L-form of Proteus mirabilis by affinity chromatography. Non-competitive inhibition of the enzyme by penicillins and low stability of the enzyme-inhibitor complex. Membrane-bound DD-carboxypeptidase of the unstable L-form of Proteus mirabilis was solubilized by the non-ionic detergent Genapol X-100 and purified to protein homogeneity by affinity chromatography on ampicillin bound to succinyl-aminododecyl-cellulose. The purified enzyme with a molecular weight of 43000 is inhibited non-competitively by penicillin G and carbenicillin, indicating a function of the penicillins as allosteric inhibitors. Sensitivity of the enzyme to penicillins is only moderate with a Ki of 1 muM for penicillin G. Breakdown of.the enzyme-inhibitor complex EI with different penicillins occurs rapidly with reappearance of active DD-carboxypeptidase. The half-life of EI with penicillin G is 5.5 min at 30 degrees C and 3.5 min at 37 degrees C, 10--1000-fold shorter than EI half-lives of DD-carboxypeptidases in several other bacteria. The low stability of the enzyme-inhibitor complex and the moderate penicillin sensitivity appear to be the basis for the continued activity of DD-carboxypeptidase during growth of the L-form and synthesis of peptidoglycan in the presence of high concentrations of penicillin.


[Microtubule inhibitors].

[Microtubule inhibitors]. This lecture is divided into 3 parts. The first one is a general survey of mitoclasic agents with a review of the main categories and a summary of the conclusions obtained from a comparative study of the antimitotic properties of various compounds on animal cells and plant cells. The second part describes the results of personal ultrastructural studies carried out on plant cells. They include researches on the normal behavior of microtubules in the cells and an analysis of various kinds of inhibition. The last part deals with the action mechanism of mitoclasic compounds, especially colchicine.


[Role of catecholamines and serotonin in regulating cyclic and tonic secretion of gonadotropic hormones]. In female rats with normal estrous cycle, in the proestrus phase, a significant decrease of the serotonine level was revealed in the anterior and medio-basal parts of the hypothalamus which coincided with ascent of the releasing factor for gonadotropic hormones in the hypothalamus and for gonadotropins in the hypophysis. The dophamine level in the hypothalamic anterior and medio-basal parts changed in different ways attaining during oestrus its maximal values in the anterior and medio-basal parts of the hypothalamus. No regular changes were revealed in noradrenaline content in the course of estrous cycle in any hypothalamic area. For purposes of studying the mechanisms of regulation of the gonadotropins tonic secretion, the same parameters were recorded in androgensterile and ovariectomized rats. Levels of serotonine and noradrenaline revealed no regular connection with changes in the gonadotropins production, while the anterior part of hypothalamis dophamine concentration in the was in reverse proportion to the extent of gonadotropic secretion. Serotonine seemed to participate in regulation of the cyclic but not the tonic secretion of gonadotropins, while dophamine seemed to exert an inhibitory effect on regulation of tonic secretion of gonadotropins.


[Somatostatin, a new hormone? (author's transl)]. Somatostatin, or SRIF (Somatotropin Release Inhibiting Factor), is a tetradecapeptide of hypothalamic origin, which inhibits the secretion of growth hormone. It has also been recognized in other parts of the central nervous system, in the islets of Langerhans, and the mucosa of the upper digestive tract. Parenteral administration of synthetic SRIF inhibits the release of growth hormone, basal and stimulated by muscular exercise, arginine, L-DOPA, insulin-induced hypoglycemia, and sleeping. It also inhibits insulin and glucagon secretion, basal and stimulated, and several other secretory processes in endocrine and exocrine glands. It may have a depressor effect on some neurons in the central nervous system. Considerable interest has been prompted in the field of diabetology by the demonstration of somatostatin-induced suppression of growth hormone and glucagon : both hormones are over-secreted in many diabetic patients, and both may be noxious for small blood vessels in the diabetic. The eventual therapeutic use of somatostatin in humans is restricted, for the moment, by the unavaibility of long-acting SRIF preparations and the possibility of some adverse effects mainly affecting hemostasis. Evaluation of the physiological role (s) for this newcomer, and of the eventual pathophysiology of endogenous somatostatin, represent an unexpected and exciting field of neuro-endocrinology.


The localization of skeletal light meromyosin in cells of myogenic cultures. Fluorescent antibodies against skeletal light meromyosin were used to study the localization of this muscle-specific antigen in myotubes, myoblasts, presumptive myoblasts and fibroblasts found in six-day myogenic cultures. The labelled antibody bound only to the lateral edges of the A-bands in myofibrils. The antibody did not bind to antigens in the nucleus, cytoplasm or in the microfilaments beneath the plasmalemma in any of the cell types examined. Similarly, the external face of the cell surface of unfixed, living myotubes and mononucleated cells did not bind the antibody. Immunodiffusion tests confirm these results: high salt extracts of myotube-containing cultures reacted against anti-skeletal light meromyosin, whereas extracts of fibroblasts and presumptive myoblast cultures failed to precipitate the antibody. It is proposed that if myosin is present in the plasmalemma of these cells, as is suggested bhe myofibrils of definitive muscle.


[Intramuscular injection of lidocaine in prevention of complications and mortality in acute myocardial infarction: double blind study on 246 cases (author's transl)]. 246 cases of acute myocardial infarction in subjects aged 70 or less, with clinical symptoms beginning no more than 8 hours before, altered enzymes, abnormal Q wave, ST segment and T wave, were studied. 99 of them received, on hospital admission, an intramuscular injection of 250 mg of lidocaine, 147 an injection of saline. In the following 3 weeks, significant differences were observed in the appearance of severe arrhythmias (no cases in the treated group and 18 cases in the placebo group, p less than 0,001) and of heart failure (39 cases in the treated group and 79 in the placebo group, p less than 0,05) and in mortality (3 cases in the treated group and 18 in the placebo group, p less than 0,05).


[The effects of verapamil on angina threshold during exercise (author's transl)]. The efficacy and duration of the effects of verapamil on angina threshold during exercise were examined in five patients with proved angina pectoris, with the experimental model of a latin square 5 X 5. The effects of verapamil (0,1 mg/kg i.v.) were compared with those of a placebo (20 ml saline i.v.), of isosorbide dinitrate (5 mg, sublingual), of propranolol (0,1 mg/kg i.v.), and of dipyridamol (20 mg i.v.). All the treatments were studied within 20 minutes after administration; the efficacy of the "drugs" was checked against placebo. Verapamil and isosorbide dinitrate significantly prolonged the duration of exercise and total amount of work done before angina and EKG alteration appearance. Similar changes were observed after propranolol, but not significantly different from the effects of placebo. Dipyridamol reduced thw work performance, without significant difference from placebo. The effects of verapamil, though less marked than its peak effects, were still present 70 minutes after administration. The possible mechanism of work performance improvements induced by verapamil in angina pectoris is briefly discussed on the basis of the Triple Product changes observed during exercise.


[Evaluation of the parameters concerning the method for determination of the sinus node recovery time. Personal experience (author's transl)]. On the basis of the personal experience of 36 patients with sick sinus syndrome, the role of length, seat, frequency of atrial stimulation (AS) on the duration of correct sinus node recovery time (CSNRT) were examined. The length of AS has little importance for values over 2 minutes. In 2 cases the CSNRT were pathological only if the position had been changed from the lateral wall of the right atrium to the middle right atrium. The frequency of AS is very important for the duration of pathological and maximal CSNRT. Therefore it is better to give every patient the major range of frequencies of AS as for a correct quantitative determination of sinus dysfunction so as not to lose some falsely negative results.


[Physiopathogenetic hypotheses in peripheral block (author's transl)]. The authors discuss the aetiological, electrophysiologic and diagnostic problems pertinent to peripheric blocks. Using the current ECG and VCG tests, it is often difficult, and sometimes impossible, to identify the ventricular wall affected by the block, the intraparietal localization and the seat of the block. Purkinje fibres, junction or muscle fibres. Because of the scarcity of experimental data, some of the diagnostic-differentials exposed have more the feature of logical hypothesis than that of certainty. The commonly held concept that the peripheric blocks do not alter, but that the terminal phases of ventricular depolarization do, is confirmed. The initial phases can also be altered, in that the Purkinje-junction-muscular fibres may be damage even in the septum, while the large ducts of conduction remain undamaged. Several discrepancies found between the presence of electric patterns of septal necrosis and that of infarctions in autopic examinations can possibly be explained as peripheric blocks localized in the left septum.


Studies on the mechanism of transduction by bacteriophage phi gamma. I. Genetic characterization of the transducing segment. The bacteriophage phi gamma, though related to the lambdoid phage phi80, has unusual features in its specialized transduction and is being investigated to determine the mechanism of the transduction process. Genetic analysis of the transducing element gives evidence for a relatively long and uniform linear segment, up to about 1% of the E. coli chromosome, extending in either direction from the prophage attachment site, e.g., on the right side: att80-tonB-trpABCDE-cysB-pryF. The att end includes a variable amount of phage genome, probably very short in most particles. In a small fraction of the transducing particles the phage segment may be more extensive and, conversely, the bacterial segment is shorter, ending around cysB. The transducing segment from modificationless bacteria carries a site susceptible to the K-restriction system which affects the efficiency of transduction.


Some genetic consequences of changes in the level of recA gene function in Escherichia coli K-12. Genetic recombination was studied in E. coli mutants that carry lesions in the recA gene but retain some capacity for generating recombinant progeny. We observed that recombination was detectable only at a very low level during the incubation of leaky RecA- merozygotes in broth. However, recombination appeared to occur at much higher frequencies when recombinant progeny were assayed by selection on minimal agar. Analysis of the recombinants obtained with Hfr donors revealed a deficiency of multiple exchanges per unit length of DNA in leaky RecA- strains. In many of these crosses recombinants that inherited donor alleles close to the transfer origin were much reduced in frequency, except when the recipient was also RecB-.


Gene conversion and intragenic recombination at the SUP6 locus and the surrounding region in Saccharomyces cerevisiae. Spontaneous secondary mutations of the ochre suppressor SUP6 were selected in a haploid strain of Saccharomyces cerevisiae. Unselected tetrads were dissected from crosses heterozygous for one of three allleles of SUP6 and for three other loci in this region which span a length of 14 map units (his2, cdc14 and met10). The study showed that all of these markers were characterized by high frequency of meiotic gene conversion and long conversion lengths which frequently extended into adjacent marked loci. Despite the high conversion frequency of SUP6, recombination between alleles of this locus reached a maximum frequency of only 2 x 10(-3) protrophs/spore. Although the allelic recombination frequencies were not distance dependent and consequently could not be used to order the alleles, the inequality between the two recombinant outside marker combinations among selected intragenic recombinants produced an internally consistent map of the suppressor locus. Recombination at SUP6 (whether detected as conversion in tetrads or the production of recombinants among random spores) was accompanied by significantly less than 50% outside marker recombination.


[Reversion of RNA-polymerase mutations affecting F'-factor stability]. Ts+ reversions of amber mutations tsR on the beta-subunit of Escherichia coli RNA polymerase (79 min on the genetic map) were investigated. TsR mutants are viable due to the partial suppression of amber mutations by su2 suppressor. Three types of reversions were isolated in the course of the selection for Ts+ character, namely, intragenic reversions and two types of extragenic reversions, located at different regions of the bacterial chromosome. The mutation N5, located between 0 and 15 min. on the map, increases practically to normal the amount of RNA polymerase beta-polypeptide, which is diminished as a result of ts22 amber mutation action, and increases the plating efficiency of several T4 phage amber mutants. The mutations designated as D are located on the chromosome near the spcA locus (64 min. on the E. coli map). These mutations are characterized by pleiotropic effect. They have properties of a weak suppressor increasing the efficiency of su2 action on amber mutations of E. coli and phage T4. At the same time D mutations are capable to decrease sharply the efficiency of the crosses with F' strains. Both these characters determined by D mutations do not segregate in transduction. It is suggested that the decrease of sexduction by D mutations depends on their influence on replication of episomes in the recipient cells.


[Transmissible hybrid plasmid RP4-ColE1]. Hybrid plasmid RP4-ColE1 was obtained by joining DNA plasmids RP4 and ColE1, each of which possessed only one site of restriction for EcoR1. These plasmid molecules were restricted by endonuclease EcoR1 and then treated by ligase. The hybrid plasmid retained the property of transmissibility typical for drug factor resistance RP4. Non-transmissible mutant of the hybrid plasmid selected by the character of the resistance of Escherichia coli C600 (RP4-ColE1) to the phage PRR1 is used for subsequent investigations.


[Detailed model of semi-conservative DNA replication. Possible role of DNA-polymerase errors in fragmented copying of both matrix strands]. A model of semiconservative DNA synthesis is considered. The model is based on a suggestion that the synthesis proceeds in the course of one-directional stretching of both matrix strands through the replicase complex (replisome), or of one-directional movement of the replisome along the chromosome. Each nucleotide link of the matrix strand find itself successively in the section of RNA primer synthesis, in the section of DNA polymerase and in the control section of a proofreading 3' leads to 5' exonuclease. Deoxynucleoside incorrectly joined by DNA polymerase of the replisome to the 3'-OH end of growing chain are, as a rule, removed when transferred in the control section. Since the reverse movement is prohibited, the DNA polymerase loses the 3'-OH end of new DNA chain and proves to be incapable to continue the synthesis. It can be renewed only after a new RNA primer formation. For continuation of the DNA synthesis after an incorrect deoxynucleotide addition, replicase must take two more mistakes in succession: to retain the incorrect nucleotide and to join the next one to this unproperly arranged primer. Only in that case the non-complementary nucleotide in a new DNA chain can escape the action of proofreading exonucleases. Hence, according to the model under discussion, the frequency of mutations should be many times lower than that of incorrect nucleotide joinings. The latter results, as a rule, in DNA chain synthesis cessation and in fragments formation. Taking into consideration the frequency of spontaneous mutations in bacteria (about 10(-9) per base-pair), the frequency of non-complementary joinings during semiconservative synthesis can be suggested of the order 10(-3) in accordance with the average Okazaki fragments size of 1000 nucleotides. Another possible reason for periodical interruptions of semiconservative DNA synthesis and for fragments formation is a distortion of replisome structure, accompanying the release of the matrix strand being copied in the direction of already replicated chromosome region. Both mechanisms can participate concurrently in DNA fragments formation. If replisomes are attached to a cell membrane, the sites of chromosome that are also fastened in the membrane, in particular, the site of "origin", are to be replicated using some mobile replicase complex.


[Mechanism of genetic recombination during bacterial recombination. VI. Single-stranded conjugation]. The mutation BT43 in the dnaB gene inhibits conjugational DNA synthesis in the recipient cell at 42 degrees C. Since only one DNA strand is transferred from the donor to the recipient in these conditions, this single strand is integrated into the recipient chromosone. This is characterized by a high increase of recombination frequency per length unit, an effect well known in the case of transformation. This peculiar genetic process is proposed to be called "single stranded conjugation". It is more efficient in recipient cells recB-recC-sbcB-lacking two main degrading enzymes, exonucleases I and V. The proof of single strandedness was given by means of clonal analysis in a special experiment. The transfer of the selected marker into the thermosensitive recipient took place at 37 degrees C and the transfer of the non-selected marker -- at 42 degrees C. Thhe progeny of one merozygote must be mixed i.e. consist of cells with both alleles of the non-selected marker. This was confirmed by experimental data.