Estimation of pancreatic IRI output rate and its relations to glucose tolerance in normal anasthetized dogs. The pancreaticoduodenal and portal venous blood flows were recorded electromagnetically in anaesthetized dogs. Blood glucose and IRI were measured in the arterial, portal, and peripheral venous as well as in the intestinal venous blood. By a mathematical model the actual net IRI output of the whole pancreas was estimated. Under basal conditions it is 10.2 +/- 2.4 mU/min (n = 30; 26 kg mean body wt.). After i.v. glucose injection, IRI output is rapidly enhanced. The biphasic nature of this reaction was unequivocally demonstrated by consideration of the ratio IRI output : blood glucose. Pancreaticoduodenal blood flow increases transiently in relation to the increased blood glucose concentration. The IRI secretion rate is well correlated with the blood glucose concentration and to the amounts of glucose or of blood reaching the whole pancreas. It is also correlated with the portal IRI Concentration. The overall peripheral venous or arterial IRI concentrations are correlated with the IRI secretion rate, but not in all individual experiments. The different phases of IRI output (basal rate, stimulated output 1-10 min and 10-60 min) show no influence on each other, nor are they correlated with the peripheral IRI concentration area. Basal IRI output is negatively correlated with the glucose assimilation constants. These constants or the peripheral BG areas, however, are independent of the stimulated IRI output rate. However, both the assimilation constants and the peripheral BG areas are related to the peripheral IRI concentration areas. Hepatic uptake of insulin and dynamics of pancreatic blood flow seem to contribute considerably to the estimated correlation pattern.


Tolbutamide-induced changes of the DNA, protein and insulin content and the secretory activity of isolated rat pancreatic islets. Following prolonged administration of tolbutamide the DNA- and protein content per islet was enhanced but the IRI content per islet was diminished. Glucose-induced (2.0, 8.0 or 16.6 mM) and leucine-induced (12.5 or 25.0 mM) IRI release from isolated islets, as well as 14C02-production from U-14C glucose, were decreased. Theophylline (5.0 mM) restored the glucose sensitivity of the islets towards normal. The results indicate that tolbutamide-induced islet cell hyperplasia does not entail islet hyperfunction, as previously thought. Decreased IRI release may partially be explained by a tolbutamide-induced alteration of the adenylate cyclase/phosphodiesterase system of the B-cell.


Immuno-fluorescent reactivity to anti-somatostatin in the gastro-intestinal mucosa of the dog. Evidence is presented that somatostatin-containing cells are present in the gastro-intestinal tract of the dog. Thus immuno-fluorescent cells were detected by the use of antiserum to cyclic somatostatin. These cells were mainly encountered in the antral mucosa and in the neighbourhood of gastrin-producing cells. No cross reaction was observed between gastrin and somatostatin. It is suggested that locally produced somatostatin controls gastrin secretion, and, more generally, that somatostatin-containing cells, multifocally distributed, modulates secretion of a large number of glands.


Diabetes in the mouse due to two mutant genes -  a bibliography. A bibliography is presented of work involving the two diabetes-producing autosomal recessive mutations in the mouse, db and db2j.


Influence of anoxia on glucose metabolism in pancreatic islets: lack of correlation between fructose-1,6-diphosphate and apparent glycolytic flux. When equilibrated with O2-CO2 (95:5), pancreatic islets of non-inbred ob/ob-mice exhibited a sigmoidal dependence of 3H2O production on D-(5-3H)-glucose concentration; the rate was most sensitive to changes of glucose concentration around 5mM and tended to be maximum above about 15mM glucose. 3H2O production from more than 5 mM D-(5-3H)-glucose was about twice as fast as the production of 14CO2 from equimolar D-(U-14C)-glucose. Islets equilibrated with N2-CO2 (95:5) did not exhibit a sigmoidal dose-response curve for 3h2o production, the process being inhibited by anoxia at glucose concentrations above 5mM. Pieces of exocrine pancreas had a slower aerobic 3H2O production than the islets and showed a clear enhancement of the process during anoxia. In comparison with oxygenated islets, anoxic islets exhibited decreased concentrations of glucose-6-phosphate and increased concentrations of furctose-1,6-diphosphate. The concomitant inhibition of glycolytic flux may be due to a low lactate dehydrogenase activity in islets yielding a slow reoxidation of NADH and a slow phosphoglyceraldehyde oxidation under anaerobic conditions.


Monoamine oxidase inhibitors: nature of their interaction with rabbit pancreatic islets to alter insluin secretion. Using an in vitro rabbit pancreas system, we studied the effect of monoamine oxidase (MAO) inhibitors on flucose-stimulated insulin secretion. We evaluated the effect of both brief (15 min) and prolonged (60 min) exposure of pancreas segments to non-hydrazine (harmine, alpha-methyltryptamine, tranylcypromine and pargyline) and hydrazine (phenelzine, nialamide, iproniazid) type MAO inhibitors. All of the hydrazine type MAO inhibitors potentiated glucose-stimulated insulin secretion. Of the non-hydrazine inhibitors, only harmine and alpha-methyltryptamine potentiated glucose-stimulated insulin secretion. Hydrazine, although not itself an MAO inhibitor, also potentiated insulin secretion. Sixty minutes of exposure to tranylcypromine or alpha-methyltryptamine caused a decrease in insulin secretion. These MAO inhibitors are primary amines and primary amines can inhibit insulin secretion. The dopamine (DA) or serotonin (5-HT) content of the B-cells was increased by incubating rabbit pancreas with L-3, 4-dihydroxyphenylalanine (L-Dopa) or 5-hydroxytryptophan (5-HTP) for forty-five minutes prior to stimulation with glucose. Non-hydrazine MAO inhibitors increased dopamine inhibition of insulin secretion and either did not alter, or decreased serotonin inhibition of insulin secretion. Rabbit pancreatic islets were isolated using the collagenase digestion technique. The MAO activity of islet homogenates was determined using 5-HT and DA as substrates. Rabbit islet MAO has only one-tenth the specific activity against 5-HT (35 +/- 8.7 mumumoles/mg/min, M +/- SEM) that it has against DA (357 +/- 62.3 mumumoles/mg/min). This suggests that one reason that MAT inhibitors do not increase serotonin inhibition of insulin secretion is because MAO is not the major pathway for 5-HT inactivation in rabbit pancreatic islets. These studies suggest that MAO inhibitors alter insulin secretion, by both decreasing B-cell monoamine degradation and by mechanisms that do not involve MAO inhibition.


[Morphologic characteristics of lysogenic phages with conversion activity in Shigella flexneri]. The morphology of three lysogenic bacteriophages which induce antigenic conversion in various Shigella flexneri serotypes (II and V type antigen and 7.8 group antigen) has been investigated. A quite different morphology appeared with respect to that of Salmonella.


Recurrent peptic ulcer.

Recurrent peptic ulcer. From 1 to 5% of patients can be expected to develop recurrent ulceration following current surgical therapy for peptic ulcer disease. The development of recurrent ulcer frequently reflects an inadequacy of the initial procedure. The nature of the inadequacy is often difficult to delineate because of alterations in anatomy and physiology and the lack of accurate diagnostic procedures. Incomplete vagotomy and inadequate gastric resection account for the vast majority of surgical deficiencies. Gastrinoma, retained gastric antrum, and hyperparathyroidism are the most frequently encountered endocrine causes. A thorough evaluation must include gastrointestinal X-rays, fiberoptic endoscopy, multiple serum calcium and gastrin determinations, and provocative testing. Medical management of recurrent ulcer fails in the vast majority of cases. Reoperation is successful in about 70% of cases and has a mortality rate of 4%. Recurrent ulcer after simple gastroenterostomy is best treated by gastric resection or vagotomy and resection. After initial adequate gastric resection, vagotomy alone usually suffices. Antrectomy and, if necessary, re-vagotomy should be done for recurrent ulcer after vagotomy and drainage. Re-vagotomy alone is usually effective therapy for recurrent ulcer after initial vagotomy and resection. Non-acid reducing operations should not be done, as they result in high mortality and high second recurrence rates.


Histological sexing of a reticulum cell sarcoma arising after liver transplantation. Nuclear sex determination, based on both X and Y chromatin counts in a reticulum cell sarcoma which arose in the liver 5 months after orthotopic grafting, clearly showed that the tumor was of host origin. The Kupffer cells of the graft were also found to be of host origin, demonstrating that their replacement may occur within 6 months of operation.


Genetic and physiological characterization of met15 mutants of Saccharomyces cerevisiae: a selective system for forward and reverse mutations. One hundred and thirty-three spontaneous and induced mutants of the met15 locus in Saccharomyces cerevisiae were characterized with respect to temperature sensitivity, osmotic remediability, interallelic complementation, and suppressibility by amber and ochre suppressors. Forty mutants are osmotic remedial; 17 of these, and no others, are also temperature-sensitive. Seven of 133 mutations are suppressible by an amber suppressor and 11 are suppressible by an ochre suppressor. Seventy percent of the mutants exhibited interallelic complementation, suggesting that the functional gene product of the met15 gene is a multimeric protein. Relative map positions of 30 met15 were estimated from the frequencies of X-ray-induced mitotic reversion of various heteroallelic diploids. All complementing nonsense mutations are located near one end of the gene in contrast to other nonsense mutations which span most of the gene, thus relating the direction of translation of the mRNA with respect to the fine-structure map. Recombination studies indicated that two of 30 mutants contained deletions of the entire met15 locus. -- It was established that a variety of mutational types, including missense, nonsense, and deletions, are recovered with this unique system in which both forward and reverse mutations can be selected on the basis of methyl mercury resistance and methionine requirement of the met15 mutants.


Regulation of newly evolved enzymes. II. The ebg repressor. The ebg (evolved beta-galactosidase) regulatory locus has been mapped. The map order is argG-ebgA-ebgR-tolC-metC, and there is 1.6% recombination between ebgR and ebgA. Studies with ebgR-/ebgR+ merdiploids have shown that ebgR- is recessive, and it is concluded that the synthesis of ebg enzyme is under negative control.


Genetic analysis of regulatory mutants of alkaline phosphatase of E. coli. A fine structure map of the phoR region of E. coli, mutations of which affect the rate of alkaline phosphatase synthesis, was constructed by Hfr X F- crosses. Mutations causing three different phenotypes (previously reported as phoRa, phoRb, phoRc (Garen and Echols 1962a,b) are clustered in three closely linked genetic loci. PhoR mutants of all three types, including the phoRb type not previously tested, are recessive to wild-type phoR+. In addition, phoRa and phoRc complement each other, while phoRa and phoRb do not. Our results support the hypothesis of Morris et al. (1974) that phoRc mutants represent a cistron (phoB) different from phoR.


Relationship between DNA repair capacity and cellular aging. The experimental evidence is reviewed which bears on the theory that aging in mammalian cells may be related to a decline in the efficiency of normal DNA repair processes. Although the data are as yet fragmentary, they do suggest that there is an age-associated decline in the capacity of cells to perform at least certain types of repair. This is particularly noticeable in human diploid cells as they reach terminal senescence in vitro. Whether this decline is causally related or even contributory to normal aging remains, however, an open question.


Cancer-related aspects of regeneration research: a review. Tissue regeneration is simply the replacement of lost cells of a tissue by those remaining. Epimorphic regeneration involves dedifferentiation of many tissues and their organization into a blastema which eventually differentiates into the missing part, usually an appendage. A detailed comparison of the cell membrane changes occurring in epimorphic regeneration, tissue regeneration and cancer can contribute to greater understanding of the differences between normal and tumor cells. Further, there is evidence that epimorphic regeneration fields may in some instances suppress tuomr induction and control existing tumors. This influence may be mediated by bioelectric fields, which are ubiquitous in nature and appear to control many cellular events. Disruption of these bioelectric fields suppresses epimorphic regeneration and may lead to cancer in mammals, while applied electric fields alter regenerative events and cause tumor regression.


[Pathogenetic model-theories of endogenous psychoses (author's transl)]. The multiplicity of frequently conflicting views in the psychiatry of today inspires the wish for a uniform way thinking about the thorough knowledge and the many hypotheses that have become almost boundlessly vast. Such ideas of models as have played a part for quite some time in the mathematical and natural sciences, and of late in the sociology, are also to be found in the field of psychiatry, without however always being called so expressis verbis. Guided by the historical development an attempt has been made to expose model-like ideas about the etiology and pathogenesis of endogenic psychoses, first of all of schizophrenia, as they have become decisive for modern psychiatry. It appears that in the abbreviation of the "model" the basis for a discussion already overdue is to be found.


Characteristics of DNA fractionated on benzoylated DEAE-cellulose. Chromatography on BD-cellulose columns with a salt gradient and formamide separates cellular DNA into two fractions (fraction I eluted within the salt gradient, fraction II with formamide), the proportions of these two fractions (ca. 2:1) being similar for DNA from a number of eucaryotic organisms. Yeast DNA was chosen for a detailed study of the mode of fractionation. Several physicochemical parameters, binding to nitrocellulose filters, sensitivity towards nuclease S1, labelling properties in vivo, and hybridization properties of the two DNA fractions were compared. It was shown that both fractions are native DNA and that the fractionation does not depend on the size or the (G + C) content of the DNA. Fraction I DNA contains only a small portion of molecules having single-stranded ends. Fraction II DNA is a heterogeneous population, containing molecules with peculiar structural characteristics: (a) It contains DNA molecules with single-stranded ends and/or gaps sensitive to nuclease S1; labeling experiments suggested that these are molecules undergoing repair and replication. (b) Another portion of fraction II is molecules sensitive to nuclease S1 in regions which are not single-stranded. (c) A third portion is DNA which, after treatment with nuclease S1, is still strongly bound to the resin. Indications that the segregation may be due to the presence of specific DNA sequences comes from the above experiments and from the finding that fraction I DNA is enriched in ribosomal genes and fraction II DNA in tRNA genes.


Purification of tDNA from yeast.

Purification of tDNA from yeast. tRNA-tDNA hybrids from yeast have been isolated. The main step in purification was chromatography on a BD-cellulose column with salt gradients and formamide, which separates the hybrid material from excess DNA. The hybrids were characterized by density centrifugation in CS2SO4 and by treatment with alkali and pancreatic ribonuclease. Experiments in which DNA that had been sheared to different molecular weights was used for hybrid formation suggest that the tRNA cistrons are tandemly arranged and that the external spacer DNA is preserved in the tDNA.


Isolation, chemical and electron microscopical characterization of neutral-salt-soluble type III collagen and procollagen from fetal bovine skin. Extraction of fetal bovine skin at neutral pH and in the presence of protease inhibitors solubilized substantial amounts of type I and type type III collagen and Type III procollagen. Type I and Type III collagen were separated from each other by salt precipitation, and DEAE-cellulose chromatography was used to separate collagen from procollagen. The main chain constituents in type III collagen and procollagen were disulfide-bonded gamma and pgamma components, respectively. Amino acid composition, cross striation banding as observed using electron microscopy, cyanogen bromide peptide patterns in disc electrophoresis gels and resistance of the disulfide regions to pepsin digestion indicated a close similarity to previously described insoluble type III collagen, which was solubilized by limited pepsin digestion. Electron microscopy of long-spacing-segment crystallites and evidence for an extended form of the disulfide-bonded cyanogen bromide peptide suggested that neutral-salt-soluble type III collagon is longer at its C-terminal end by about 10 to 20 amino acid residues than pepsin-treated material. A small elongation was also indicated in the N-terminal portion of the molecule. Procollagen has an additional N-terminal extension with a length of about 160 A, but no difference was observed between collagen and procollagen at their C-terminal ends.


[Reactivity of the 3'-terminal oligonucleotide Sequence C-A-C-C-A of tRNAPhe and tRNAVal from baker's yeast upon N-oxidation with monoperphthalic acid as compared to the oligonucleotides C-A-C-C-A and A-A-A-U-C-A-C-C-A (author's transl)]. The nucleobases adenine and cytosine were subjected to N-oxidation by monoperphthalic acid at pH 7 and 0 degrees C in tRNAPhe, in the 3'-terminal pentanucleotide C-A-C-C-A from tRNAPhe, as well as in tRNAVal and in the 3'-terminal nonanucleotide A-A-A-U-C-A-C-C-A from tRNAVal. In the tRNAs, the oxidative attack occurs stepwise from the 3'-end. First the 3'-terminal adenine is oxidized, then the following two cytosines, then the following adenine. The oligonucleotides are far more reactive than the identical sequences in tRNA and are not oxidized according to a sequential mechanism.


Blastogenesis of human lymphocytes by endotoxin. Lipopolysaccharides of S. typhimurium, S. enteritidis and E. coli at wide range of concentrations were used to induce blastogenesis in human and mouse (nude) lymphocytes. Human lymphocytes from peripheral blood showed positive response to at least one source of LPS (stimulation index of 2-9). The optimum concentration resulting in maximum stimulation varied with different individual, sources and concentrations of LPS used. Lymphocytes from cord blood failed to respond to LPS, but had positive response to PHA. All three LPS produced about equally strong mitogenic effects on mouse spleen cells.


Phagocytosis as a surface phenomenon. V. Contact angles and phagocytosis of rough and smooth strains of Salmonella typhimurium, and the influence of specific antiserum. The angle made by a drop of saline in contact with a monolayer of Salmonella typhimurium or phagocytic cells, the contact angle, is a measure of their relative interfacial tension, and is predictive of a successful phagocytosis. Smooth strains of S. typhimurium possess a contact angle lower than the phagocytic cells and resist phagocytosis. Rough strains have an angle higher than the phagocytes and are readily engulfed. The lower contact angle of smooth strains can be increased by treatment with specific antibody resulting in more efficient phagocytosis.


Antibodies to heat-labile Escherichia coli enterotoxin in Apaches in Whiteriver, Arizona. Antitoxin titers to heat-labile Escherichia coli enterotoxin were measured in Apache children hospitalized with acute diarrhea and in Apaches of different age groups without diarrhea in Whiteriver, Ariz. The study suggests that in this locale, exposure to enterotocigenic E. coli is probably widespread and occurs early in life. Antitoxin titer rises after idarrheal disease associated with enterotocigenic E. coli infection, however, were not regulary found.


Extracellular iron acquisition by mycobacteria: role of the exochelins and evidence against the participation of mycobactin. Mycobacterium bovis var. BCG was grown under iron-deficient conditions in the presence and absence of 1% Tween 80. Mycobactin, the iron iron ionophore of mycobacteria, was found solely within the bacteria grown in the absence of Tween, but low concentrations (0.75 mug/ml) of it appeared in the medium in the presence of the surfactant. Both types of medium contain agents, named exochelins, which could solubilize iron. 55Fe added to spent culture media was recovered only chelated to these compounds. Two exochelins were detected, isolated, and purified. Neither were precursors or breakdown products of mycobactin. In the desferri-form, exochelin MB-2, the major component, reversed the inhibitory effect of serum on the growth of BCG, and in their ferri-forms exochelins MB-1, MB-2, and MS (from Mycobacterium smegmatis) stimulated the growth of their producing organism in the presence of serum. Exochelin MB-2 could physically remove iron from ferritin, and BCG used ferritin as a source of iron during growth even when ferritin was separated from the bacteria by a dialysis membrane. As solutions of the exochelins were freely dialyzable, whereas solutions of mycobactin, even in Tween, were not, only exochelin could have been active in this experiment. The exochelins are proposed as the functional extracellular iron-binding agents of BCG and other mycobacteria, the role of mycobactin being confined to that of a cell wall iron transporter.


Induction of heat-labile enterotoxin synthesis in enterotoxigenic Escherichia coli mitomycin C. Introduction of heat-labile toxin (LT) synthesis in enterotocigenic strains of Escherichia coli by mitomycin C (MTC) was demonstrated. Six enteropathogenic strains which produce LT were inducible, exhibiting an 896-fold increase in LT when compared to ininduced cultures. On the other hand, four nonenteropathogens and three other pathogens which produce only the heat-stable toxin were not induced to produce LT. Gel filtration chromatography, antibody neutration, and heat lability studies suggest that the toxin synthesized in the presence of MTC is the same as the toxin synthesized in the absence of MTC.


Effects of BCG, Corynebacterium parvum, and methanol-extration residue in the reduction of mortality from Staphylococcus aureus and Candida albicans infections in immunosuppressed mice. An immunosuppressed mouse model was devised to test the effects of immunopotentiators on the prevention of bacterial and fungal infections. The effects of BCG and Corynebacterium were tested against Staphylococcus aureus and Candida albicans infection. The effect of methanol-extraction residue (MER-BCG) was tested against S. aureus septicemia. CDF mice were given various doses of BCG, 1.0 mg of C. parvum, or 0.5 mg of MER intraperitoneally at varying intervals before injection of an intravenous bacterial challenge. Four days before challenge, 300 mg of cyclophosphamide per ml was given intraperitoneally. BCG (106 colony-forming units) reduced mortality due to S.aureus at pretreatment intervals of 3, 7, 14, and 28 days. Isonicotinic acid hydrazide treatment elimated the protective effect of the live BCG. C. parvum was as effective as BCG against S. aureus septicemia when given 3 days before infection, but lost most of its protective effect after that time. MER protected at doses as small as 0.25 mg when given 25 days prior to challenge. Both BCG and C. parvum exerted a protective effect against Candida albicans infection.


Production and properties of an extracellular bacteriocin from Streptococcus mutans bacteriocidal for group A and other streptococci. An extracellular bacteriocidal substance is produced by a serotype c strain of Streptococcus mutans in liquid meduim during the stationary phase of growth. The lethal effect of the substance was demonstrated by the decrease in viable counts of a standardized suspension of group A streptococci in broth. No lysis of affected cells was observed and no changes in appearance of these cells was seen in electron micrographs. The material was effective against certain strains of immmunological groups A, C, D, G, H, L, and O streptococci. It was inactive against strains of S. mutans belonging to the a, b, c, and d serotypes, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. The factor was purified 273-fold from the culture fluid by column chromatography. It was sensitive to trysin and Pronase and resistant to catalase. It possessed a molecular weight of more than 20,000 and was not dialyzable. The properties of this substance indicate that it is a bacteriocin. Group A streptococci, which had been treated with antiserum specific for the cell wall group and type antigens, were susceptible to the bacteriocin. Streptococcal strains resistant to the lethal action of the bacteriocin adsorbed the bacteriocin from the solutions, as did the sensitive cells. The bacteriocin was not adsorbed at 0 C.


Decontamination of gnotobiotic mice experimentally monoassociated with Candida albicans. Gnotobiotic AKR mice, experimentally monoassociated with Candida albicans, were successfully decontaminated by oral treatment with amphotericin B incorporated in the drinking water. Germfree mice first were swabbed orally with viable C. albicans and then were allowed to acclimatize for 4 weeks. The log10 of number of C. albicans per gram of organ (with luminal contents) was 7.9 and 7.7 in the stomach and cecum, respectively. Direct fecal smears, as well as impresssion smears of stomach and cecum mucosal surfaces, revealed yeastphase cells, many with germ tubes, but no hyphal forms. No illness or mortality was observed over this period. The mice then were given amphotericin B DISsolved in the drinking water and offered ad libitum. At levels of 0.1 and 0.2 mg/ml, the number of fecal C. albicans was decreased but not eliminated completely. However, 0.3 mg/ml was sufficient to decontaminate the mice completely and return them to the germfree state. Residual amphotericin B was detected in the feces of the mice only while they were receiving the 0.3 mg/ml dose level. These mice remained germfree until the termination of the experiment, 10 weeks after the antibiotic had been discontinued and replaced by plain drinking water.


Ineffectiveness of Vi and chemically treated endotoxins as typhoid vaccines in mice challenged with a Salmonella typhosa-Salmonella typhimurium hybrid. Purified Vi antigen, acetic anthydride-treated Salmonella typhosa endotoxin, and potassium methylate-treated S. typhosa endotoxin employed as vaccines in Swiss white mice failed to protect these animals against challenge with a  virulent S. typhimurium hybrid expressing S. typhosa antigens.


Cephamycins: a review, prospects and some original observations. The cephamycins are a group with great potential. The first member of the group intended for therapeutic use offers the following advantages over existing cephalosporins: 1. Stability to various beta-lactamases; in an environment increasingly threatened by R-factors, this property may be of increasing value as time passes. 2. Possible lack of cross-allergenicity with other beta-lactam antibiotics. 3. Activity against anaerobic strains. Cefoxitin is only the first semi-synthetic derivative; presumably there are other compounds awaiting assessment which have even more favourable properties.


Lymphokines. II. Use of horse monocytes as indicator cells for human MIF. Peritoneal exudate macrophages in guinea pigs and peripheral blood monocytes in man are the most readily available cells sensitive to the migration-inhibiting factor(s) (MIF) induced by tuberculin or insoluble concanavalin A in supernatants of stimulated lymphocyte cultures. The scarcity of MIF-sensitive cells is probably the main reason for the unsatisfactory results obtained with direct and indirect MIF tests when using white blood cells as indicator cells. Isolated horse monocytes represent an alternative sensitive source of indicator cells for human MIF assays, whereas guinea pig peritoneal exudate macrophages appear to be less sensitive and to show large individual variations in sensitivity to human MIF. The species specificity of MIF from various origins shows various patterns and is briefly discussed.


Antibodies against Escherichia coli O antigen. Antibody amounts and avidities measured with ammonium sulfate precipitation technique. The ammonium sulfate precipitation (ASP) technique, modified Farr assay, has been utilized to study antibodies to Escherichia coli O2, O4, O6 and O75 antigens the induction of which could not be ascribed to the effect of a defined antigen. It was noted that the antibody titers against the O6 antigen in children, 0.1-2.5 years of age, were of lower magnitude than the others, significantly for the O2 and O4 antigens. Comparison between boys and girls under and over 1.0 year of age revealed higher titers in sera from girls and in the older children. However, significant differences were obtained only in a few instances. The antibody avidities were in most cases low and no correlation was found between antibody quantity and avidity. In consecutive samples from adults a varying pattern of antibodies to the O6 and O75 antigens was observed.


Fraction A: a new immunotherapeutic approach for ragweed pollinosis. Fraction A, a concentrate derived from short ragweed extract by (NH4)2SO4 precipitation, contains all major allergens including antigen E. Therapeutic efficacy and safety of this alum-adsorbed product was evaluated in a multicenter double-blind clinical trial by comparing it to placebo in 131 randomly matched ragweed hay fever patients who were not treated for at least 1 year. Therapeutic response to fraction A injections was more significant than placebo (p less than 0.05) by investigators' evaluation, by average daily symptom score (1.097 vs. 1.378, p less than 0.05) during the peak ragweed season, or by average proportion of days in which medication was required (0.411 vs. 0.584, p = 0.01). Fraction A patients exhibited a significantly higher (p less than 0.0005) increase in hemagglutinating titer after treatment than placebo controls. Radioallergosorbent binding, evaluated in a subgroup of 31 patients before and after the ragweed season, was unchanged or decreased in 12 of 15 patients treated with fraction A. In contrast, 9 of 16 placebo-treated patients showed an increase in this index. Systemic effects were noted after 42 (1.5%) of 2.641 injections, while local reactions occurred at the rate of 2.3%. It is concluded that a single course of preseasonal immunotherapy wih alum-adsorbed fraction A is effective and relatively safe.


Effects of contact sensitization and delayed hypersensitivity reactions on immune responses to non-related antigens. Modulation of Immune responses. Guinea pigs were immunized intracutaneously into the ears with sheep red blood cells (SRBC). Application of a sensitizing dose of the contact allergen dinitrochlorobenzene (DNCB) onto the same ears was shown to suppress or enhance the humoral response to SRBC depending on the time of application. When guinea pigs were sensitized to a contact allergen, application of a sensitizing dose of a non-related allergen on the same ears either had no effect or caused a clear enhancement of the development of delayed type hypersensitivity (DTH). Strongest enhancement was found when both sensitizations were performed on the same day. Further experiments on the effects of a concomitant DTH reaction elicited at the site of application of a contact allergen showed a strong potentiation of DTH when B-cell suppression was minimized by pretreatment with cyclophosphamide (CY). It was considered that CY-DTH-immunopotentiation might be a useful tool for achieving a higher level of sensitivity after epicutaneous sensitization.


The industrialization of fetishism or the fetishism of industrialization: a critique of Ivan Illich. This article critically assesses the ideology of industrialism in light of Ivan Illich's Medical Nemesis. The paper is divided into three sections. The first section is a description of the main features of that ideology, the most prevalent and influential one used in sociological literature to explain the state both of Western societies and of our health services. Also in this section, it is shown how these features appear in Illich's analysis of our societies, of our health services, and of the different clinical, social, and structural iatrogeneses that health services create. The second section examines the assumptions underlying Illich's analysis and discusses their validity to explain the nature and function of our Western health services and their iatrogenic effects. Where Illich's explanations are considered invalid, alternative explanations are presented. Among them, it is postulated that it is not industrialism, but the assumedly transcended category of capitalism that is the cause of the social and structural iatrogeneses. The third section discusses the political implications of Illich's analysis, in a moment when our Western societies are supposedly in crisis.


1974 and after: a general practitioner's views of the reorganization of the British National Health Service. Living and working through the period since the British National Health Service began in 1947, the author describes his experiences as a family and general practitioner and in particular notes the effects and non-effects of the reorganization that took place in the National Health Service in 1974.


The correlation between the clinical effectiveness and ratio of blood concentration to minimum inhibitory concentration of sulfamethoxazole against Escherichia coli isolates. The determination of a parameter in a dosage equation by the pharmacokinetics. The correlation between the clinical effectiveness and ratio of blood concentration to minimum inhibitory concentration of sulfamethoxazole against Escherichia coli isolated from clinical specimens were studied. This value is one of important parameters in a dosage equation by the pharmacokinetics, it showing sigma in an equation, were determined by clinical trial for acute simple cystitis of women. The sigma values were justified as 4 or more.


Plastic surgery of the ureteropelvic junction with a pedicle stomach flap. Experiments were carried out on dogs on the application of a pedicle flap cut from the stomach in plastic surgery of the ureteropelvic junction. The experiments showed that the flap healed well without any stenosis at the site where it was sewn in. An analysis of microscopic patterns showed that there was primary repair of the ureter wound which was the result of an adequate supply of blood to the flap.


Cell-mediated tumour immunity in patients with transitional cell carcinoma of the urinary bladder. Some recent data on problems of cell-mediated immune reaction directed against tumour-associated antigens (TAA) of transitional cell carcinomas of the urinary bladder (BTCC) are reviewed. All of the 13 samples of BTCC examined in our laboratory using a lymphocytotoxicity microassay contained the characteristic TAA of urinary bladder carcinomas. TAA were absent in 14 control tissue samples. Forty-seven out of 70 (67%) patients with BTCC developed cytotoxic lymphocytes reacting with BTCC cells. However, cytotoxic lymphocytes were also found in the blood of 8 out of 165 (4.8%) control lymphocyte donors.


Significance of anaerobic bacteria isolated from the urinary tract. II. Experimental studies. An attempt to cause retrograde urinary tract infection with Bacteroides fragilis (a strain subcultured in artificial media) failed to produce any significant renal infection in rats. Intravenous inoculation with Fusobacterium (a strain subcultured in artificial media) also did not cause demonstrable renal infection in rats. Nor could the anaerobic organism be demonstrated in the kidneys of these animals. Animals receiving Bacteroides (a strain subcultured in artificial media) inoculated directly into the renal medulla developed no renal infection. The anaerobic organism however, continued to be demonstrable in the kidneys of 78 per cent of these animals even on the 3rd day after inoculation. Rabbits receiving Bacteroides (a fresh clinical isolate) which was injected into the subcutaneously fixed kidney with ureteral obstruction all developed overt renal infection. There was also evidence of marked proliferation of the injected anaerobic bacteria and pyuria in these cases.


Enfranchisement and rationing: effects of Medicare on discretionary hospital use. This study examines the effects of Medicare on hospital output using an economic model in which market demand for hospital care is treated as the sum of demands in separable markets. Results indicate that Medicare affected utilization patterns by the elderly, but in terms of an increased level of admissions and increased lengths of stay, not in terms of case mix or (apparently) in the level of the nondiscretionary component of care. However, the results also show the Medicare resulted in rationing of hospital services to patients under 65 years of age: case-specific lengths of stay declined, and there appears to have been a distinct decline in the degree of discretionary hospital use by this patient group.


Response to LH-RH and HCG in two brothers with the Reifenstein syndrome. Two brothers with Reifenstein syndrome underwent LH-RH and HCG tests at various ages ranging from 13 to 17 years. We found that at age 13 the plasma LH and FSH response to one LH-RH injection was normal. After the age of 14, the basal plasma concentration of LH and FSH and their response to LH-RH became elevated. Concomitantly the plasma testosterone levels rose to abnormal levels. These findings are compatible with progressive development of primary gonadal dysfunction and with peripheral insensitivity to testosterone.


[Surgery of injuries of the facial nerve on the lateral region of the face (author's transl)]. Injuries of the lateral facial region frequently cause a partial or complete destruction of the trunk or individual branches of the facial nerve. Operative methods are presented, which have proved to be especially successful for reconstruction of the facial nerve. For a successful outcome of the nerve suture together with a good functional result later on, an exact suture tehcnique is mandatory. The procedure is discussed. On the other hand, the indication for operation raises a number of problems, which mainly concern the extent of nerve injury and the period elapsed after the damage.


[Angiography in the diagnosis of nasopharyngeal tumours (author's transl)]. The angiography of tumours of the nasopharynx is an important contribution to preoperative diagnostis. Applying modern, improved techniques (superselective catheterisation, magnification angiography, subtraction methods) it is possible to have an idea of the size and the localization of the tumour as well as of afferent and efferent vessels. Often a differential diagnosis is achieved; in some tumours (angiofibromas, haemangiomas, cysts, vascular malignant tumours) the angiographic findings are pathognomonic.


A spectrophotometric assay for gentamicin. A rapid and accurate spectrophotometric assay has been developed for the determination of blood werum levels of gentamicin and related antibiotics. The assay uses a purified enzyme from Escherichia coli JR88/C600 that acetylates gentamicin with the production of coenzyme A, linked to a chemical reaction with a sulfhydryl reagent to produce stoichiometric amounts of a sensitive chromophore, monitored in the visible spectrum. The system provides advantages of speed, cost, convenience, accuracy, and enzyme stability to the desirable characteristics encountered with previous enzymatic methods.


Cognitive processing and linguistic reference in autistic children. Mutism or echolalia and failure to analyze sentences for their deep structures are characteristics of the language behavior of autistic children. The experimental literature indicates that cognitive processes which underlie the construction of a lexicon or the learning of ordering rules may be disturbed in these children. The inability to coordinate visual and aural stimulation inhibits lexical development, while difficulties in sequencing and use of inappropriate sequencing codes inhibits the growth of a productive grammar. It is suggested that remedial programs which include training in these processes may prove beneficial, and that substitute modes of information processing which the autistic child uses may be exploited to teach language forms.


Genetic control of multiple pathways of post-replicational repair in uvrB strains of Escherichia coli K-12. The effect of the recA, uvrD, exrA, and recB mutations and of post-irradiation treatment with chloramphenicol on the survival and post-replication repair after ultraviolet irradiation of uvrB strains of Escherichia coli K-12 was examined. Each of these mutations or treatments was found to decrease survival and the extent of repair. The interactions of the inhibitory effects of the uvrD, exaA, and recB mutations and chloramphenicol treatment were determined by examining the survival and repair characteristics of the several multiple mutants. The survival results suggest that the post-replication repair process in uvrB strains may be subdivided into at least five different branches. These include three branches that are blocked by the exrA, recB, or uvrD mutation, a fourth branch that is blocked by any of these mutations and is also sensitive to chloramphenicol treatment, and at least one additional branch that is not sensitive to either of these mutations or to chloramphenicol treatment. The extent of post-replicational repair observed with each of the strains is in general agreement with the pathways postulated on the basis of the survival data, although there are several apparent exceptions to this correlation.


Inactivation of adenosine 5'-triphosphate synthesis and reduced-form nicotinamide adenine dinucleotide dehydrogenase activity in Escherichia coli by near-ultraviolet and violet radiations. Near-ultraviolet (near-UV) light (300 to 380 nm) is a significant component of sunlight and has a variety of effects on biological systems. The present work is an attempt to identify chromophores (molecular absorbers of light) and targets (critical damaged molecules) for inhibition of adenosine triphosphate (ATP) synthesis in Escherichia coli by near UV. The fluence of 334 nm required for 37% survival of net ATP synthesis (F37) in E. coli AB2463 in succinate medium is 140 kJ/m2. The action spectrum for this inactivation is almost structureless, exhibiting a smooth transition from high efficiency at 313 nm to low efficiency at 405 nm. The action spectrum for inhibition of net ATP synthesis is consistent with the chromophore being either ubiquinone Q-8 or vitamin K2. The fluence required is consistent with ubiquinone Q-8 also being a target molecule. The activity of reduced nicotinamide adenine dinucleotide dehydrogenase in extracts of E. coli B is also inactivated by near UV and shows an F37 of about 40 kJ/m2. The action spectrum for this effect is quite structureless; it shows high efficiency at 313 nm and low efficiency at 435 nm. The data do not suggest a target molecule for this action, although it is possible that ubiquinone Q-8 absorbs the near-UV energy and then passes it on to some other target molecule. The data further indicate that inactivation of the oxidative phosphorylation system is not a primary factor in near-UV-induced growth delay in E. coli.


Plasmid-determined beta-lactamase indistinguishable from the chromosomal beta-lactamase of Escherichia coli. A plasmid, derived from a naturally occurring strain of Proteus mirabilis, conferred resistance to cephalosporins, apparently mediated by a beta-lactamase indistinguishable from that determined by the chromosomal gene of Escherichia coli K-12. There was evidence for a recombination event between the wild-type plasmid and a defective F factor (Fsp) in the Escherichia coli K-12 culture in which it was stored.


Deoxyribonucleic acid polymerase II activity in an Escherichia coli mutator strain. The polB gene encoding deoxyribonucleic acid (DNA) polymerase II has been located close to a mutator gene, mutT1, in Escherichia coli. We find the DNA polymerase II prepared from mutT1, strains to be normal in reaction requirements, heat stability, and ability to remove mismatched bases at termini. Recombinants formed from a mutant defective in DNA polymerase II (polB100) and mutT1 are deficient in polymerase II and have the same mutator phenotype as mutT1. Our linkage analysis indicates that mutT1 and polB100 are not isoallelic.


Inhibition of nucleoside Q formation in transfer ribonucleic acid during methionine starvation of relaxed-control Escherichia coli. The elution profiles of Asp-tRNA from unstarved and starved cultures of a relaxed-control (Rel-) strain of Escherichia coli were compared by reversed-phase chromatography. Methionine starvation results in the appearance of several additional species of Asp-tRNA which are not observed with starvation for leucine or histidine. By the criterion of cyanogen bromide-effected shifts in chromatographic elution position, a large portion of the tRNAAsp synthesized in methionine-starved cells lacks the normal Q nucleoside. By the same criterion, virtually all of the tRNAAsp from unstarved, leucine-starved, and histidine-starved cells contain Q. We conclude that methionine starvation prevents the formation of the norma Q nucleoside in Rel- E. coli.


Excision of pyrimidine dimers in toluene-treated Escherichia coli. Toluene-treated cells were used for examining excision of pyrimidine dimers in Escherichia coli strains W3110, DM845 (uvrA-), P3478 (polA-), and KS5064 (polAex1). Excision occurring in toluene-treated cells is rapid, adenosine 5'-triphosphate dependent, and requires the uvrA gene function. In strains lacking either the polymerizing or 5' leads to 3' exonucleolytic activity of deoxyribonucleic acid polymerase I, excision does occur. However, both in vivo and in vitro, the excision in such strains is initially slower than wild type.


cis-Dominant, transfer-deficient mutants of the Escherichia coli K-12 F sex factor. Rare conjugational progeny formed by crossing each of five Hfr strains with a recA-F- strain have been characterized. Selection was made for a proximal Hfr marker, taking strict precautions to prevent transfer of recA+ to the zygotes. Most of the progeny were found to be F' strains containing deletion mutant plasmids. With two exceptions, these mutant plasmids have lost all of the tra genes, which are required to confer conjugational donor ability upon a host. In addition, all but the exceptional mutant plasmids were found to be very poorly transmissible from transient heterozygotes which also contain a wild-type F' plasmid. The poor transmissibility is a cis-dominant transfer-defective phenotype which may result from deletion of all or part of the origin of transfer replication (ori), or of a gene determining a cis-acting protein. The two exceptional mutant plasmids may carry short deletions of some of the tra genes or polar tra mutations. The remaining progeny were nonmutant F' strains and F- strains. The frequency with which the F- strains were recovered permits us to estimate that the maximum amount of recombination possible in a recA56 zygote is 10(-6) that of a recA+ zygote.


Morphological analysis of nuclear separation and cell division during the life cycle of Escherichia coli. Quantitative electron microscope observations were performed on Escherichia coli B/r after balanced growth with doubling times (tau) of 32 and 60 min. The experimental approach allowed the timing of morphological events during the cell cycle by classifying serially sectioned cells according to length. Visible separation of the nucleoplasm was found to coincide with the time of termination of chromosome replication as predicted by the Cooper-Helmstetter model. The duration of the process of constrictive cell division (10 min) appeared to be independent of the growth rate for tau equals 60 min or less but to increase with increase doubling time in more slowly growing cells. Physiological division, i.e., compartmentalization prior to physical separation of the cells, was only observed to occur in the last minute of the cell cycle. The morphological results indicate that cell elongation continues during the division process in cells with tau equals 32 min, but fails to continue in cells with tau equals 60 min.


Thermosensitive mutations affecting ribonucleic acid polymerases in Saccharomyces cerevisiae. Among 150 temperature-sensitive Saccharomyces cerevisiae mutants which we have isolated, 15 are specifically affected in ribonucleic acid (RNA) synthesis. Four of these mutants exhibit particularly drastic changes and were chosen for a more detailed study. In these four mutants, RNA synthesis is immediately blocked after a shift at the nonpermissive temperature (37 C), protein synthesis decays at a rate compatible with messenger RNA half-life, and deoxyribonucleic acid synthesis increases by about 40%. All the mutations display a recessive phenotype. The segregation of the four allelic pairs ts-/ts+ in diploids is mendelian, and the four mutants belong to three complementation groups. The elution patterns (diethylaminoethyl-Sephadex) of the three RNA polymerases of the mutants grown at 37 C for 3.5 h show very low residual activities. The in vitro thermodenaturation confirms the in vivo results; the half-lives of the mutant activities at 45 C are 10 times smaller than those of the wild-type enzymes. Polyacrylamide gel electrophoresis shows that the synthesis of all species of RNA is thermosensitive. The existence of three distinct genes, which are each indispensable for the activity of the three RNA polymerases in vivo as well as in vitro, strongly favors the hypothesis of three common subunits in the three RNA polymerases.


Direction of deoxyribonucleic acid replication in Escherichia coli under various conditions of cell growth. The direction of chromosome replication in a temperature-sensitive initiation mutant of Escherichia coli (CT28) is shown autoradiographically to be bidirectional. This mode of replication persists even when the rate of replication is reduced by slow growth in succinate minimal medium or in the presence of chloramphenicol. Therefore, although the rate of replication can be affected by certain physiological stimuli, the topology of replication need not be.


Actin-like properties from Escherichia coli: concept of cytotonus as the missing link between cell metabolism and the biological ion-exchange resin. A protein fraction (A-L fraction) with characteristics reminiscent of muscle actin has been isolated from Escherichia coli. The A-L fraction undergoes reversible aggregation under the same conditions in which actin is polymerized and depends primarily on potassium for its polymerization. This fraction, upon electrophoresis on acrylamide gels in the presence of sodium dodecyl sulfate, exhibits a distinct peak at the characteristic molecular weight of 45,000. Passage of skeletal muscle myosin through the A-L fraction specifically removes this 45,000-molecular weight peak. Examination of the myosin by sodium dodecyl sulfate electrophoresis after the passage reveals a new band at the proper molecular weight. The A-L fraction from wild-type E. coli is compared with the protein from a potassium transport mutant. Important catalytic differences exist between the A-L fractions of the two strains. The A-L fraction from the mutant fails to polymerize in low-K media in the K+ concentration range in which the mutant fails to take up to K+. In low-K+ media, the parent strain accumulates potassium and the A-L fraction from this organism polymerizes. The cell swelling reaction of both strains has been studied. Parent cells swell during low-K+ uptake, whereas the mutant does not. It is construed from this that the differences in the characterization of the A-L fraction relative to that of the wild type are related to the loss of cell swelling in the mutant and hence to the loss in alkali cation selectivity. The possible role of contractile proteins in biological ion exchange is discussed.


Isolation of Escherichia coli K-12 mutants with altered level of beta-N-acetylglucosaminidase. Eight mutants with less than 25% of the wild-type level of beta-N-acetylglucosaminidase activity have been isolated from Escherichia coli K-12. Studies on these mutants suggest that less than 1% of the wild-type level of this enzyme may be adequate for the normal growth and division of E. coli cells.


Cell cycle-specific incorporation of lipoprotein into the outer membrane of Escherichia coli. A cell cycle-specific incorporation of free lipoprotein into the outer membrane of Escherichia coli was observed, with a maximal rate of incorporation occuring at the time of septation.


Repression of diaminopimelic acid decarboxylase in Escherichia coli: gene dosage effects and escape synthesis. Gene dosage and escape synthesis experiments support the hypothesis that diaminopimelate decarboxylase repression by lysine involves a repressor molecule in a negative control system.


Effect of GAL4 gene dosage on the level of galactose catabolic enzymes in Saccharomyces cerevisiae. Lack of GAL4 gene dosage on the level of uridine diphosphogalactose epimerase (EC 5.1.3.2) activity suggests the positive regulatory role for this locus on the control of galactose catabolic enzymes in Saccharomyces cerevisiae.


Increased loss of duplicated genes in streptomycin-resistant (strA) mutants of Escherichia coli k-12. The recombination-dependent loss of a duplicated portion of the Escherichia coli chromosome is five- to tenfold greater in strains containing streptomycin resistance (strA) mutations than in the strA+ parental strain. Streptomycin (500 mug/ml) partially reverses the increase. These results suggest an interaction between strA mutations and recombination.


Plasmid-determined ability of a Salmonella tennessee strain to ferment lactose and sucrose. The ability of a Salmonella tennessee strain to ferment both lactose and sucrose was attributed to a conjugally transmissible plasmid, deoxyribonucleic acid molecular weight 164 x 10(6), bearing the genetic determinants of both fermentation characters.


Genetic and physical studies of recombinant plasmids formed between an R plasmid of compatibility group FI and sex factor F of HfrH. Recombinant plasmids between an R plasmid of the FI group (R162/3) and the sex factor F or HfrH were produced after the conjugal transfer of this R plasmid into HfrH. Three types of recombinant plasmids were identified after the mating of HfrH (R162/3) with recA and rec+ recipients. One specimen of each type (pIP218, pIP222, pIP226) was studied in this report. All three recombinant plasmids carry the same genetic information for resistance to antibiotics (CSSuT) retained from R162/3. pIP218 retained all the other properties from F of HfrH: derepression for pilus synthesis, mobilization of the chromosome for the proximally transferred HfrH genes (thr, leu, proA), interference with T7 propagation, and ability to be cured by acridine orange. pIP222 retained from F of HfrH the derepression for pilus synthesis and the same polarity of chromosome transfer (thr, leu, proA), while pIP226 retained the interference with T7 propagation and acridine orange curing. Physical studies revealed that replication control and/or recovery of F and pIP218 as covalent circles of deoxyribonucleic acid are similar, and are different from R162/3. The new plasmids are more likely the result of a substitutive recombination event than a fusion. We propose genetic maps of these recombinant plasmids, showing the unequal participation of the parental plasmids in their formation.


Coupling of rates of transcription, translation, and messenger ribonucleic acid degradation in streptomycin-dependent mutants of Escherichia coli. The growth rates of streptomycin-dependent mutants varied in proportion to the level of streptomycin supplied; growth also varied characteristically from one dependent strain to another at a given streptomycin concentration. When cells growing at different rates (over a threefold range) were treated with rifampin, direct proportionality was observed for three parameters: (i) the rates of shutoff of transcription of total ribonucleic acid (RNA) and ribosomal RNA, as measured by pulse labeling at later times; (ii) the translation time for molecules of beta-galactosidase; and (iii) the rate of chemical degradation of messenger RNA. In contrast, the rate of functional inactivation of both total and beta-galactosidase messenger RNA was about the same at all growth rates. None of the variations of growth or other parameters were observed in an otherwise isogenic streptomycin-resistant strain treated with streptomycin. Since the mutational change in strd mutants and the site of action of streptomycin are in the 30S ribosomal subunits, it is suggested that the rate of ribosome function is set by the dependent lesion (and the level of streptomycin). One possibility is that the other correlated effects are mechanistically "coupled" to ribosome function, but the apparent coupling could also be an indirect result of differential effects of streptomycin on variables such as ribosomal miscoding and nucleotide pool size. However, since the rate of functional inactivation of messenger RNA is constant even when the RNA is broken down two- to fourfold more slowly, translation yield tends to be proportional to the growth rate of the dependent strains.


Regulatory mutants and control of cysteine biosynthetic enzymes in Salmonella typhimurium. The cysB region in Salmonella typhimurium regulates in a positive manner the noncontiguous structural genes for the enzymes responsible for sulfate reduction in cysteine biosynthesis. We treated three cysB mutants with chemical mutagens and selected 81 secondary mutants in which the inability to utilize sulfate was suppressed. Growth experiments on the suppressed mutants showed that the original loss of sulfate utilization had been corrected to varying degrees and that portions of the pathway had been established in abnormal relationship to one another. Sixty of the suppressed mutations were mapped via transductional analysis, and each was very closely linked to the original cysB mutation. We demonstrated that the cysB product functions in the regulation of the cysteine biosynthetic enzymes during both logarithmic growth and stationary phase. Mutation can alter the regulatory response of one enzyme in either an upward or downward direction while the regulation of other enzymes in the pathway remains unchanged. These data are consistent with the idea of a multivalent or multisite regulator molecule.


Cytochrome abnormalities and cyanide-resistant respiration in extranuclear mutants of Aspergillus nidulans. The cytochrome spectra of two extranuclear mutants of Aspergillus nidulans and the double-mutant recombinant formed from them have been examined both at room temperature and at the temperature of liquid N2 and compared with those of the wild-type strain. The oligomycin-resistant, slow growing mutant contained an increased amount of cytochrome c without any loss of cytochromes b and a,a3. The cold-sensitive mutant, apparently normal when grown at 37 C, showed an increased amount of cytochrome c and a partial loss of cytochromes b and a,a3 when grown at 20 C. A combination of these effects was observed in the double-mutant recombinant. Cyanide-resistant respiration was present in both mutant strains and in the recombinant at much higher levels than in the wild-type strain. In the oligomycin-resistant mutant, this was usually present together with cyanide-sensitive respiration, whereas in the cold-sensitive mutant and recombinant grown at 20 C cyanide-resistant approached 100%. Inhibitor and growth yield studies indicated that the cyanide-resistant pathway was not used by the cold-sensitive mutant during growth at 20 C.


Extrachromosomal deoxyribonucleic acid in R factor-harboring Enterobacteriaceae. Extrachromosomal deoxyribonucleic acid (DNA) from 24 different R factor-harboring Enterobacteriaceae was isolated and characterized by analytical ultracentrifugation and electron microscopy. The R factors represented 15 different patterns of transferable drug resistance found in enterobacteria from an enclosed geographic area. All of the strains contained extrachromosomal, circular DNA molecules within the range of 0.4 to 52 mum. More than one size class of circular DNA molecules was observed in the majority of the extrachromosomal DNA preparations. The buoyant density of the extrachromosomal DNA ranged from 1.700 to 1.720 g/cm3. The majority of the bacteria contained extrachromosomal DNAs of various densities. Three-fourths of the R factors were classified as fi+. The investigation illustrates the extensive variability in the physical characteristics of plasmid DNA from R factor-harboring strains.


H2-dependent anaerobic growth of Escherichia coli on L-malate: succinate formation. Escherichia coli grew anaerobically on L-malate only in the presence of H2; 91% of the L-malate utilized was converted to succinate. Anaerobically isolated membrane vesicles catalyzed the reduction of fumarate with H2 and contained a b-type cytochrome. Cytochrome c552 was present in the "periplasmic space."


Selection and properties of Escherichia coli mutants defective in the synthesis of cyclopropane fatty acids. Mutants of Escherichia coli K-12 defective in the synthesis of cyclopropane fatty acids (CFA) have been selected and isolated by a L-[methyl-3H]methionine suicide procedure. Two mutants were isolated. Stationary-phase cultures of both mutants contain less than 0.7% of the CFA content found in the parental strain. The CFA deficiency is attributed to a deficiency of CFA synthetase activity. Extracts of both mutants contain less than 10% of the CFA synthetase activity found in extracts of the parental strain. Experiments in which parental and mutant extracts were mixed indicate that the lack of activity in the mutant strains is not due to an inhibitor of CFA synthetase present in the mutant extracts. We have not yet detected a physiological phenotype for these mutants. These strains grow normally at various temperatures in a variety of media. We have tested survival (colony-forming ability) in response to (i) prolonged incubation in stationary phase, (ii) exposure to drying, and (iii) exposure to detergents, heavy metals, low pH, high salt concentration, and a variety of other environmental conditions. The survival of both mutants is identical to that of the parental strain under all conditions tested. The compositions (excepting the CFA deficiency) and metabolic turnover rates of the phospholipids of both mutant strains are indistinguishable from those of the wild-type strain. The transport of several amino acids also seems normal in these mutants.


Evidence for the involvement of ribonucleic acid in the production of F pili. The effects of rifampin and streptolydigin, inhibitors of ribonucleic acid (RNA) synthesis, on the production of F pili by Escherichia coli were studied by electron microscopy. The inhibition of RNA synthesis reduces the number of new pili produced by depiliated cells, but does not affect their length or the number of pili present at the time of inhibition or the retraction of pili. We suggest that the rifampin-sensitive step may be linked to the establishement of a site for pili production. Evidence is provided that chloramphenicol inhibits retraction. We suggest that retraction requires some protein whose pool size is limited.


Turnover of polyadenylate-containing ribonucleic acid in Saccharomyces cerevisiae. We examined the kinetics of incorporation of [3H]adenine into polyadenylate-containing ribonucleic acid [poly(A)-containing RNA] in yeast. The total poly(A)-containing RNA from spheroplasts and intact cells and the polysomal poly(A)-containing RNA exhibited similar incorporation kinetics. At 30 C half-saturation of the pool of poly(A)-containing RNA with label occurred in approximately 22 min. Since precursor pools appeared to require 5 min to saturate with label, we conclude that at 30 C messenger RNA molecules in yeast decay with an average half-life of 17 min.


Isolation and characterization of Escherichia coli chromosomal mutants affecting plasmid copy number. We have isolated chromosomal mutants of an Escherchia coli K-12 strain that maintain higher levels of an F' plasmid. The mutants are designated as plasmid copy number (pcn) mutants. They were detected by selecting for increased lactose fermentation in bacteria deleted for the lac operon but harboring an F'lacI,P pro+ plasmid. When examined for the amount of F' plasmid deoxyribonucleic acid (DNA) by the dye-CsCl isopycnic technique, the mutants show two to seven times as much covalently closed, circular (CCC) DNA as does the parental strain. The increased plasmid level in one mutant strain (pcn-24) was confirmed by DNA-DNA hybridization; however, this latter technique indicated about a twofold lower increase when compared with the increase measured for pcn-24 by the dye-CsCl technique. In mutant pcn-24 the increased amount of F' DNA reflects a proportional increase in monomeric-size plasmid molecules because oligomeric forms are not found. Also, in mutant pcn-24 the extra CCC plasmid copies do not seem to be randomly distributed throughout the cell's cytoplasm but appear complexed in situ with their host's folded chromosome. In all pcn mutants examined to date, the classical sex factor F is maintained at normal levels, whereas the viral plasmid Pl CM is maintained at two to three times the normal level. In all 17 pcn mutants isolated, the pcn mutation maps on the chromosome and not on the plasmid. Finally, the absolute amount of CCC F' DNA detectable in lysates of the six different pcn mutants examined decreased 50 to 90% upon incubation of the lysate at 37 C. In contrast, no loss of CCC DNA occurs when lysates of the parental F' strain are incubated at 37 C.


Adaptation of membrane lipids to alcohols. The effects of alcohols of different chain lengths on the fatty acid composition of Escherichia coli K-12 have been examined. My results indicate that these cells radically change their fatty acid composition when grown in the presence of alcohols. These changes represent an adaptive membrane alteration compensating for the direct physicochemical interaction of alcohols with the membrane. Similar adaptive responses of membrane lipids are proposed as a possible biochemical basis for tolerance to alcohol and related drugs.


Analytical isoelectric focusing of R factor-determined beta-lactamases: correlation with plasmid compatibility. R factor-determined beta-lactamases have been investigated by analytical isoelectric focusing. The enzymes such as those specified by the R6K and RP4 plasmids (TEM-type enzymes) are notably homogenous in biochemical tests (Hedges et al., 1974), but two subclasses can be distinguished by isoelectric focusing. Three subclasses can be distinguished among the oxacillin-hydrolyzing enzymes, in good agreement with the classification based upon biochemical characteristics (Dale and Smith, 1974). The TEM-type beta-lactamases are promiscuously distributed among plasmids of a wide variety of compatibility groups, whereas the various oxacillin-hydrolyzing enzymes show some degree of correlation with compatibility.


Physical properties of L-asparaginase from Serratia marcescens. Purified L-asparaginase from Serratia marcescens had an apparent-weight average molecular weight of 171,000 to 180,000 as determined by electrophoresis on polyacrylamide gels and by sedimentation equilibrium at low speed in an analytical ultracentrifuge. A subunit molecular weight of 31,500 +/- 1,500 was estimated for the enzyme after treatment with sodium dodecyl sulfate and urea and electrophoresis on polyacrylamide gels; a similar value was obtained by high-speed sedimentation equilibrium in the presence of guanidine hydrochloride. Our data indicate that the Serratia enzyme could have five or six subunits of 32,000 daltons, compared to four subunits of 32,000 daltons in the Escherichia coli enzyme. The Serratia L-asparaginase also appears to be a larger molecule than the enzyme from Erwinia carotovora, Proteus vulgaris, Acinetobacter glutaminasificans, and Alcaligenes eutrophus. The Serratia enzyme, like that from E. caratovora, was more resistant than the E. coli enzyme to dissociation by sodium dodecyl sulfate. This resistance could be due to the finding that the Serratia enzyme had a relatively high hydrophobicity, similar to the enzyme from E. caratovora, when compared with the hydrophobicity of the E. coli enzyme. The isoelectric point of the Serratia enzyme was approximately 5.2. The influence of certain physical characteristics of the enzyme on the biological properties is discussed.


Construction of intergeneric hybrids using bacteriophage P1CM: transfer of the Klebsiella aerogenes ribitol dehydrogenase gene to Escherichia coli. Study of many of the interesting properties of Klebsiella aerogenes is limited by the lack of a well-characterized genetic system for this organism. Our investigations of the evolution of the enzyme ribitol dehydrogenase (EC 1.1.1.56) in K. aerogenes would be greatly facilitated by the availability of such a system, and we here report two approaches to developing one. We have isolated mutants sensitive to the coliphage P1, which will efficiently tranduce genetic markers between such sensitive strains and which will thus make detailed mapping studies possible. Derivatives of K. aerogenes lysogenic for P1 can be readily isolated by using the specialized transducing particle P1CMclr100. Bacteria lysogenic for this phage are chloramphenicol resistant and temperature sensitive. Phage particles produced by temperature induction of such lysogens can be used to transfer K. aerogenes genes to the natural host of P1 phage. Escherichia coli. We have used this method to prepare derivatives of E. coli K-12 carrying the K. aerogenes genes conferring the ability to metabolize the pentitols ribitol and D-arabitol. We have shown that these E. coli-K. aerogenes hybrids synthesize a ribitol dehydrogenase with the properties of the K. aerogenes enzyme and have mapped the position of the transferred gene on the E. coli chromosome. The ramifications of this methodology are discussed.


Escherichia coli glyoxalate carboligase. Properties and reconstitution with 5-deazaFAD and 1,5-dihydrodeazaFADH2. Glyoxalate carboligase (EC 4.1.1.47) has been purified to electrophoretic homogeneity from Escherichia coli. The enzyme was found to be a dimer of subunits of identical molecular weight of 68,000. Resolution of the holoenzyme into apoenzyme and FAD led to a dissociation of the dimer into monomers. The apoenzyme could be reconsitituted to full catalytic activity with FAD or the flavin coenzyme analogue 5-deazaFAD. Reconstitution of the apoenzyme with the reduced flavin analogue 1,5-dihydro-5-deazaFADH2 led to the recovery of 50% of enzymatic activity. The reconstitution of apoglyoxalate carboligase with all three coenzymes followed Michaelis-Menten kinetics with Km values of 0.25, 0.74, and 0.72 muM for FAD deazaFAD, and deazaFADH2, respectively.


Actin, alpha-actinin, and tropomyosin interaction in the structural organization of actin filaments in nonmuscle cells. During the spreading of a population of rat embryo cells, approximately 40% of the cells develop a strikingly regular network which precedes the formation of the straight actin filament bundles seen in the fully spread out cells. Immunofluorescence studies with antibodies specific for the skeletal muscle structural proteins actin, alpha-actinin, and tropomyosin indicate that this network is composed of foci containing actin and alpha-actinin, connected by tropomyosin-associated actin filaments. Actin filaments, having both tropomyosin and alpha-actinin associated with them, are also seen to extend from the vertices of this network to the edges of the cell. These results demonstrate a specific interaction of alpha-actinin and tropomyosin with actin filaments during the assembly and organization of the actin filament bundles of tissue culture cells. The three-dimensional network  they form may be regarded as the structural precursor and the vertices of this network as the organization centers of the ultimately formed actin filament bundles of the fully spread out cells.


Studies of muscle proteins in embryonic myocardial cells of cardiac lethal mutant mexican axolotls (Ambystoma mexicanum) by use of heavy meromyosin binding and sodium dodecyl sulfate polyacrylamide gel electrophoresis. In the Mexican axolotl Ambystoma mexicanum recessive mutant gene c, by way of abnormal inductive processes from surrounding tissues, results in an absence of embryonic heart function. The lack of contractions in mutant heart cells apparently results from their inability to form normally organized myofibrils, even though a few actin-like (60-A) and myosin-like (150-A) filaments are present. Amorphous "proteinaceous" collections are often visible. In the present study, heavy meromyosin (HMM) treatment of mutant heart tissue greatly increases the number of thin filaments and decorates them in the usual fashion, confirming that they are actin. The amorphous collections disappear with the addition of HMM. In addition, an analysis of the constituent proteins of normal and mutant embryonic hearts and other tissues is made by sodium dodecyl sulfate (SDS) gel electrophoresis. These experiments are in full agreement with the morphological and HMM binding studies. The gels show distinct 42,000-dalton bands for both normal and mutant hearts, supporting the presence of normal actin. During early developmental stages (Harrison's stage 34) the cardiac tissues in normal and mutant siblings have indistinguishable banding patterns, but with increasing development several differences appear. Myosin heavy chain (200,000 daltons) increases substantially in normal hearts during development but very little in mutants. Even so the quantity of 200,000-dalton protein in mutant hearts is significantly more than in any of the nonmuscle tissues studied (i.e. gut, liver, brain). Unlike normal hearts, the mutant hearts lack a prominent 34,000-dalton band, indicating that if mutants contain muscle tropomyosin at all, it is present in drastically reduced amounts. Also, mutant hearts retain large amounts of yolk proteins at stages when the platelets have virtually disappeared from normal hearts. The morphologies and electrophoresis patterns of skeletal muscle from normal and mutant siblings are identical, confirming that gene c affects only heart muscle differentiation and not skeletal muscle. The results of the study suggest that the precardiac mesoderm in cardiac lethal mutant axolotl embryos initiates but then fails to complete its differentiation into functional muscle tissue. It appears that this single gene mutation, by way of abnormal inductive processes, affects the accumulation and organization of several different muscle proteins, including actin, myosin, and tropomyosin.


Selection of chinese hamster somatic cell mutants after irradiation of BUdR-labelled cells. The selection of somatic cell mutants after irradiation of BUdR-labelled cells is improved with the use of a mercury lamp combined with a plastic sheet which filters the unwanted wave lengths below 303 nm. New somatic cell mutants defective for galactose metabolism or auxotrophe for fetal calf serum were thus isolated.


Distribution of lipid in the lamellate endocuticle of Rhodnius prolixus (Hemiptera). The lamellate appearance of the cuticle in the abdomen of the Rhodnius larva conforms to the conception of Bouligand in being an optical artifact which results from the spiral arrangement of successive layers of oriented fibrils. But superimposed on this structure is an actual lamination of bound lipid with the same spacing. The relation of the lipid layers to the optical lamination changes with the aspect from which the system is viewed. There must therefore be a cyclical secretion of lipid by the epidermal cells. Since the period of this cycle agrees with the cycle of rotation of the fibrous layers, which is supposedly inherent in the chemistry of the system, it is possible that it is the lipid which controls or initiates this helicoidal 'cholesteric crystallization'. There is evidence of a cyclical change in the secretion of lipid by the microvilli; it is suggested that there may be alternating cycles of eccrine and apocrine secretion, and that the lipid laminae represent the apocrine phases. The pore canals in Rhodnius are roughly cylindrical in cross-section, with lipid-impregnated walls. The contents of the lumen become slightly more electron opaque before the cuticle is stretched by feeding. There is probably some enzymic dissolution of the cuticle which precedes stretching; and this may concern particularly the lipid fraction. After the great distension and expansion of the cuticle which occur at feeding, lipid laminae can no longer be demonstrated in the old cuticle.


Incorporation of lipid into the epicuticle of Rhodnius (Hemiptera). The incorporation of lipid into both the outer and inner epicuticle during deposition is described. Waterproofing of the epicuticle by secretion of the wax layer, and sclerotization with or without melanization, are controlled from a distance by the epidermal cells by way of the pore canals. The pore canals gradually narrow as they approach the epicuticle. On reaching the inner epicuticle the canal ends in a conical projection from the apex of which a permeable lipophilic channel about 20-25 nm in diameter runs vertically to the apex of which a permeable lipophilic channel about 20-25 nm in diameter runs vertically to the surface. Shortly before ecdysis, silver-binding material (perhaps protein rich in tyrosine, or other precursors concerned in sclerotization) spreads radially from a point in the cuticular channels just below the outer epicuticle, and gradually impregnates the outer two thirds or more of the inner epicuticle. The precise pattern varies in different cuticular structures. Argentaffin materials (polyphenols) first appear in these same sites at the time of ecdysis and increase rapidly during the next 24 h. Lipid appears in the lumen of the distal parts of the pore canals (with a patchy distribution) shortly before ecdysis. When digestion and absorption of the old endocuticle are almost complete, minute lipid droplets appear on the surface of the epicuticle, apparently exuded from the epicuticular channels, and spread to make a uniform layer. When first formed this layer strains readily with Sudan B, but the lipid becomes incorporated in a delicate non-lipid silver-binding membrane (also exuded from the epicuticular channels) and hardens just before ecdysis, to form the so-called 'wax layer' which then no longer stains with Sudan B. Within half an hour after ecdysis the alcian blue-staining cement layer is poured out by the dermal glands, and forms a continuous but somewhat irregular covering over the 'wax layer'. Changes in the epicticle that accompany the repair of abrasions are described.


Isolation and quantitation of picomole quantities of digoxin, digitoxin and their metabolites by high-pressure liquid chromatography. The following high-pressure liquid chromatographic (HPLC) separations are described: (1) isocratic separation of digoxin and its metabolites, (2) isocratic separation of digitoxin and its metabolites, (3) gradient elution separation of digoxin, digitoxin and their metabolites, and (4) gradient elution separation of gitoxin from digoxin and its metabolites. These methods utilize a multi-wavelength UV detector set at 220 nm and a reversed-phase column with various mixtures of acetonitrile and water as the mobile phase. The feasibility of using these HPLC methods as qualitative and quantitative techniques for digitalis glycosides is discussed.


Analytical aspects of capillary gas chromatography of lower fatty acids [up to C18]. The principal aspects influencing analytical capillary gas chromatography of fatty acids up to C18 have been evaluated. Selected fundamental problems of interlaboratory exchange of retention data were problems of defined temperature in commercial thermostated air baths, of capillary tubing, and of stationary phases. A modification of commercial thermostats has been proposed in order to secure a defined temperature for glass capillary columns. It has been found that retention data of fatty-acid methyl esters can be measured under standard conditions with the same accuracy as retention data of hydrocarbons on squalane. Metal capillary columns coated with Apiezon L were found to be unsuitable for the analysis of fatty-acid methyl esters when compared with the results of their quantitative analysis in packed Apiezon L and polar capillary columns. Possibilities of a nontraditional statistical evaluation of the results of measurements are suggested. A program in FORTRAN IV language is given for the calculation of Kovats' retention indices for fatty-acid methyl esters.


Strength-duration characteristics of estrogen effects on gonadotropin response to gonadotropin-releasing hormone in women. I. Effects of varying duration of estradiol administration. This study was designed to investigate the effects of increased serum concentrations of estradiol of varying durations upon the gonadotropin responses to synthetic gonadotropin-releasing hormone (GnRH or LRF). Beginning at 8:00 PM on the first day of the menstrual cycle, subjects received im injections of estradiol benzoate (E2B), 5 mug/kg initially, followed by 2.5 mug/kg every 12 h for a total of 3, 5, 7, 9, or 11 injections. Twelve h after the last E2B injection, or 36, 60, 84, 108, or 132 h after the first injection of E2B (2 subjects at each time interval), each subject received 100 mug GnRH, iv. In addition, each subject received 100 mug GnRH iv during one of the seven days of the antecedent (control) menstrual cycle during which no exogenous estradiol was administered. When GnRH was infused 36 h after the initiation of E2B pretreatment, there was no significant LH or FSH increase. In contrast, LH and FSH responses were augmented and prolonged when compared with control cycles when GnRH was administered at 84, 108, or 132 h. At 60 h, responses of LH were augmented, although not to as great a degree. FSH responses were not augmented at 60 h. Expressed as maximal increase from baseline, gonadotropin responses following E2B were 1 1/2 to 9 times those achieved during control cycles (without E2B). Since mean serum estradiol concentrations at 36 h (185.9 +/- 20.0), when gonadotropin responses were absent, were similar to those at 60 (157.7 +/- 31.6), 84 (186.2 +/- 38.1), 108 (181.3 +/- 46.7), and 132 h (128.0 +/- 43.0 pg/ml), when responses were augmented, these results support the concept that the modulating effect of estradiol on pituitary response is dependent upon the duration of exposure of the hypothalamic-pituitary system to increased concentrations of estradiol. It is probable that the duration of the late follicular phase rise in serum estradiol is responsible, at least in part, for the augmented gonadotropin response seen at midcycle.


Serum LH and FSH responses to the repetitive administration of gonadotropin-releasing hormone in patients with idiopathic hypogonadotropic hypogonadism. The serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) responses to the repetitive administration of synthetic gonadotropin-releasing hormone (GnRH) were studied in six male patients with idiopathic hypogonadotropic hypogonadism. Each patient received 50 mug of GnRH intravenously every four hours for 43 consecutive doses, i.e., for one week. Five of the six patients had substantially greater maximum serum LH concentrations in response to the 43rd dose than to the first dose. The mean (+/- SE) maximum serum LH concentration of all six patients following the 43rd dose (14.6 +/- 3.0 mIU/ml) was significantly (P less than 0.01) greater than that to the first dose (8.3 +/- 2.6 mIU/ml. These results suggest that the LH response of the human gonadotroph to GnRH depends on the prior exposure of the gonadotroph to GnRH. All six patients had substantially higher basal serum FSH levels prior to the 43rd dose (13.6 +/- 2.6 mIU/ml) than prior to the first dose (2.8 +/- 0.4; P less than 0.01). Four patients had increases in basal FSH level to within the normal adult male range (5-15 mIU/ml), and two had increases to above it. This uniform elevation of the serum FSH level to normal or above normal suggests that GnRH may be the primary FSH-releasing hormone as well as the primary LH-releasing hormone.


Gonadotropin secretion in prepubertal and pubertal primary hypogonadism: response to LHRH. Plasma LH and FSH were measured before and after LHRH administration in 10 patients with Turner's syndrome, in 7 with anorchia, in 2 castrates, in 18 with Klinefelter's syndrome, and in 11 prepubertal subjects with unilateral cryptochidism used as a control group. Basal LH was elevated in 4 and basal FSH in 8 of 10 patients with Turner's syndrome. Four patients with anorchia showed elevations of LH while FSH had increased in all of them. The two castrates had normal or slightly increased basal LH and definite elevations of FSH. Prepubertal subjects with Klinefelter's syndrome had normal plasma LH and FSH levels, but showed a marked elevation when they developed puberty. After LHRH administration, mean LH increased by 297% and FSH by 81% in Turner's syndrome, while in anorchia LH increased 757% and FSH 104%. After LHRH administration, patients with unilateral cryptorchidism had an LH increment of 316% and a FSH increment of 164%. Patients with prepubertal Klinefelter's syndrome showed elevations of 261% for LH and 221% for FSH after LHRH treatment. Adolescent subjects with Klinefelter's syndrome had an increment of 352% for LH and only 13% for FSH after LHRH administration. We have concluded that patients without functioning gonads fail to suppress gonadotropin secretion even before puberty while the gonads of the prepubertal Klinefelter's syndrome are able to control LH and FSH release. After puberty, in spite of the hypersection of LH and FSH observed in all subjects with agonadism there is a large pituitary reserve of the gonadotropins. We suggest that the relative inability of pubertal patients with Klinefelter's syndrome to increase FSH after LHRH treatment might be due to the presence of an abnormal compound secreted by the gonads.


Carbohydrate metabolism in pregnancy: XIII. Relationships between plasma insulin and proinsulin during late pregnancy in normal and diabetic subjects. To assess the effects of pregnancy on the relationships between plasma insulin and proinsulin, studies were performed during late gestation in women with normal carbohydrate metabolism or diabetes mellitus. Plasma was secured after overnight fast and 1, 2, and 3 hours following oral glucose (100 g). Samples were analyzed directly for total immunoreactive insulin (TIR) and for insulin and proinsulin following plasma fractionation by gel filtration. Fractionation disclosed that most of the normal gestational increase in basal and glucose-stimulated TIR can be ascribed to insulin rather than disproportionate increments in proinsulin-like components. Normal proinsulin/insulin relationships were also preserved in mild diabetics despite greater variability in their TIR response to glucose. Thus, mild carbohydrate intolerance during pregnancy is not attended by abnormalities in plasma proinsulin. In contrast basal proinsulin levels were elevated in 4 of 9 pregnant subjects with diabetes sufficiently severe to necessitate subsequent insulin therapy. Following glucose administration in the severe diabetics, the relative contribution from proinsulin to TIR was altered so that ratios of circulating proinsulin/insulin were increased at all levels of blood sugar. Postpartum tests of glucose tolerance in some of the normal and mildly diabetic subjects confirmed that pregnancy per se does not modify appreciably the relationships between plasma insulin and proinsulin although there may be some tendency for proinsulin to account for a smaller proportion of TIR.


LH and FSH response to synthetic LHRH after consecutive administration of clomiphene citrate in normal males. LHRH tests (100 mug iv) were performed in 11 normal male volunteers before and after administration of 100 mg clomiphene citrate per os for 7 days. Serum FSH, both pre and post LHRH levels (peak value and area under response curve) were significantly increased (P less than 0.01) by clomiphene citrate, while the maximum increments of FSH above baseline (deltaFSH) and area under response curve from basal level (deltaarea) were unchanged. On the other hand, basal serum LH levels increased significantly (P less than 0.005), post LHRH levels did not change by clomiphene citrate, whereas the increments after LHRH administration (both deltaLH and deltaarea) decreased significantly (P less than 0.05) on the second LHRH test. Basal levels of testosterone increased following clomiphene citrate treatment (P less than 0.005). The increased testosterone is considered to suppress the effect of LHRH on LH secretion.


Disappearance of biologic activity of synthetic LRH in normal and hypogonadal men. The disappearance rates of the biologic activity of synthetic LRH have been measured in normal and hypogonadal men before and during estrogen treatment. Biologic activity was assayed by the change in radioimmunoassayable rat LH levels induced by injection of human plasma into ovariectomized rats pretreated with estrogen and progesterone, and compared with a dose-response curve using synthetic LRH. Following rapid iv injection of 100 micrograms synthetic LRH, peripheral blood peak levels of biologic activity occurred in less than 2-6 minutes, and the disappearance curve contained 2 exponential components. In 3 normal men the initial component had a t1/2 of 2 minutes and the second component a t1/2 of 10 minutes. Six hypogonadal men had first and second components of disappearance no different from normals whether or not they responded to LRH injection with increased endogenous hLH. While diethylstilbestrol 2 mg orally for 7 days suppressed pituitary hLH response to exogenous synthetic LRH (mean max.deltahLH 236 VS 46 NG/ml), it did not alter the disappearance rates in normal or hypogonadal men. These studies show that the biologic activity of synthetic LRH disappears from the blood after iv administration at rates similar to radioimmunoassayable and radioactive-labeled LRH. They further suggest that the lack of pituitary responsiveness to LRH in hypogonadal and estrogen-treated men is not due to more rapid clearance of the releasing hormone.


Direct inoculation procedure for the rapid classification of bacteria from blood culture. A procedure was developed for 4-h identification of bacteria from blood culture. From a turbid blood culture bottle a 10- to 15-ml aliquot was drawn off and centrifuged. The pellet was utilized to inoculate a series of enzymatic and physiologic reactions. Three hundred eight positive blood cultures were tested including 222 strains of Enterobacteriaceae, 40 strains of facultative grampositive cocci, 26 strains of anaerobes, and 20 assorted strains. There was over 96.5% agreement between 4-h and conventional methods with the Enterobacteriaceae, 98% with facultative gram-positive cocci, 100% with anaerobes, and 99% with the assorted strains.


Isolation of Yersinia enterocolitica from swine. Data pertaining to 20 strains of Yensinia enterocolitica isolated from the cecal content of swine slaughtered at Toronto Abattoirs are presented. Fifteen of 20 cultures belonged to the two predominant human serotypes in Ontario (i.e., serotype O:3 and O:5,27.) Seven cultures of Y. enterocolitica O:3 belonged to the "Canadian" human phage type 9b. These findings are further proof that swine are reservoirs for Y. enterocolitica human infection. Seventeen Y. enterocolitica cultures were isolated by the cold enrichment method only. Difficulties encountered in isolating Y. enterocolitica from feces specimens are discussed. It would appear that the reported rate of Y. enterocolitica isolation from feces specimens is far below the actual incidence. Fourteen isolates of Y. pseudotuberculosis serotype III were isolated during the same survey.


Evaluation of a microscopy method for rapid detection and identification of Mycoplasma pneumoniae. A microscopy test that used the typical shape of Mycoplasma pneumoniae cells growing on glass was investigated for its value for diagnostic purposes. Suspensions from 108 throat swabs were infected artificially with 102, 103, and 104 colony-forming units of three M. pneumoniae strains per ml. Agar medium, a diphasic medium, and the microscopy method with liquid medium in cover slip chambers were compared for isolation of the mycoplasmas. The mycoplasms were detected first by the microscopy method in nearly all concentrations tested. Typical M. pneumoniae cells could often be detected after 48 h. No differences were found between a laboratory strain and two low-passage strains. The experimental results suggest that under special circumstances the microscopy method could be a useful tool for isolation and identification of M. pneumoniae.


Clinical isolation of Yersinia enterocolitica: cold temperature enrichment. A cold-temperature enrichment procedure was used to isolate Yersinia enterocolitica serotype 6 from a clinical stool specimen. The use of conventional laboratory media and enrichment procedures failed to isolate this organism.


Skin test and blastogenic responses to Sporotrichun schenckii. In vivo skin testing and in vitro lymphocyte blastogenesis were evaluated in a young adult population as methods for detecting cellular immunity to Sporotrichum schenckii. Similar procedures for Candida albicans and Coccidioides immitis were also investigated. 5 of 143 subjects had positive skin tests and 14 had positive blastogenic responses to S. schenckii. These 14 subjects also exhibited unusually high responses to C. albicans in vitro and 11 of the 14 were female. Data demonstrated a correlation coefficient of 0.89 when comparing the blastogenic assays for S. schenckii and C. albicans, suggesting cross antigenicity. Intact cellular immune mechanisms in combination with exposure to C. albicans may protect the host from systemic infection with S. schenckii. Although a limited number of subjects were studied, as a group, females had more vigorous cellular immune responses to C. albicans than males. The rare occurence of sporothrix infection in females as compared to males may be the result of antigenic stimulation from commonly observed vaginal colonization with C. albicans. The present data indirectly support this hypothesis.


Pathogenesis of shigella diarrhea. Serum anticytotoxin antibody response produced by toxigenic and nontoxigenic Shigella dysenteriae 1. The serum antitoxin response to the cytotoxin contained in preparations of Shigella dysenteriae 1 (Shiga's bacillus) exotoxin was studied in natural and experimental infections of man. Natural infection resulted in the rapid appearance of toxin-neutralizing antibody, which disappeared some time between 9 and 18 mo after infection. Experimental infection of human volunteers provided the opportunity to study immunoglobulin class of the antibody in sera obtained serially from 7 to 50 days after infection. Neutralizing antibody was present only in the IgM fraction isolated by sucrose density gradient ultracentrifugation. This was confirmed by the use of solid-phase immunoaffinity chromatography. Even though the time-course and immunoglobulin class of the antitoxin antibody response was similar to that previously observed for anti-O polysaccharide antibody, the biologically active cytotoxin was shown to be highly susceptible to destruction by proteolytic enzymes. Sera from subjects infected with a virulent invasive chlorate-resistant Shiga mutant thought to be "nontoxigenic" also contained antibody which was similarly restricted to the IgM fraction. Biologically active cytotoxin was recovered when this mutant organism was grown in liquid media with controlled ion concentration. The mutant cytotoxin was heat labile, neutralized by antiwild-type cytotoxin antibody, and was separable by isoelectric focusing into two fractions with pI 7.2 and 6.1 like the wild-type toxin. These studies show that cytotoxin antigen is produced during in vivo infection with Shiga bacilli, resulting in a serum antitoxin antibody response. Without explanation is the restriction of the antibody to the IgM class and lack of evidence for an IgG antibody to the protein cytotoxin. Finally, mutant strain 725, previously designated "nontoxigenic," was shown to produce biologically active cytotoxin in vitro and, in experimentally infected volunteers, to result in a serum IgM antibody similar to that observed during infection with the wild-type strain.