Frequencies of twelve ascus-types and arrangement of three genes from tetrad data. Equations expressing the theoretical frequencies of twelve ascus-types in the tetrad analysis of a triply heterozygous diploid are described. Using these equations, a mapping procedure for a gene X, is proposed. The procedure requires that two genes, X and Y, of the same phenotype be heterozygous and that the map position of Y be known, and that another standard gene, Z, show an independent phenotype from X and Y. This procedure does not require the laborious allelism test of the segregants to determine the allelic 2:2 segregation in tetrads for the X and Y genes, which is indispensable for mapping by the conventional procedure. The exact placement of the X gene on a chromosome is possible by the chi2 minimization procedure in comparison with the expected frequencies of the six ascus-types or four spore-types deduced from the twelve expected ascus-types to give the optimal fit with the observed data.


[Identification of 3 genes in the F-plasmid of Escherichia coli K-12]. The F'argG plasmid and its two transfer-deficient (tra-) analogues have been used to analyse the pleiotropic effect of a mutation in the integrated F-factor of HfrC strain. This mutation has been shown to disturb the functioning of at least three plasmid genes constituting, probably, a single regulon: the rsf gene determining the production of recombination-stimulating factor via conjugation (RSF); the prt gene responsible for the protective effect of the plasmid against N-methyl-N'-nitro-N-nitrosoguanidine, methyl methanesulphonate and UV-irradiation; the rep gene the product of which can be involved in the control of Hfr-chromosome replication. Possible location and order of the genes in the F-plasmid are discussed.


[Mutation of bacteriophage T4 restoring phage supressor psul+ activity in bacterial strain Escherichia coli BN]. The mutant of bacteriophage T4psu1+XF2 carrying a mutational aleration in the central region of proline-serine tRNA precursor is isolated. The mutational alteration results in the recovery of amber suppressor activity of phage psu1+ serine tRNA in Escherichia coli BN in which the synthesis of this tRNA is normally blocked. Since the amber suppressor activity of mutant serine tRNA becomes sensitive to a restrictive action of strR mutations, its structure seems to be different from that of parental suppressor serine tRNA.


[Genetic control of recombination processes in Aspergillus nidulans. I. Effect of uvs-mutations on the frequency of spontaneous and ultraviolet ray-induced intergenic recombination]. Effect of 3 uvs mutations (uvs 12, 19 and 25) on recombination processes in Aspergillus nidulans is studied. All the mutations are found either to affect the fertility of carp bodies and germination ability of askospores, or result in complete inability of heterokaryons to form cleistocarpia. Two mutations change the frequency of spontaneous meitotic crossing-over at pro-paba region of the chromosome I and do not affect the rate of mitotic recombination at w-centromeric region of the chromosome II: uvs 12 mutation increases, and uvs 19 mutation decreases the frequency of meiotic recombination. One mutation (uvs 25) decreases the rate of spontaneous mitotic crossing-over. All uvs mutations decrease the frequency of VU light induced mitotic recombination at w-centromeric region of the chromosome II. The data obtained, together with earlier reported characteristics of uvs mutants, suggest that recombination mechanisms in yeast participate in reparation processes more actively than in prokariotes. Different effects of the same uvs mutations on spontaneous frequency of meiotic and mitotic crossing-over draw to the conclusion that genetic control and molecular mechanisms of these processes in A. nidulans are not identical.


[Isolation and properties of merodiploid strains with F-factor including the pts-region of the Escherichia coli K-12 chromosome]. A technique of hybridization of haploid methanol-utilizing yeast Pichia pinus MH4 is worked out using UV- and N-nitrosoguanidine-induced auxotrophic mutants. Vegetative diploid cultures are isolated. Tetrad analysis and random spore analysis have revealed a meiotic nature of spores, recombination of genetic material in the process of sporulation and the chromosomal nature of some mutations. A possibility to construct a genetic map of the yeast Pichia pinus MH4 is demonstrated on the basis of tetrad analysis. Three linkage groups are revealed. The life cycle in a homothalic haploid yeast, Pichia pinus, was demonstrated. They are capable to form zygotes and meiotic spores under conditions preventing vegetative growth.


[Recombination of amber mutants of bacteriophage T4B. II. Localization of amber mutants on maps of genes 30, 34, 35, 36 and 38]. Localization of 275 amber mutants of five genes of phage T4B (30, 34, 35, 36 and 38) on genetic maps allowed us to determine the recombination length of these genes. Gene 34 substantially differs from the rest studied genes by numbers of amber mutants isolated in each gene and by recombination frequency. In particular, according to the results of crossing the flank markers, the recombination length of gene 34 is 10 times greater than in gene 38; using the summary value of recombination frequencies between elementary intervals a 20-fold excess of the length of gene 34 compared with the length of gene 38 was receieved. Molecular weight of the product of gene 34 is only 6 times as great as in gene 38. An elevated recombination frequency was also detected in gene 35. The data obtained indicate a local recombination anomaly at the region of genes 34--35 of bacteriophage T4 genome.


[Effect of amino acid substitutions in the polypeptide chain of DNA-polymerase on manifestation of the mutator effect]. Thin map of gene 43, controlling the synthesis of T4 DNA polymerase, is obtained by mapping experiments performed with 39 amber mutants, and is used for analysis of the sites of DNA polymerase gene from the point of view of displaying the mutator effect. The mutant sites studied possessed different reaction on amino acid substitutions in the polypeptide chain of the enzyme. Most of sites of the DNA polymerase gene, with the exception of two "supersensitive", responsed only on the apparent type of the amino acid substitutions: the mutator effect of amber mutations, which are located at these sites, was exhibited only in the case of insertion of the definite amino acid in the respective point of polypeptide chain. The proposed system of amber mutations for studying the mutator effect, allowed the authors to obtain the data on the effect of concrete alterations in the polypeptide chain of the enzyme on the development of its mutator properties.


[IS-elements and their role in genetic recombination]. The data concerning the biological functions and properties of short specific polynucleotide sequences (so called insertion sequences--IS) are reviewed. IS elements integrated in a genome can lead to strongly polar mutations in Escherichia coli, its bacteriophages and plasmids, while some IS (IS2) being integrated in inverted orientation turn on the gene activity. Several copies of the IS elements are present in the E. coli chromosome. A characteristic feature of IS is their ability to recA-independent migration along the bacterial chromosome. Possible mechanisms of IS integration are discussed. IS elements play the key role in the majority of recA-independent recombinational events: F-prime and partially Hfr-formation, plasmid recombination and dissociation, some cases of deletion formation etc. IS elements participate in recombination in the form of direct or inverted repeats. Direct repeats probably determine the processes of dissociation of the complete multicomponent R-factors and other plasmids. Inverted repeats (some of them are palindromes) are responsible for the migration of several drug-resistance determinants called transposons. Possible mechanisms of IS-dependent and probably IS-controlled recombination are discussed.


Antihypertensive therapy in elderly patients. Pilot trial of the European Working Party on High Blood Pressure in the Elderly. A study protocol for a double-blind randomised control trial of hypotensive treatment in elderly hypertensive patients has been tested in a number of pilot centres throughout Europe. It was shown that this study is possible from the logistic point of view. In these elderly patients, hydrochlorothiazide + triamterene treatment with or without methyldopa, maintained a significant hypotensive effect in the absence of major electrolyte disturbances. The initiation of hypotensive therapy did not provoke an excess of cardiovascular complications. The influence of hypotensive therapy on the general well-being and on the morbidity and the mortality of elderly patients with high blood pressure, is thereby being assessed. The study will continue for 5 years and other centres are invited to join.


Effect of prolonged feeding with chenodeoxycholic acid on bile in patients with and without gallstones. Nineteen patients who received chenodeoxycholic acid 750 mg/day for six months had duodenal bile aspirated before and after treatment. In five patients with hypertriglyceridaemia but no gallstones cholesterol saturation was reversed in every case, the mean cholesterol saturation index (SI +/- standard deviation) changing from 1-38 +/- 0-31 to 0-68 +/- 0-06 (P less than 0-005). In 14 patients with gallstones there was also an improvement in bile cholesterol content, but this was not sufficient to produce mean unsaturation, saturation index changing from 1-55 +/- 0-52 to 1-13 +/- 0-43 (P less than 0-05). Only seven of 14 patients with gallstone achieved cholesterol unsaturation. In four patients with hypertriglyceridaemia and gallstones, mean unsaturation was produced and the saturation index changed from 1-70 +/- 0-45 to 0-86 +/- 0-47 (P less than 0-05). When all nine patients with hypertriglyceridaemia were grouped, the mean saturation index fell from 1-52 +/- 0-40 to 0-76 +/- 0-30 after therapy (P less than 0-001). In contrast the 10 patients without hypertriglyceridaemia showed no significant fall in saturation index which was 1-50 +/- 0-54 before and 1-24 +/- 0-40 after therapy. The ability of chenodeoxycholic acid feeding to improve bile saturation with cholesterol correlated with the presence of hypertriglyceridaemia whether or not gallstones were present. It did not correlate with gallstone dissolution or body weight.


Double-blind clinical trial on gastroduodenal ulcer healing with prostaglandin E2 analogues. Seventy-seven patients with gastroduodenal ulcer were treated with two methyl-prostaglandin E2 analogues, m-PGE2, in a double-blind clinical trial. Each of three groups was given 15 S-15 methyl PGE2 methyl ester, 15 R-15 methyl PGE2 methyl ester, and placebo, respectively. Both forms of m-PGE2 analogues appeared to reduce gastric acid secretion, to shorten ulcer healing, and also to produce some side-effects, form 'S' being the more potent. Prompt healing of the ulcer with these agents did not prevent the recurrence of the disease. As the serum gastrin response to a meal after m-PGE2 administration was not reduced, this agent seems directly to affect oxynthic cells.


Metronidazole in the treatment of chronic proctitis: a controlled trial. Twenty-two patients with chronic proctitis were treated with metronidazole for 28 days in a double-blind controlled trial, but they did not appear to benefit from the drug.


Opposition replacement: illustrated by a new technique. Preliminary report of cases. A modification of le Coeur's method of restoring opposition of the thumb is described. An outrigger is built upon the thumb metacarpal to give a suitable direction of force, an enhanced mechanical advantage and an improved appearance.


Cock another hat at the thumb.

Cock another hat at the thumb. This paper discusses the attributes necessary to preserve function of the thumb, particularly length and opposability. The details of an operation to lengthen the short stump with sensitive skin are illustrated.


Split thickness hypothenar grafts for skin defects of the hand. Split thickness hypothenar grafts provide excellent coverage for fingertips as well as other areas of the hand. The method is simple and readily applied in the emergency room with little additional equipment.


Simultaneous and successive induction of synthesis of beta-galactosidase and tryptophanase in Escherichia coli K 12 in the chemostat. beta-Galactosidase and tryptophanase were induced either simultaneously or successively during continuous cultivation of the inducible strain Escherichia coli K 12 in the chemostat. Growth was limited by glycerol and the dilution rate was 0.1 h-1. During both the simultaneous and successive induction specific rates of synthesis, as well as maximum enzyme levels, were identical with those obtained after independent induction of individual enzymes. As compared with batch cultivation, beta-galactosidase reached the same specific rate of synthesis in the chemostat, whereas the specific rate of synthesis of tryptophanase in the chemostat was up to five times higher.


Cytochemical and biological properties of Mycobacterium bovis BCG. It was the aim of the present communication to find a simple test for a reliable discrimination of Mycobacterium bovis BCG from Mycobacterium tuberculosis. A total of 26 BCG strains, out of them 10 Czechoslovak strains (2 lyophilized cultures of BCG of different batch, 6 strains isolated from abscesses of children after BCG-vaccination and 2 strains from fatal cases after BCG-vaccination) and 16 strains obtained from foreign laboratories, were used. Of the tested characteristics a combination of 3 tests, sensitivity to 1 microgram of 2-thiophene carbonylhydrazide (TCH), activity of 3 acylamidases (urease, nicotinamidase and pyrazinamidase) and a quantitative nitrate test, was found to be most advantageous. The Czechoslovak strains of Mycobacterium bovis BCG were fully sensitive to TCH, of the 3 acylamidases mentioned above only urease was positive and nitrate was reduced only little or not at all. On the other hand, strains of Mycobacterium tuberculosis were always resistant to TCH, had positive urease, nicotinamidase and pyrazinamidase and reduced nitrate very intensively.


Spontaneous and induced variability in Streptomyces nogalater producing nogalamycin. Variability in the production of nogalamycin by Streptomyces nogalater var. nogalater was followed in untreated and mutagenized populations of the standard strain NRRL 3035 and its spontaneous variant K-18 using the method of agar blocks with subsequent tests under submerged conditions. In both strains the most active variants were obtained by natural selection without mutagenic treatment; in this way productivity increased by 108% after two selection steps. Treatment with UV-radiation did not yield variants with a highly increased activity. Gamma-radiation extended the variability but, at the same, substantially increased the number of non-producing and low-producing isolates. Relatively high yields of (+)-variants were obtained after treatment with nitrous acid but their activity did not reach that observed in the most active spontaneous variants.


Antifungal antibiotic of the Basidiomycete Oudemansiella mucida. II. Life cycle and fructification of the producing fungus. The developmental cycle of the fungus Oudemansiella mucida, the producer of a new antifungal antibiotic, was found to be controlled by the mechanism of homogenic tetrapolar incompatibility; under our conditions, the cycle took about 12 weeks to completion. Optimum conditions for a laboratory-scale fructification were investigated. Normal fruiting body formation required sufficient illumination, temperatures below 20 degrees C, and relative humidity in excess of 70%. Flask-grown fruiting bodies did not differ from the naturally occurring ones. The basidiospores of the produced fruiting bodies yielded the reference monokaryons necessary for physiological, cytological and genetical studies.


Antifungal antibiotic of the Basidiomycete Oudemansiella mucida. III. Nuclei in the hyphae of mono- and dikaryons. Nuclear ratios were studied in terminal and subterminal cells of various mycelia of the basidiomycete Oudemansiella mucida, the producer of the antifungal antibiotic mucidin (MuciderminR Spofa). The dikaryon, the monokaryon, and the mucidin-producing strain that had been cultivated for a long time under submerged conditions were compared. Dedikaryotization was found to have taken place in the producing strain. The originally dikaryotic culture with characteristic clamp connections on the mycelium and with two nuclei in every hyphal cell lost permanently the clamp connections, probably owing to continuous intense agitation. The hyphae contained solely mononuclear cells. Mating with a compatible monokaryon yielded a dikaryon capable of normal fructification.


Primary structure of protein S13 from the small subunit of escherichia coli ribosomes. The experimental details which led to the determination of the complete primary structure of protein S13 from the small subunit of Escherichia coli ribosomes are presented. S13 consists of 117 amino acid residues and has the following composition: Asp6, Asn2, Thr6, Ser6, Glu6, Gln2, Pro4, Gly11, Ala11, Cys1, Val7, Met2, Ile12, Leu9, Tyr2, Phe1, His3, Lys11 and Arg15. Tryptophan was not found. The molecular weight of protein S13 as derived from the sequence shown in Fig. 1 is 12970. The amino acid sequence of the protein was determined by combining the results obtained from liquid phase Edman degradation of the intact protein with those from the peptides isolated after enzymatic digestions with trypsin, Staphylococcus aureus protease and thermolysin. Additional information about the primary structure was derived from analysis of the chymotryptic peptides of protein S13 and from its digestion with carboxypeptidase C. The amino acid sequence of protein S13 was compared with the published sequences of the other ribosomal proteins of E. coli and predictions for the secondary structure of this protein were made.


Rupture of a tuberculous pseudoaneurysm of the innominate artery into the trachea and esophagus: report of a case and review of the literature. Spontaneous rupture of a tuberculous pseudoaneurysm of the innominate artery, undiagnosed until necropsy, occurred simultaneously into the trachea and the esophagus in a patient receiving anti-tuberculosis chemotherapy. This is the first reported instance of a tuberculous innominate artery aneurysm, the third tuberculous aneurysm with rupture into the trachea, the sixth into the esophagus, and the first with rupture into both structures. A review of the literature relating to tuberculous aneurysms of the major arteries is provided.


The origin and mechanism of the allograft reaction. Previous explanations for the allograft reaction have been based on the concept that antigen causes immunocyte activation, following engagement of the immunocyte's specific receptor. This notion lead to the concept of immune surveillance, the idea that the evolutionary pressure responsible for the development of the vertebrate immune system involved in allograft rejection was a need to recognize and destroy tumor cells that carried novel antigens. Allografts were rejected because they were, in effect, mistaken for tumor cells. At the practical level, these ideas suggested that a solution to the allograft problem required treatment of the recipient in a way that would reduce or eliminate the recipient's immune response to the grafted tissue. We have rejected these ideas on the grounds that the basic premise, the notion that antigen alone drives T cell differentiation, is invalid. To explain the origin of the allograft response, we have developed a theory of allogeneic reactivity based on the concept that a stimulator cell is required for the activation of blood cells involved in both nonspecific inflammatory reactions and specific cellular immunity. This theory provides a conceptual link between invertebrate and vertebrate alloreactivity and explains why the MHC and factors controlling the expression of T cell activity map in the same region of the genome. According to this theory, it is blood cells carried within the transplanted tissue and not transplantation antigen on the surface of graft parenchymal cells, that constitute the major barrier to allotransplantation. Experimentally we have presented evidence for a two-signal mechanism for T cell activation. Both antigen and an inductive stimulus are required for T cell activation, and neither factor alone induces detectable T cell activation. Organ culture of thyroid tissue for 4 weeks renders it non-immunogenic without altering its antigenic composition. Furthermore, cyclophosphamide pretreatment of the thyroid donor, a procedure that does not destroy the vascular bed of the donor tissue, also reduces its immunogenicity. These findings are of both theoretical and practical importance. They show that transplantation antigen carried on the parenchymal cells of a transplant do not constitute the major barrier to allotransplantation and, at least in the case of thyroid and parathyroid transplantation, indefinite allograft survival can be achieved by treatments directed at the transplanted tissue and not the recipient.


Replica-plating virulence assay for Agrobacterium tumefaciens. Replica plating onto wounded tobacco leaves provides a convenient method for screening for avirulent mutants of Agrobacterium tumefaciens.


Impaired serum antibody response to inactivated influenza A and B vaccine in renal transplant recipients. Before and after vaccination with a commercial inactivated influenza vaccine containing A/Aichi/2/68 (H3N2) and B/Massachusetts/1/71 antigens, the serum hemagglutination inhibition antibody titers to homologous and heterologous strains of A and B influenza viruses were measured in 45 renal transplant patients and 66 healthy controls (62 for the B strains). At least a fourfold titer rise to the homologous A strain occurred in 14 of 45 transplant patients (31%) versus 37 of 66 controls (56%). Fourfold or greater heterologous A rises occurred in only 8 of 45 transplant patients (18%) compared with 40 of 60 controls (61%). In both the homologous and heterologous B responses, at least fourfold hemagglutination inhibition titer rises were seen in significantly fewer transplant patients than control subjects. In the transplant group, no correlation was observed between degree of antibody response and age, previous influenza vaccination, percentage of patients initially seronegative, time since transplantation, dose of immunosuppressive drugs, level of renal function, or nature of original renal disease.


Humoral immune response to the heat-labile enterotoxin of Escherichia coli in naturally acquired diarrhea and antitoxin determination by passive immune hemolysis. Acute- and convalescent-phase sera from 132 students attending a university in rural Mexico were assayed for antibody against the heat-labile enterotoxin (LT) of Escherichia coli by neutralization of LT activity in the Y-1 adrenal cell assay and by passive immune hemolysis of LT-sensitized sheep erythrocytes. The two titration methods produced comparable results with respect to antitoxin responses detected. An inverse relationship was found between acute geometric mean antitoxin titer and the occurrence of diarrhea associated with LT-producing E. coli, especially in newly arrived students from the U.S.A. A significant correlation (P less than 0.00 5) was found between a rise in antitoxin titer detectable by the passive immune hemolysis technique and diarrhea with LT-producing E. coli isolated. Thus, humoral antitoxin titers appear to be a useful indicator of immune status with respect to enterotoxigenic (LT) E. coli diarrhea.


Prevention of recrudescent malaria in nude mice by thymic grafting or by treatment with hyperimmune serum. Nude mice died when infected with the normally avirulent malarial parasite Plasmodium berghei yoelii. Furthermore, malaria recrudesced in Nu/Nu mice after the termination of acute disease by treatment with clindamycin. Recrudescence was not observed in Nu/Nu mice that had been grafted with thymic tissue or treated with hyperimmune serum. Mice mad B cell deficient by treatment with anti-mu-chain serum also died when infected with P. berghei yoelii. The data suggest that a crucial role of the thymus in preventing recrudescent malaria in this model system is to provide a helper function in the production of protective antibody.


Modification by soluble antigen of the immune response to mycobacterial infection. After inoculation with viable BCG organisms, spleen cells from mice developed cellular immune reactivity to purified protein derivative antigen by day 10, which declined by day 25; after rechallenge on day 25, a vigorous secondary response rapidly developed (by day 29). The inclusion of autochthonous serum (from the BCG-infected mice) in the in vitro cellular immune reactivity tests altered this pattern considerably: the primary response was shortened, and the secondary response totally abated, suggesting the presence of blocking factors active against BCG-specific cellular immune reactivity in the sera of infected animals. Subsequent experiments indicated that BCG-specific serum blocking factor activity could be generated in the serum of control mice by the intravenous inoculation of purified protein derivative.


Antibody response to rabies virus in Syrian hamsters. Syrian hamsters were injected with inactivated, attenuated, and virulent rabies virus (RV), and the antibody response was quantified by a neutralization test and the immunoglobulin class of the virus antibody was characterized by indirect fluorescent microscopy. Serum antibodies to RV were found to be predominantly of the immunoglobulin G2 (IgG2) class, although IgG1 anti-RV also were detected in high-titered sera obtained after secondary challenge. Brain extracts of hamsters inoculated intracerebrally with RV contained only IgG2 anti-RV. IgA and IgM anti-RV were not detected. The preferential IgG2 response to RV is in marked contrast to the isolated IgG1 response detected after inoculation of hamsters with soluble purified protein antigens.


Gastrointestinal antibody responses in axenic mice to topically administered Escherichia coli. Immunoglobulin levels were determined in gastrointestinal secretions of both conventional and axenic mice, as was the stability of the immunoglobulin classes in these secretions. Axenic animals were then administered nonviable Escherichia coli O111:B4 by topical application into the oral cavity. Immunoglobulin (Ig) A, IgG1, AND IgG2 were detected in the gastrointestinal secretions, with an apparent gradation in stability (IgA greater than IgG1 greater than IgG2) under the conditions investigated. Specific antibodies (IgA and IgG) to both the somatic and the flagellar antigens of E. coli were demonstrated. The time course of the responses to the polysaccharide and protein antigens suggested that a secondary response to lipolysaccharide was being observed in the secretions. Examination of the autoclaved diet fed to the germfree animals indicated the presence of antigenically intact polysaccharide materials (i.e., lipopolysaccharides), whereas antigenic bacterial proteins were uniformly undetectable. Therefore, endotoxin in the diet was apparently causing a primary type of sensitization in the mice, whereas the active oral immunization regimen induced a secondary antibacterial response in the secretions.


Effect of cyclophosphamide on experimental Nocardia asteroides infection in mice. It was found that cells of Nocardia asteroides GUH-2 (virulent) were approximately 10 times more virulent  than cells of N. asteroides 14759 (intermediate) and greater that 500 times more virulent than N. asteroides 10905 (avirulent) cells when early-stationary-phase cultures suspended in saline were injected intravenously into "normal" mice. There appeared to be a specific organ tropism for each strain. Thus, N. asteroides GUH-2 infected primarily the kidneys, N. asteroides 14759 infected the lungs and heart, and N. asteroides 10905 (in large doses) infected the lungs. Cyclophosphamide treatment of the mice 72 h prior to infection dramatically increased host susceptibility to nocardial infection, especially to N. asteroides 14759. However, cyclophosphamide treatment did not significantly alter the organ specificity for each strain. Cyclophosphamide greatly enhanced the ability of the nocardial strain to grow within its target organ and significantly altered normal host clearance from these organs.


Effect of zinc ion on the hemolytic activity of thermostable direct hemolysin from Vibrio parahaemolyticus, streptolysin O, and Triton X-100. The effect of Zn2+ on hemolysis induced by the thermostable direct hemolysin from Vibrio parahaemolyticus (vibriolysin) streptolysin O, and Triton X-100 was studied. We found that in certain buffers, such as tris(hydroxymethyl)-aminomethane-hydrochloride, boric acid-borax, and N-hydroxyethyl piperazine-N'-2-ethanesulfonic acid-sodium hydroxide, hemoglobins released from erythrocytes were easily precipitated by addition of Zn2+, thus resulting in a false inhibition of hemolysin by Zn2+ when hemolysis was assayed by measuring absorbance at 540 nm of released hemoglobins. Under experimental conditions in which hemoglobin was not precipitated, hemolysis induced by streptolysin O was inhibited by Zn2+, whereas that induced by vibriolysin and Triton X-100 was not. Thus, we concluded that the mode of inhibitory action of Zn2+ on hemolysis was not due to a reversible alteration in the state of the lipid bilayer of erythrocytes membranes as proposed by Avigad and Bernheimer (Infect. Immun. 13:1378-1381, 1976).


Enhancement of the antibody response in vitro by adherent cells from mice infected with Mycobacterium lepraemurium. Spleen cells from mice systemically infected 4 to 6 weeks previously with Mycobacterium lepraemurium gave an enhanced primary antibody response in vitro to sheep erythrocytes, but responded normally to dinitrophenylated polyumerized flagellin. The ability to enhance the response was associated with the glass-adherent spleen cell population and with peritoneal cells. Similar cells obtained from infected mice depleted of T lymphocytes failed to enhance the antibody response. These studies suggest that macrophages that became activated during the development of cell-mediated immunity to infection can also stimulate antibody responses to thymus-dependent antigens.


Infection of HeLa cells with Salmonella typhimurium 395 MS and MR10 bacteria. After interaction with HeLa cells cultured in vitro, the fraction of adhering extracellular and that of internalized smooth Salmonella typhimurium 395 MS and rough 395 MR10 have been determined by two different techniques. (i) By using the indirect fluorescent-antibody technique on unfixed and acetone-fixed HeLa cell preparations, intracellular bacteria were considered to become stained only after acetone fixation. (ii) Based on the assumption that gentamicin affects only extracellular bacteria, disintegration of the infected HeLa cells and viable count allowed the determination of internalized bacteria. Both techniques showed that MS as well as MR10 bacteria gained intracellular access, the fraction of MR10 cells doing so being much greater. The net increase in the intracellular bacterial population was small within 3 h of incubation.


Kinetics of phagocytosis and intracellular killing of Candida albicans by human granulocytes and monocytes. The study of the phagocytosis and intracellular killing of Candida albicans by granulocytes and monocytes has been hampered by the budding and pseudomycelium formation of this yeast during a relatively short incubation period at 37 degrees C and by the similar density of candida cells and phagocytes, which makes differential centrifugation impossible. In the present study, C. albicans was used after 5 days of preculture at 30 degrees C, after which the number of candida cells remained constant during incubation at 37 degrees C for 90 min. On this basis, phagocytosis and intracellular killing were limited to a period of 60 min. Phagocytosis of C. albicans by granulocytes and monocytes was measured with a hemocytometer, the number of extracellular candida being a measure of the ingestion of these microorganisms. After 60 min, 96% of the candida cells were ingested by normal human granulocytes and monocytes. This process was dependent on the opsonin concentration and temperature and was inhibited by mono-iodoacetic acid. Heat-inactivated serum was less active than fresh serum, reflecting the role of complement factors with respect to opsonization. Intracellular killing was measured by a microbiological assay. After 60 min of incubation of phagocytes together with C. albicans and serum, human granulocytes and monocytes killed 58 and 50% of the ingested candida, respectively. This process was inhibited by phenylbutazone. Phagocytes from patients with chronic granulomatous disease showed impaired intracellular killing.


Enhanced susceptibility of mice to combinations of delta 9-tetrahydrocannabinol and live or killed gram-negative bacteria. Combinations of delta 9-tetrahydrocannabinol (delta 9-THC) and bacterial endotoxin were shown to be hyperadditively toxic for mice. A variety of purified lipopolysaccharide (LPS) preparations elicted enhanced mortality in combination with delta 9-THC. Escherichia coli O26:B6 LPS (Boivin preparation) at an essentially nonlethal dose of 2.5 mg/kg reduced the dose of delta 9-THC required to kill 50% of the treated mice from ca. 350 to 150 mg/kg. Inbred BALB, DBA, and C3H/HeCr mice, noninbred ICR mice, and hybrid CDF1 and BDF1 mice were hyperreactive to combinations of delta 9-THC and LPS. Moreover, a variety of heat-killed intestinal and gram-negative bacteria, live E. coli, and complexes of lipid A with a variety of proteins substituted for LPS in the synergistic toxicity of LPS and delta 9-THC. Extracts of marijuana also elicited hyperreactivity to LPS. The hyperadditive lethality of combinations of delta 9-THC and LPS was markedly less in mice rendered refractory to LPS or delta 9-THC by repeated administration of LPS or delta 9-THC, respectively.


Local immune response to lipoprotein of the outer membrane of Escherichia coli in experimental pyelonephritis. The local immune response to the lipoprotein of the outer membrane of Escherichia coli O6:K13:H1 was determined in experimental hematogenous pyelonephritis in rabbits. Local antibody was analyzed with the enzyme-linked immunosorbent assay on newly synthesized protein from kidney. Local or intrarenal antibody was detected by day 7 of infection, a few days later than serum antibody. The synthesis of antibody in immunoglobulin G class was present 6 days before the synthesis of immunoglobulin M antibody. Antibody activity to this antigen was also present in urine, and antibody could be eluted from bacteria coated with antibody. Local antibody to the lipoprotein was synthesized in equal quantities by animals infected with a heterologous species of E. coli. This antigen was not as mitogenic for splenic or kidney lymphocytes as was the O antigen. Hence, the lipoprotein of the organisms is a potent immunogen but a weak mitogen locally in pyelonephritis. This antibody response probably does not induce protection against infection, but represents a marker for presence of infection.


Regulation of endotoxin-induced inhibition of macrophage migration by fresh serum. Purified endotoxin (LPS) caused macrophage migration inhibition (MMI) in capillary tube cultures of guinea pig peritoneal macrophages in medium prepared with 15% fresh-frozen guinea pig serum. The inactivation of serum by heating at 56 degrees C for 30 min or by zymosan absorption prevented LPS-induced MMI. LPS was fully inhibitory in fresh C4-deficient guinea pig serum. Heat treatment of normal serum at 50 to 52 degrees C for 30 min to inactivate the alternate complement (C) pathway prevented or significantly decreased LPS-induced MMI, but heating C4-deficient serum at 50 to 52 degrees C for 30 min prevented LPS-MMI in all instances. These results suggest that the reaction was effected via the alternate C pathway but that some inhibition of migration was permitted via the classical C pathway, presumably due to antibodies for LPS in some normal sera. Pretreatment of normal serum with cobra venom factor decreased or prevented LPS-MMI in most instances, but similar results were obtained with C4-deficient serum. Experiments with chelated sera were unsuccessful because of the immobilization of macrophages by 10 mM ethylenediamine-tetraacetic acid and by 10 mM Mg-ethyleneglycol-bis (beta-aminoethyl)-N,N-tetraacetic acid. Low doses of concanavalin A and staphylococcal enterotoxin B and large doses of pokeweed mitogen caused MMI in "inactivated serum" medium, but MMI was enhanced in fresh serum.


Effect of alkali-treated lipopolysaccharide on the intracellular cations of human erythrocytes. The adsorption to human erythrocytes of Escherichia coli lipopolysaccharide treated by mild alkaline hydrolysis (h-LPS) stimulated an increase in the intracellular Na+ concentration and a decrease in the intracellular K+ concentration of the erythrocytes. Erythrocytes treated by h-LPS remained responsive to the membrane adenosine triphosphatase inhibitors ouabain and ethacrynic acid, indicating that hLPS did not alter erythrocyte cations be depleting energy intermediates or uncoupling energy metabolism from active cation transport. The h-LPS-treated erythrocytes became non-agglutinable by the lectin concanavalin A prior to the development of changes in intracellular cations. In addition, h-LPS-treated erythrocytes demonstrated a three-fold greater cation response to ethacrynic acid than the untreated erythrocytes; this greater response was probably due to local membrane effects by h-LPS on the ethacrynic acid-sensitive adenosine triphosphatase. It is suggested that the h-LPS-induced alteration of erythrocyte cation content was secondary to an increase in ion permeability localized to the concanavalin A receptor regions of the erythrocyte membrane, possibly combined with indirect effects of membrane-bound h-LPS on ethacrynic acid-sensitive adenosine triphosphatase.


Insect immunity. III. Purification and partial characterization of immune protein P5 from hemolymph of Hyalophora cecropia pupae. We previously showed that in pupae of Hyalophora cecropia, eight hemolymph proteins (P1 through P8) were selectively synthetized after immunization (Faye et al., Infect, Immun. 12:1426-1438, 1975). We also showed that a gross fractionation was obtained by a series of ammonium sulfate precipitations (designed A through D) and that protein P5 was enriched in fraction A. Starting from fraction A, we have now purified protein P5 by using dialysis, isoelectric focusing, and hydroxylapatite chromatography. The final product gave a single band in both gradient gel and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using the latter method, proteins P5 and P8 were found to be enriched in fraction A, but they were absent in fraction Ac prepared from nonimmunized pupae. Protein P5 was found to have a pI of 6.6 and a molecular weight of 24,000 in sodium dodecyl sulfate and 96,000 in tris (hydroxymethyl) aminomethane-borate, pH 9.0. These data suggest a structure for P5 composed of four subunits of equal size. Protein P5 stimulated the killing of Escherichia coli by hemolymph fractions B and D, but it had neither killing nor phenol oxidase activity of its own.


Antagonistic effect of extremely oxygen-sensitive clostridia from the microflora of conventional mice and of Escherichia coli against Shigella flexneri in the digestive tract of gnotobiotic mice. Two extremely oxygen-sensitive strains of Clostridium sp., designated Clostridium E and P, were obtained from digestive microflora of conventional mice and found to constitute a barrier against Shigella flexneri SF-2 when associated in vivo with Escherichia coli K-12. These and other simplified fractions of the conventional microflora were demonstrated to have an effect comparable to that of the total flora. When K-12 and Clostridium E were established in gnotobiotic mice before the introduction of SF-2, the latter was reduced to a level below detection in the digestive tract. Whe SF-2 was established first, the antagonistic effect exerted by Clostridium E and K-12 was variable and, apparently, related to the rate of establishment of Clostridium E. Mutants of SF-2 resistant to the barrier effect of Clostridium E and K-12 appeared at the end of 3 months when SF-2 was established in gnotobiotic mice alone or with K-12, and after only a week when SF-2 was associated only with Clostridium E. These results suggest that the bacterial antagonsim in this model is related to the production in vivo of an antibiotic substance active against SF-2. It appears that the substance may be produced by Clostridium E, stimulated by K-12.


Suppression of babesiosis in BCG-infected mice and its correlation with tumor inhibition. Infection of mice with Bacillus Calmette-Guérin (BCG) provided good protection against Babesia species. The intensity and duration of this protection was similar to that established after natural recovery from babesiosis. It developed too soon after the first exposure to the parasite, and was too radioresistant, to be attributable to specific antibody production. In addition, the parasites degenerated within circulating erythrocytes. This phenomenon is inconsistent with phagocytosis or lysis of parasites or parasitized cells, or prevention of entry of parasites into erythrocytes, causing the observed protection. Hence the phenomenon is best explained by the release of a nonspecific mediator that can limit multiplication of parasites within erythrocytes. These results not only throw light on mechanisms of immunity against hemoprotozoa. There are many points of similarity between the nonspecific protection BCG and Corynebactium parvum provide against Babesia species and inhibition of tumor growth by these agents. Therefore, babesiosis in mice may be a convenient experimental model for assessing stimulation of the mononuclear phagocyte system, which appears to be the basis of nonspecific immunity against bacteria, parasites, and tumors.


Development of delayed-type hypersensitivity during Mycobacterium lepraemurium infection in mice. Various preparations of Mycobacterium lepraemurium were used to elicit delayed-type hypersensitivity in the footpad of mice infected with this organism. With a sonicated preparation of the mycobacterium, a significant increase in footpad swelling was elicited in mice infected with M. lepraemurium 5 weeks previously, but not in BCG-infected animals or uninfected controls. This footpad reaction was shown to peak at 24 h and to be associated with an infiltration of mononuclear cells. The kinetics of footpad swelling, its association with lymphoproliferation, and its dependence on T lymphocytes were each examined. The results support the hypothesis that this is a delayed-type hypersensitivity reaction. The ability to transfer this reactivity to normal mice with cells but not serum offers further confirmation that this hypersensitivity is dependent on cell-mediated immunological mechanisms rather than humoral antibody. The relevance of this to the study of the immunological response of mice to murine leprosy is discussed.


Ultrastructural localization of sucrases in Streptococcus mutans GS-5 and an extracellular polysaccharide mutant: a comparative cytochemical and immunocytochemical study. Electron microscopy and cytochemical and immunocytochemical procedures were used to study the ultrastructural distribution of sucrase enzymes in two strains of Streptococcus mutans. In a strongly adherent and virulent parent strain, GS-5, most of the invertase and fructosyltransferase activities were demonstrated extracellularly or bound to the cell surfaces. Intracellularly, enzymatic sites were detected near the plasma membrane on the periphery of the nucleoid and central mesosome. In GS-511, a mutant of diminished virulence and adherence, most of the enzymatic activity was not located on the cell surfaces, but was found away from the cell walls and associated with extracellular polysaccharides. Intracellularly, GS-511 manifested the same distribution of invertase and fructosyltransferase as did GS-5; however, the close association of these enzymes with the plasma membrane was not shown in GS-511. In both strains, extracellular areas near regions associated with cross wall formation appeared to show localized concentrations of these sucrases. Antibodies against partially purified glucosyltransferase (GTF) enzymes from GS-5 were used to localize GTF by immunocytochemical techniques. Indirect ferritin localization procedures showed that the extracellular and cell-bound GTF enzymes were distributed in similar locations as the fructosyltransferase and invertase enzymes. By absorption of the antiserum with whole GS-511 cells, the location of extracellular GTF and surface antigens unique to GS-5 was demonstrated. The dramatically reduced levels of cell-bound sucrase activity in GS-511 indicates the significant role of these enzymes in adherence and cariogenicity.


Blastogenic transformation by lipopolysaccharide of blood leukocytes from immunized but not normal cattle. Escherichia coli lipopolysaccharide produced blastogenic transformation in whole-blood leukocytes from heifers that had been infected and immunized with Campylobacter fetus, but not in cells from control animals. This suggests that lipopolysaccharide dose not function as a B-cell mitogen in cattle and that its stimulation of cells from immunized animals occurred through another mechanism.


Detection of antibodies to embryonic antigens in sera of multiparous or colon tumor-bearing rats by a new indirect immunofluorescence assay. An indirect immunofluorescence assay using antigen coupled to agarose beads detected high titers of antibody to embryonic antigens in sera from multiparous rats and rats bearing colon carcinomas. Sera from pregnant rats had antibody titers greater than 10(3) and some rats still had titers greater than 10(2) 30 weeks after the end of pregnancy. Rats which developed colon carcinomas after treatment with 1,2-dimethylhydrazine were bled monthly between the end of treatment and detection of carcinoma. Antibody to embryonic antigens appeared in their sera at least 2 months before roentgenologic diagnosis of tumor.


The age old question of old age: was it really so much better back when? The belief that the elderly in pre-industrial societies fared considerably better than the elderly in modern mass societies has received nearly universal acceptance. Utilizing literary, historical and archaeological reports from early Greece to the present, the present paper offers data which challenges popular conceptions. Close examination of evidence dealing with societal evaluation, family and kin support reveals prejudicial stereotypes as pervasive as those associated with modern urban industrialized societies. While a gradual transition of the basis of authority and esteem is noted from nomadic and agrarian to industrial societies, there is substantial correspondence in the dominant cultural imagery of old age, life expectancy notwithstanding.


Effect of oxygen on bacteria and cultured mammalian cells irradiated in the frozen state. The oxygen effect for bacteria and cultured mammalian cells at -196 degrees C was studied, using suspensions to which were added cryoprotective and chemical protective agents. The oxygen enhancement ratio (o.e.r.) in the frozen state was dependent on the chemicals added to the suspensions. When the chemicals had a high competitive reactivity with oxygen to the damage, the o.e.r. in the frozen state was comparable to that in the liquid suspensions. Without chemicals, the o.e.r. in the frozen state decreased significantly, probably because of the low competitive activity of endogenous SH compounds. In general, the systems with a higher o.e.r. in the liquid state had a lower o.e.r. in the frozen state.


Inhibition of unscheduled DNA synthesis and repair of potentially lethal X-ray damage by 2-deoxy-D-glucose in yeast. The effects of the glucose antimetabolite, 2-deoxy-D-glucose (2-DG), on DNA repair (assayed by unscheduled DNA synthesis) and on the repair of potentially-lethal damage (assayed by cell viability after irradiation) have been studied in X-irradiated respiratory-deficient yeast cells (auxotroph for 5'-thymidine-monophosphate). Experimental results show that: (a) both these phenomena can be inhibited by 2-DG; (b) the repair of potentially-lethal damage occurs after the unscheduled DNA synthesis is almost complete; and (c) the repair of potentially-lethal damage can be inhibited by 2-DG even after the completion of the unscheduled DNA synthesis.


[Double-blind comparison of the effects of a new tricyclic antidepressant (Lofepramine) and a tetracyclic antidepressant (maprotiline)]. Two random groups of depressed out-patients were treated with either Maprotiline or the new antidepressant Lofepramine. The depressive state was assessed by using the Hamilton Depression Scale (HAM-D) before and after 1, 3 and 6 weeks of treatment. Both drugs produced remarkable improvement globally in total HAM-D score as well as in the symptom clusters. The differences in both groups are not significant, but compiling the results of adverse reactions and the therapeutic effect, the treatment outcome in the Lofepramine group was slightly superior.


Trazodone by intravenous infusion in depressions secondary to organic disease. 16 patients with organic depression were treated with low doses of trazodone (100 mg/day) by i.v. infusion. This drug was shown to have a marked antidepressant and anxiolytic action and did not produce any significant side-effects. The authors suggest that on the basis of the diminished 24-hour urinary excretion of 5-HIAA, trazodone seems to have an antiserotonin action at low doses.


Plasma aldosterone and magnesium in serum and urine during lithium treatment. Serum magnesium, urinary magnesium excretion and plasma aldosterone were determined in 8 manic-depressive before and at intervals during 3 months of lithium treatment (longitudinal study), and in 27 manic-depressive patients who had been treated with lithium for 3 months to 20 years (transversal study). In the longitudinal study urinary magnesium excretion and urine flow increased slightly after 1-3 months of therapy; serum magnesium and plasma aldosterone were unchanged. In the transversal study urinary magnesium excretion, serum magnesium and plasma aldosterone were within normal limits, and they did not differ significantly from levels in the longitudinal study. No correlation was found between serum magnesium or urinary magnesium excretion and plasma aldosterone in either study. The results indicate that changes in urinary magnesium excretion during lithium therapy are not mediated via changes in plasma aldosterone, but may be secondary to diminished renal water reabsorption induced by lithium.


Effect of imidazole on ionophore-induced ocular hypertension. The effect of intraperitoneal injections of imidazole on the elevation of intraocular pressure produced by the topical application of the cation ionophores A23187 or X537A was studied in rabbits. Pretreatment with 200 mg./kg. imidazole completely inhibited the ocular hypertension induced by 1.0 percent A23187. The elevation of intraocular pressure produced by 0.5 percent X537A was not blocked by pretreatment with imidazole.


[Modern burn care].

[Modern burn care]. This paper is intended to present an approach to the modern care of burns out of the experience of a dermatological burn unit for the dermatologist in the outpatient clinic. It is the first burn unit in Austria.


Joint scaling tests.

Joint scaling tests. It is shown that a joint scaling test developed by Tan (1974) is closely related to the widely used standard Cavalli joint scaling test (described by Mather and Jinks, 1971) which was not referred to in Tan's paper. With the numbers of individuals per generation observed in practice, the two tests give essentially similar results. The Cavalli procedure also provides estimates of genetical parameters and is more readily extended to a wider range of situations.


What chance did Mendel's experiments give him of noticing linkage? The a priori probability of noticeable linkage among all conceivable experiments of the size reported by Mendel cannot reasonably be taken as greater than 24-36 per cent; and therefore, the frequently heard opinion that his chances of encountering linkage were high, approaching 99-4 per cent, appears to be mistaken.


A modified triple test-cross analysis to test and allow for inadequate testers. When the population under investigation consists of highly inbred lines the full triple test-cross of Kearsey and Jinks (1968) supplemented by the selfed progenies of the population allows unambiguous and independent tests for epistasis and the adequacy of the pure-breeding testers, L1 and L2. This can also be achieved by supplementing the simplified triple test-cross of Jinks, Perkins and Breese (1969) with the selfed progenies of the L1i and L2i families. If the L1 and L2 testers prove to be inadequate due to the presence of common loci, modifications of the analyses are proposed which correct the resulting biases in the genetical components of variation.


Light and electron microscopic observations of the autonomic innervation of the mouse gallbladder mucosa. A histochemical, cytochemical, and secretory study. The autonomic innervation of the mouse gallbladder mucosa was studied using histo- and cytochemical methods. In a light microscopic investigation the distribution of acetylcholinesterase (AChE) activity and formaldehyde-induced fluorescence was studied histochemically. Nerve fibres and small varicosities showed concentrations of AChE activity very close to the epithelium in the subepithelial connective tissue. No adrenergic nerves were observed in the mucosa. When using the electron microscope and employing the potassium permanganate fixation/staining technique only one sort of axonal enlargement was encountered, viz. the cholinergic type. These varicosities contained numerous agranular vesicles (500-600 A in diameter). No varicosities of the adrenergic (dense-cored vesicles) type were observed. Signs of increased secretory activity in the epithelium were observed in the first few minutes after cholinergic stimulation. After repeated in vivo stimulation, there was an almost total depletion of glycoprotein granules, best seen when using the cytochemical PA-CrA-silver technique. The findings suggest that the subepithelial connective tissue and the epithelium of the mouse gallbladder mucosa have a cholinergic innervation.


Penicillin G or ampicillin for oral treatment of canine urinary tract infections. Penicillin G or ampicillin was administered orally to 144 dogs with urinary tract infections. The daily dosage of penicillin G ranged from 110,000 to 165,000 U/kg (50,000-75,000 U/lb), and the dosage of ampicillin varied from 77 to 110 mg/kg (35-50 mg/lb). The daily dose of each antibiotic was divided into 3 or 4 doses and given at approximately 8- or 6-hour intervals for 10 to 14 days. Response to treatment, based on results of urine culture, varied from no response for infections caused by Pseudomonas spp to 100% response for those caused by Staphylococcus aureus and Streptococcus spp. About 50% of infections caused by Escherichia coli were eliminated, as were about 80% of those due to Proteus mirabilis. Mean concentrations of penicillin G and ampicillin in urines collected at 6-hour intervals after oral administration to clinically normal adult dogs were approximately 350 microgram/ml for both drugs when each was given individually in daily dosages (divided QID) of 55 mg/kg (25 mg/lb). The minimum inhibitory concentration of penicillin G for a number of the bacteria isolated from the urine of the infected dogs was compared with the results of the clinical trials and to the minimum inhibitory concentration of a larger number of urinary bacterial isolates.


Controlled randomized study comparing amoxycillin and pivmecillinam in adult out-patients presenting with symptoms of acute urinary tract infection. A comparative study of amoxycillin and pivmecillinam was performed on 298 out-patients with acute urinary tract infection, receiving either 375 mg amoxycillin three times daily or 400 mg pivmecillinam three times daily. The primary cure rate was 90% in the pivmecillinam group compared to 82% in the amoxycillin group. Resistant enterobacteriaceae emerged in approximately 5% of patients receiving amoxycillin but not after treatment with pivmecillinam. No serious side effects were observed in patients receiving picmecillinam and the tolerance was generally good. Upper gastrointestinal side effects were more frequent in the pivmecillinam group whereas lower gastrointestinal side effects predominated in the amoxycillin group. 200 mg pivmecillinam three times daily compared with 400 mg three times daily showed no differences in cure rate and side effects were lower (11% compared to 19%).


Clinical evaluation of pivmecillinam in acute simple cystitis: a comparative study with amoxycillin by a randomized double-blind technique. The comparative efficacy of pivmecillinam and amoxycillin in 243 patients with acute simple cystitis was determined in a randomized double-blind trial. Pivmecillinam was found to be significantly more effective on both clinical and bacteriological grounds. The superiority of pivmecillinam was primarily due to its efficacy in infections due to ampicillin resistant Escherichia coli. Fewer side effects were reported in patients who received pivmecillinam.


Pivmecillinam and amoxycillin as combined treatment in purulent exacerbations of chronic bronchitis. One hundred and thirty-two patients with purulent exacerbations of chronic bronchitis were randomly allotted to treatment in three groups. They received (a) amoxycillin 250 mg and pivmecillinam 200 mg; or (b) amoxycillin 500 mg; or (c) amoxycillin 500 mg and pivmecillinam 400 mg: three times daily for 10 days. By the 7th day of treatment there was significant improvement over amoxycillin alone for both groups given combined chemotherapy in conversion of sputum to mucoid and in general improvement; at the end of treatment results in patients given the higher doses of both antibiotics were still superior to amoxycillin alone. Patients were observed 2 to 4 weeks later, when those given amoxycillin alone relapsed much more frequently. The three treatments were well tolerated and succeeded equally in clearing potential pathogens from the sputum. Combined treatment may be superior due to synergy against Haemophilus influenzae or to the elimination of beta-lactamase producing organisms and should be investigated further.


Effect of cold exposure on water requirements on three species of small mammals. Water turnover rate (WTR) was determined from tritiated water (3H2O) loss in the short-tailed shrew (Blarina brevicauda), red-backed vole (Clethrionomys gapperi), and white-footed mouse (Peromyscus leucopus). When given ad libitum water at Ta of 20 degrees, B. brevicauda, C. gapperi, and P. leucopus turned over 16.2, 13.8, and 6.2 ml/day, respectively; minimum WTR was 9.9, 7.8, and 3.5 ml/day, respectively. When they were given ad libitum water at 5 degrees C, WTR was 1.4 to 1.6 times higher than at 20 degrees C. On minimum water rations, WTR at 5 degrees C was 1.7 to 1.9 times higher than at 20 degrees C. Since increases in VO2 at 5 degrees C and at 20 degrees C were of about the same magnitude, increased metabolic rates may have caused increased water requirements. Total body water (TBW) was calculated from 3H2O dilution. On minimum water rations, the three species at both Ta's showed decreases in TBW and body weight, but percent body H2O increased.


[14C]urea and [14C]sucrose as permeability indicators in histamine pulmonary edema. To see whether, by avoiding red blood cell transport effects, [14C]-sucrose would be a more sensitive lung vascular permeability indicator than [14C]urea, we compared the effects of 4 microgram/kg-min intravenous histamine phosphate infusions on lung vascular permeability-surface area products (PS) for each indicator in the same unanesthetized sheep. Histamine caused hematocrit, PaCO2 and pHa to increase and pulmonary arterial pressure and Pa02 to decrease. [14C]Urea PS (integral extraction calculation) increased from a base line value of 14.7 +/- 1.5 SE ml/s to 17.7 +/- 1.3 (P less than 0.05) with histamine, but [14C]sucrose PS did not change (base line, 6.5 +/- 2.9; histamine, 5.8 +/- 1.2). We conclude that [14C]urea is probably a more useful permeability indicator in the lung circulation than [14C]sucrose, because urea is more sensitive to moderate increases in permeability.


Splanchnic blood flow, O2 consumption, removal of lactate, and output of glucose in highlanders. An impairment of gluconeogenesis has been proposed to explain the low arterial blood glucose of highlanders. Therefore, we studied splanchnic blood flow, splanchnic uptake of oxygen and lactate, and  output of glucose in nine normal and six anemic highlanders at an altitude of 3,750 m. Splanchnic blood flow, arteriovenous difference for oxygen, and oxygen consumption were comparable at rest in both groups and in lowlanders from the literature, whereas splanchnic output of glucose, and uptake of lactate were approximately twice those in lowlanders. After 10 min of mild exercise in 12 subjects (7 normals, 5 anemic), no significant changes in splanchnic hemodynamics and metabolism were found. During 29% oxygen breathing in 8 subjects (5 normals, 3 anemics), arterial lactate, splanchnic uptake of lactate and output of glucose fell to normal sea-level values. We concluded that splanchnic hemodynamics are similar in adapted highlanders and in lowlanders, and that there is no evidence of an impaired gluconeogenesis at the altitude of the present study.


Collaborative study comparing the spiral plate and aerobic plate count methods. The spiral plate count method is a semiautomated plating technique that greatly reduces manpower and material costs normally associated with the pour plating technique. In this collaborative study, 8 laboratories compared the spiral and pour plating techniques, using 4 samples each of 3 products: frozen pumpkin pie, frozen chicken pot pie, and shampoo. The results show that 10 of the 12 comparisons of means of the pour and spiral methods were not significantly different; 2 values were significant at alpha = 0.01. Overall, the components of variance were less than that of the current milk standard, and the replicate per cent coefficient of variation was satisfactory. This study indicates that the spiral plate method is an acceptable alternative to the pour plate method; the spiral plate method has been adopted as official first action.


Microbiological plate and turbidimetric assays of chlortetracycline in feeds: collaborative study. The manual and automated turbidimetric assays and a modified official plate assay for chlortetracycline (CTC-HCl) in feed were collaboratively studied. Three feed samples (swine feed, 100 g CTC-HCl/ton; premix I, 20 g each of CTC-HCl and sulfamethazine/lb, and 10 g penicillin/lb; and premix II, 50 g CTC-HCl/lb) were analyzed at 2 dilutions. Twelve laboratories conducted the plate assay; 8 laboratories the manual turbidimetric method; and 7 laboratories, the Autoturb analysis. Within a method, there was no significant difference between dilutions. Between methods, there was a significant difference between the manual turbidimetric plate assays only for swine feed. However, the same sample dilutions or the average values of the 2 dilutions for both methods showed no statistical difference. Among the collaborators, the slope of CTC-HCl standard curve varied between about 2.0 and 3.0 for the plate method. The turbidimetric assay has been adopted as official first action for feeds containing larger than or equal to 20 g CTC-HCl/lb.


Induction of colicin E1 synthesis in recomination-defective mutants in both the RecBC and RecF pathways. Colicin E1 synthesis was induced in mutants defective in both the RecBC and RecF pathways to the same extent as in Rec(+) strains.


Inhibition of thiamine transport in Saccharomyces cerevisiae by thiamine disulfides. Both thiamine disulfide and O-benzoyl thiamine disulfide, which are thiolfrom derivatives of thiamine, strongly inhibited thiamine transport in Saccharomyces cerevisiae. The inhibition appeared to be due to a high affinity of the analogs for yeast cell membranes, in which thiamine transport component(s) may be integrated.


Propionate-induced synthesis of odd-chain-length fatty acids by Escherichia coli. Exogenous propionate is incorporated in vivo by Escherichia coli as a primer to produce lipids with fatty acids of odd chain lengths. This provides a method for the specific labeling of the three terminal carbons in the fatty acyl chains of phospholipids.


Fine-structure mapping of the firA gene, a locus involved in the phenotypic expression of rifampin resistance in Escherichia. The firA (Ts)200 mutation not only eliminates the resistance to rifampin of certain genetically resistant strains, but, moreover, renders ribonucleic acid synthesis thermolabile. The firA gene has been mapped by P1 tranduction and is located extremely close to the structural gene for deoxyribonucleic acid polymerase III at 4 min on the Escherichia coli linkage map.


Killer double-stranded ribonucleic acid synthesis in cell division cycle mutants of Saccharomyces cerevisiae. The synthesis of killer double-stranded ribonucleic acid (dsRNA) in Saccharomyces cerevisiae was examined in seven different cell division cycle mutants (cdc) that are defective in nuclear deoxyribonucleic acid replication and contain the "killer character." In cdc28, cdc4, and cdc7, which are defective in the initiation of nuclear deoxyribonucleic acid synthesis, and in cdc23 or in cdc14, defective in medial or late nuclear division, an overproduction of dsRNA at the restrictive temperature was observed. In contrast to the above mutants, the synthesis of killer dsRNA is not enhanced at the restrictive temperature in either cdc8 or cdc21, which are defective in deoxyribonucleic acid chain elongation. Examination of killer sensitive strains (cdc7 K- and cdc4 K-) has shown that the complete killer dsRNA genome is essential for the overproduction of dsRNA at the restrictive temperature.


Genetic characterization of an Escherichia coli mutant altered in the structure of murein lipoprotein. Mutants defective in the structure, biosynthesis, and assembly of murein lipoprotein have been isolated. One of these mutants has been shown to synthesize a structurally altered lipoprotein. The biochemical features of the mutant lipoprotein (lipid deficiency, dimer formation, and a reduced, bound form of lipoprotein) could be attributed to a single mutation (or closely linked mutations) located at 36.4 min of the Escherichia coli map. We propose that this mutant is altered in the structural gene for murein lipoprotein (mlpA). Biochemical studies carried out with a heterogenote, mlpA/F'mlpA+, revealed the biochemical codominance of the wild-type and mutant genes.


Isolation and characterization of specialized lambda transducing bacteriophage carrying the metBJF methionine gene cluster. Secondary attachment site lysogens of Deltaatt(lambda)Deltappc-argECBH strains of Escherichia coli with lambdacI857 integrated into the bfe gene (88 min) were isolated. Of 20 such lysogens examined, 2 produce lysates with transducing phage containing the metBJF gene cluster (87 min). Reintroduction of the ppc-argECBH chromosome segment (which lies between the bfe and met genes) into these strains virtually abolishes the production of met transducing phage. All of the phage examined have lost essential genes from the left arm of the lambda chromosome. Approximately 85% of the phage appear to have the same genetic composition, containing the metBJF gene cluster, but not the closely linked gene cytR, and having lost phage genes G and J. Analytical CsCl density gradient centrifugation of five representatives of this major class of phage shows four of them to have identical densities (lighter than lambda), while the fifth cannot be resolved from lambda. The four apparently identical phage were isolated from three separate lysates, which suggests the existence of preferred sites for illegitimate recombination on the bacterial and phage chromosomes. Three specialized transducing phage that carry cytR in addition to metB, metJ, and metF have also been studied. Each of these viruses has a different amount of phage deoxyribonucleic acid. Two of them have less deoxyribonucleic acid than lambda, whereas the third has about the same amount. The metB, metF, and cytR genes of the transducing phage have been shown to function in vivo. The phage-borne metB and metF genes are subject to metJ-mediated repression.


Genetic analysis of Escherichia coli K-12 region I flagellar mutants. Flagellar mutants in Escherichia coli region I were obtained by selection for resistance to the flagellotropic phage chi. F' elements carrying this region of the E. coli genome were then constructed. Stable merodiploid strains with a flagellar defect on the exogenate and another on the endogenote were prepared. These merodiploids yielded information on the complementation behavior of mutations in this region. Region I was shown to include at least six cistrons, flaV, flaK, flaL, flaM, flaS, and flaT. Mu-induced and deletion fla mutants were also isolated. By using these mutant strains, the transcriptional order was shown to be flaV-flaK-flaL-flaM-flaS-flaT. The definition of region I fla genes and their transcriptional relationships were confirmed by genetic tests with hybrid A phage carrying fla genes in this region.


Intracellular localization of the superoxide dismutases of Escherichia coli: a reevaluation. All of the superoxide dismutase isozymes of Escherichia coli have been shown to occur in the cell matrix, and none have been found in the periplasm. This was the case with both E. coli B and E. coli K-12, whether grown on a low phosphate medium or on a Trypticase soy-yeast extract medium. Alkaline phosphatase was used as a marker of the periplasm; adenosine deaminase and glucose 6-phosphate dehydrogenase were used as matrix markers, and consistent results were obtained by osmotic shock, spheroplast formation, and use of a diazonium salt that penetrates the periplasm but cannot cross the plasma membrane. A previous report that the iron-containing superoxide dismutase of E. coli is a periplasmic enzyme is now seen to have been in error.


Major proteins of the Escherichia coli outer cell envelope membrane as bacteriophage receptors. Three Escherichia coli phages, TuIa, TuIb, and TuII, were isolated from local sewage. We present evidence that they use the major outer membrane proteins Ia, Ib, and II, respectively, as receptors. In all cases the proteins, under the experimental conditions used, required lipopolysaccharide to exhibit their receptor activity. For proteins Ia and II, an approximately two- to eightfold molar excess of lipopolysaccharide (based on one diglucosamine unit) was necessary to reach maximal receptor activity. Lipopolysaccharide did not appear to possess phage-binding sites. It seemed that the lipopolysaccharide requirement reflected a protein-lipopolysaccharide interaction in vivo, and lipopolysaccharide may thus cause the specific localization of these proteins. Inactivation of phage TuII by a protein II-lipopolysaccharide complex was reversible as long as the complex was in solution. Precipitation of the complex with Mg2+ led to irreversible phage inactivation with an inactivation constant (37 degrees C)K = 7 X 10-2 ml/min per microgram. With phages TuIa and TuIb and their respective protein-lipopolysaccharide complexes, only irreversible inactivation was found at 37 degrees C. The activity of the three proteins as phage receptors shows that part of them must be located at the cells surface. In addition, the association of proteins Ia and Ib with the murein layer of the cell envelope makes this pair trans-membrane proteins.


Isolation, genetic analysis, and characterization of Escherichia coli mutants with defects in the lacY gene. Five hundred thirty-five lacY mutants were isolated from an Escherichia coli strain carrying the lactose operon on an F' factor, either without mutagenesis or after mutagenesis with 2-aminopurine or N-methyl-N'-nitro-N-nitrosoguanidine. Crosses against 48 independently isolated deletions ending in the lacY gene divided the gene into 36 deletion groups. Suppressibility studies with 7 nonsense suppressor strains classified 276 mutants as nonsense mutants and 78 as missense (or nonsuppressible) mutants. One hundred seventy-nine mutants were "leaky" and could not be so allocated, and two were found to have small internal deletions. Nonsense mutants could in many cases be subdivided even within deletion groups on the basis of their suppressibility pattern, giving a total of 70 groups of nonsense mutants. Studies of these mutants allow the following conclusions: lactose and melibiose most probably do not have separate binding sites on the permease; the lacY region most likely consists of one cistron, and so both active transport and facilitated diffusion are functions of one protein; and finally, there is probably no small defined region of the permease responsible for energy coupling of transport. Furthermore, the strains and the analysis form the basis for a future functional study of the permease by biochemical techniques.


Glutamate transport driven by an electrochemical gradient of sodium ions in Escherichia coli. The role of Na+ in glutamate transport was studied in Escherichia coli B, strain 29-78, which possesses a very high activity of glutamate transport (L. Frank and I. Hopkins, J. Bacteriol., 1969). Energy-depleted cells were exposed to radioactive glutamate in the presence of a sodium gradient, a membrane potential, or both. One hundred- to 200-fold accumulation of the amino acid was attained in the presence of both electrical and chemical driving forces for the sodium ion. Somewhat lower accumulation values were obtained when either chemical or electrical driving forces were applied separately. A chemical driving force was produced by the addition of external Na+ to Na+-free cells. A membrane potential was established by a diffusion potential either of H+ in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone or of SCN-. These results support the hypothesis of a Na+-glutamate cotransport. Na+-driven glutamate transport was also observed in wild-type E. coli B but not in a strain of K-12.


Peptide transport in yeast: utilization of leucine- and lysine-containing peptides by Saccharomyces cerevisiae. A variety of leucine-containing di- and tripeptides and two lysine-containing dipeptides supported the growth of strain Z1-2D, a leucine, lysine auxotroph of Saccharomyces cerevisiae. However, (Lys)2, (Lys)3, (Lys)4, and (Lys)5 as well as Gly-Leu-Gly, three tetra- and one pentapeptide containing leucine were not utilized by the mutant. Cellular peptidases released leucine or lysine from all of these non-growth-supporting peptides, suggesting that the failure of strain Z1-2D to utilize these compounds reflects their failure to enter the yeast. Competition studies employing phenylalanine or non-leucine-containing peptides showed that the uptake of peptides into S. cerevisiae Z1-2D is distinct from that of amino acids and that di- and oligopeptides may share a common transport system. The failure of strain Z1-2D to utilize any peptide larger than (Leu)3 may indicate a transport size limit. Such a size limit would influence the construction of models that explain the action of yeast mating factors.


Origin and direction of replication of the drug resistance plasmid R100.1 and of a resistance transfer factor derivative in synchronized cultures. The origin and direction of replication of the resistance plasmid R100.1 and its resistance transfer factor derivative, pAR132, were studied by electron microscopy autoradiography of partially denatured molecules and partial denaturation mapping of replicative intermediates. Results of these studies indicate the existence of an origin of replication at 8.8 kilobases on the R100 map. Replication from this origin in cultures synchronized for initiation of replication is predominantly unidirectional in a single direction.


Threonyl-transfer ribonucleic acid synthetase from Escherichia coli: subunit structure and genetic analysis of the structural gene by means of a mutated enzyme and of a specialized transducing lambda bacteriophage. Threonyl-transfer ribonucleic acid synthetase (ThrRS) has been purified from a strain of Escherichia coli that shows a ninefold overproduction of this enzyme. Determination of the molecular weight of the purified, native enzyme by gel chromatography and by polyacrylamide gel electrophoresis at different gel concentrations yielded apparent molecular weight values of 150,000 and 161,000, respectively. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate yields a single protein band of 76,000-dalton size. From these results an alpha(2) subunit structure can be inferred. A mutant with a structurally altered ThrRS, which had been obtained by selection for resistance against the antibiotic borrelidin, was used to map the position of the ThrRS structural gene (thrS) by P1 transductions. It was found that thrS is located in the immediate neighborhood of pheS and pheT, which are the structural genes for the alpha and beta subunits of phenylalanyl-transfer ribonucleic acid (tRNA) synthetase, the gene order being aroD-pheT-pheS-thrS. A lambda phage that was previously shown to specifically transduce pheS, pheT, and also the structural gene for the translation initiation factor IF3 can complement the defect of the altered ThrRS of the borrelidin-resistant strain. This phage also stimulates the synthesis of the 76,000, molecular-weight polypeptide of ThrRS in ultraviolet light-irradiated. E. coli cells. These results indicate that the genes for ThrRS, alpha and beta subunits of phenylalanyl-tRNA synthetase, and initiation factor IF3 are immediately adjacent on the E. coli chromosome.


Relationship between cell coiling and motility of spirochetes in viscous environments. The lowest viscosity that stops translational motility of cells (minimum immobilizing viscosity [MIV] was determined for various spirochetes. The viscous agent used was polyvinylpyrrolidone, The MIV for either Spirochaeta halophila P1 or Spirochaeta aurantia J4T was approximately 1,000 centipoise (cp), and for Leptospira interrogans (biflexa) B16 the MIV was greater than 500 cp. In comparison, the MIV for the flagellated bacteria Escherichia coli and Spirillum serpens was 60 cp. MIV values for two S. halophila mutant strains lacking the characteristic cell coiling (Hel-mutants) were 70 and 120 cp,  approximately one-tenth the MIV for the wild-type strain. MIV values for cells of S. aurantia strains with fewer coils than comparably long cells of S. aurantia J4T were 300 to 600 cp. The average velocity of strains of S. aurantia and S. halophila decreased at viscosities higher than 2 to 3 cp. At 2 cp the average velocity of S. halophila P1 was 16 micron/s, whereas the average velocities of Hel-mutant strains were 7 to 9 micron/s. This study indicates that the coiling of spirochetes plays a role in their ability to move through environments of realtively high viscosity. Among the spirochetes we investigated, this ability is greater in the more extensively coiled strains.


Heteroduplex analysis of tra delta f' plasmids and the mechanism of their formation. Four tra delta FargG+ plasmids, derived from matings between Hfr AB312 and a recA recipient, have been shown to have deletions of at least 50% of the F genome, including the region in which the tra genes map. The mutant plasmids do contain the F genes required for plasmid maintenance. Correlations can be made between, on the one hand, the F genes present on the tradelta F' plasmids and the F genes transferred early by an Hfr donor, and, on the other hand, the F genes deleted from the tradelta F' plasmids and the F genes transferred late by an Hfr donor. A biased representation of proximally and distally transferred chromosomal markers among the tradelta F' elements was also demonstrated. Taken Taken together, the asymmetrical representation of Hfr genes and the cis dominance of the Tra phenotype of these mutants can best be explained by the hypothesis that the tradelta F' plasmids are formed by repliconation of the transferred exogenote in a recA recipient.


Mycoplasma phosphoenolpyruvate-dependent sugar phosphotransferase system: purification and characterization of enzyme I. The Mycoplasma phosphoenolpyruvate-dependent sugar phosphotransferase system consists of three components: a membrane-bound enzyme II, a soluble phosphocarrier protein (HPr), and a soluble enzyme I. The soluble enzyme I was purified by ammonium sulfate fractionation; Bio-Gel P-10 gel filtration; acid precipitation; diethylaminoethyl-Bio-Gel A; and Bio-Gel HTP column chromatography. The enzyme I was shown to be homogeneous by electrophoresis in a pH 8.9 non-sodium dodecyl sulfate gel and by isoelectric focusing. Whereas the protein moved as a single component in both the non-sodium dodecyl sulfate gel and isoelectric focusing, on sodium dodecyl sulfate gels, it moved as three subcomponents. The molecular weights of the three subunits, alpha, beta, and gamma, were 44,500, 62,000 and 64,500, respectively. The holoprotein moved as a single component, in the region of 220,000 daltons, in a Bio-Gel A 0.5-agarose column. The molar ratio of subunits was estimated to be 2alpha:1beta:1gamma. The elution characteristics on a diethylaminoethyl column at pH 7.4 and 6.8, acid precipitation data, and amino acid composition indicated that the protein is acidic. Isoelectric focusing occurred at pH 4.8. N-terminal amino acids determined by the dansyl chloride method indicated that glycine, alanine, and tyrosine are N-terminal amino acids of the three subunits. Although the protein was stable for at least 14 months at -20 degrees C, it was irreversibly inactivated by the thiol reagent N-ethyl-maleimide.


Properties of proteins produced after damage to deoxyribonucleic acid of Escherichia coli. Large amounts of extra proteins, X (in the envelope fraction) and X' (in the cytoplasmic fraction) were detected by SDS-polyacrylamide gel electrophoresis when DNA of Escherichia coli was damaged. These two proteins had the same apparent molecular weight (appproximately 40,000) and were produced under identical conditions, including requirement  for the recA" and lexA+ genotype. Sucrose density gradient centrifugation revealed that protein X' consisted of relatively large and heterogeneous aggregates in the cytoplasmic fraction; the distribution of protein X in the envelope was not determined. As proteins X and X' were shown to be equivalent, it is suggested that they are identical. Co-precipitation of the aggregates of protein X' with the envelope led to the appearance of protein X in the envelope fraction.


Relationship between messenger ribonucleic acid and enzyme levels specified by the leucine operon of Escherichia coli K-12. The levels of leucine-forming enzymes in Escherichia coli K-12 varied over a several thousand-fold range, depending upon conditions of growth. The highest levels were achieved by growing auxotrophs in a chemostat under conditions of leucine limitation. Under such conditions, enzyme levels were increased 45- to 90-fold relative to cells grown in minimal medium containing leucine (the latter values arbitrarily called 1). Leucine operon-specific messenger ribonucleic acid levels were elevated to about the same extent as enzyme levels in cells grown in a chemostat. Growth in media of greater complexity resulted in progressively lower levels of leucine-forming enzymes, reaching a value of less than 0.02 for growth in a medium containing tryptone broth and yeast extract. The levels of leucine operon-specified enzymes and messenger ribonucleic acid were also measured in strains containing about 25 copies of plasmid pCV1(ColE1-leu) per chromosome. For such strains grown in minimal medium, enzyme levels were proportional to the number of plasmids per cell. Furthermore, they followed the same trends as those described above upon derepression in a chemostat or upon repression following growth in rich media. Leucine messenger ribonucleic acid, measured both by pulse-labeling and hybridization-competition experiments, was roughly proportional to enzyme levels over this entire range. For a plasmid-containing strain grown in a chemostat under conditions of leucine limitation (about 100 plasmids per chromosome), about 27% of pulse-labeled ribonucleic acid was coded for by genes in or adjacent to the leucine operon, and 10% of the total protein was beta-isopropylmalate dehydrogenase.


Transport of sugars and amino acids in bacteria. XVIII. Properties of an isoleucine carrier in the cytoplasmic membrane vesicles of Escherichia coli. The properties of the carrier for isoleucine in Escherichia coli were studied using cytoplasmic membrane vesicles (IM vesicles) prepared by the method of Yamato, Anraku, and Hirosawa (J. Biochem. 77, 705 (1975)). The IM vesicles exhibited respiration-dependent isoleucine transport activity which was more than 30-fold higher than that of "Kaback vesicles" prepared by our hand from the same strains of E. coli K12. The isoleucine carrier activity of IM vesicles was inhibited by norleucine but not by threonine. The carrier was driven by proton motive force. Mutants were isolated which had lost the carrier activity for isoleucine, as judged by assay with IM vesicles. Using these mutants, the effects of binding proteins specific for branched chain amino acids on the translocation of substrate in IM vesicles were studied. Leucine-isoleucine-valine-threonine-binding protein (LIVT-binding protein) stimulated the initial rate of isoleucine uptake by IM vesicles only when the vesicles possessed carrier activity and it did not affect the Kt value for entry of substrate. This evidence suggests the partial reconstitution of the osmotic shock-sensitive transport reaction in which the binding protein seems to affect the carrier activity with turnover ability.


Strain specificity of outer membrane proteins in Escherichia coli. Outer membrane proteins of various strains of Escherichia coli were compared using three different systems of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The outer membranes of E. coli K-12, E. coli B, and E. coli J-5 had distinctive protein compositions. As regards proteins which interact with peptidoglycan, E. coli K-12 contained O-8 and O-9, while E. coli B possessed one protein which migrated to the position of O-9. Although E. coli J-5 possessed two such proteins, O-8' and O-9', their positions on polyacrylamide gel were different from those of O-8 and O-9. Protein O-7, which migrates slightly more slowly than O-8, was found specifically in E. coli K-12. Proteins O-10 and O-11 were found in all strains tested, although the relative amounts were different depending on the strain. Strains of E. coli K-12 and E. coli J-5 gave three major bands, O-2a, O-2b, and O-3, in the region of high molecular weight. These proteins were repressed by iron in the cultivation media. Strains of E. coli B, on the other hand, gave only O-2b and O-3. E. coli J-5 gave two other major bands in this region, but the amounts were not controlled by iron in the cultivation media.


Effects of ATP and ADP on hydrogen-deuterium exchange in heavy meromyosin. An examination of difference spectra. The exchange reaction of peptide hydrogens with deuterium has been followed by measuring the decrease of the amide II band for heavy meromyosin (HMM). The difference spectra between HMM and HMM + ATP, between HMM and HMM + ADP, and between HMM + ATP and HMM + ADP have been examined as functions of time in order to detect small differences in the kinetic behavior of these different states of HMM. It has been found that, at 14 degrees C and 26 degrees C (pH 8.0), the exchange reaction is slightly slower for HMM + ATP than for HMM, and slightly slower for HMM + ADP than for HMM + ATP. This indicates that the secondary structure of HMM changes its flexibility during the ATP splitting cycle.


Studies on bacterial chemotaxis. I. Separation of proteins from cell envelope of Escherichia coli. Radioactive proteins from Escherichia coli cell envelope fraction were separated by two-dimensional polyacrylamide gel electrophoresis. Electrophoresis was carried out under several sets of conditions, and autoradiographs were obtained. Many of the proteins were separated at well-defined positions with good reproducibility. Some of the proteins moved relative to these stationary proteins depending at least two factors, i.e. the amount of proteins applied in the first dimension and the electric current applied in the second dimension. Among more than 200 spots, methyl-accepting chemotaxis protein and flagellin were identified by using labelled or cold preparations of these proteins as markers. Some of the spots were assigned to proteins from the outer membrane of the bacteria. The results provide a good foundation for comparative studies of membrane proteins from genetically altered strains of the bacteria.


Regulation of Escherichia coli ornithine transcarbamylase by orotate. Ornithine transcarbamylase from Escherichia coli, strain W, exhibits negative cooperativity with respect to ornithine, and the enzymatic activity is further regulated by orotate. The effect of orotate on ornithine transcarbamylase is dependent not only upon the carbamylphosphate concentration, but also upon the concentration of ornithine. At high concentrations of carbamylphosphate (10 mM), a conversion from negative cooperativity to positive cooperativity is observed with 10 mM orotate. At 1 mM carbamylphosphate, however, 10 mM orotate activates the enzyme at low ornithine concentrations, but as the ornithine concentration is increased above 5 mM, inhibition is observed. Thus, a regulatory link has been established between the pathways of arginine biosynthesis and pyrimidine biosynthesis, each of which utilizes carbamylphosphate.


Cro regulatory protein specified by bacteriophage lambda. Structure, DNA-binding, and repression of RNA synthesis. The Cro protein specified by bacteriophage lambda is a repressor of the genes expressed early in phage development and is required for a normal late stage of lytic growth. We have purified Cro protein to virtual homogeneity and analyzed its structure and properties as a DNA-binding protein and repressor of RNA synthesis. To confirm that the protein is the product of the cro gene, we have also shown that a missense mutation in the cro gene leads to a product that is more temperature- and salt-sensitive in its DNA-binding property. As purified, Cro protein is a dimer of identical subunits of molecular weight 8600. The purified protein binds to lambda-DNA carrying the specific binding sites (operators oL and oR) with an estimated dissociation constant of 10(-10) M to 10(-11) M; there is also weaker binding to other sites on DNA, as found for other DNA-binding regulatory proteins. In a purified transcription system, the Cro protein is an effective and specific repressor of RNA synthesis from the N and cro genes; thus Cro is an autorepressor which regulates its own synthesis. A comparison of the properties of the two lambda repressor proteins, cI and Cro, indicates that cI is a "strong repressor" specialized for complete turnoff of lytic functions needed for the maintenance of lysogeny, whereas Cro is a "weak repressor" specialized for a gradual turnoff of early viral genes that potentiates the late stage of lytic development.


In vitro studies on the methylation of histones in rat brain nuclei. When isolated nuclei from 12-day-old rat brains were incubated with S-adenosyl-L-[methyl-3H]methionine, significant amounts of 3H-methyl were incorporated into lysyl residues in histones H3 and H4. About 0.024% of the total methylation sites on histone H3 and 0.013% of the sites on histone H4 were unmethylated at the time the nuclei were isolated. Methylation of these sites proceeded stepwise, progressing to a stable ratio of 0.93:1.0:0.17 for N epsilon-mono-, N epsilon-di-, and N epsilon-trimethyllysine in histone H3 and 0.19:1.0 for N epsilon-mono- and N epsilon-dimethyllysine in histone H4. The Km values of the enzyme for S-adenosyl-L-methionine were 11.5 +/- 1.1 micron and 12.5 +/- 1.3 micron with histones H3 and H4 as methyl acceptors, respectively. The Vmax values were 11.1 and 5.3 pmol of 3H-methyl incorporated/min/mg of histone H3 and H4, respectively. Since histone H3 contains 2 mol of N epsilon-methyllysine/mol and histone H4 contains 1 mol/mol, no difference in the overall rates of methylation can be deduced from the data. S-Adenosyl-L-homocysteine, one of the products of the reaction, was a competitive inhibitor with respect to S-adenosyl-L-methionine. The Ki values for S-adenosyl-L-homocysteine were 5.5 +/- 0.4 micron and 5.9 +/- 0.5 micron with histones H3 and H4 as methyl acceptors, respectively.


Escherichia coli protein X is the recA gene product. Escherichia coli protein X is known to be made in large amounts following DNA damage or inhibition of DNA replication. We have shown that it is identical to the recA gene product by partial proteolytic digestion of the radiochemically pure proteins and analysis by electrophoresis on polyacrylamide-sodium dodecyl sulfate gels.


Micrococcus luteus correndonucleases. II. Mechanism of action of two endonucleases specific for DNA containing pyrimidine dimers. Py pyrimidine dimers Py correndonucleases I and II from Micrococcus luteus act exclusively on thymine-thymine, cytosine-cytosine, and thymine-cytosine cyclobutyl dimers in DNA, catalyzing incision 5' to the damage and generating 3'-hydroxyl and 5'-phosphoryl termini. Both enzymes initiate excision of pyrimidine dimers in vitro by correxonucleases and DNA polymerase I. The respective incised DNAs, however, differ in their ability to act as substrate for phage T4 polynucleotide ligase or bacterial alkaline phosphatase, suggesting that each endonuclease is specific for a conformationally unique  site. The possibility that their respective action generates termini which represent different degrees of single strandedness is suggested by the unequal protection by Escherichia coli binding protein from the hydrolytic action of exonuclease VII.


Identification of UDP-glucose as an intermediate in the biosynthesis of the membrane-derived oligosaccharides of Escherichia coli. The membrane-derived oligosaccharides of Escherichia coli constitute a closely related family of oligosaccharides containing approximately 9 glucose units variously substituted with sn-glycero-1-phosphate and phosphoethanolamine residues derived from the head groups of membrane phospholipids, and also with succinate in O-ester linkage (Kennedy, E.P., Rumley, M.K., Schulman, H., and van Golder, L.M.G. (1976) J. Biol. Chem. 251, 4208-4213). Studies with mutant strains defective in the synthesis of various nucleoside diphosphate sugars have now revealed that UDP-glucose is an essential intermediate in the biosynthesis of these oligosaccharides. Mutants unable to synthesize UDP-glucose do not contain significant amounts of the membrane-derived oligosaccharides. In contrast, a strain unable to synthesize ADP-glucose, the glucosyl donor for glycogen synthesis in E. coli, contained normal amounts of the membrane-derived oligosaccharides, although with a somewhat different pattern of distribution of the various subspecies. In confirmation of these genetic studies, pulse-label isotope tracer studies have been carried out with glucose of high specific activity, under conditions in which UDP-glucose comprises a large fraction of the total radioactivity in the low molecular weight pool. Subsequent "chase" experiments clearly revealed the conversion of UDP-glucose to the higher molecular weight membrane-derived oligosaccharides.


Pseudouridine-deficient transfer RNAs from Escherichia coli B and their use as substrates for pseudouridine synthetase. Transfer RNAs isolated from Escherichia coli B grown in the presence of 2-thiouracil are deficient in pseudouridine. Much of this deficiency is from the T psi C region, which has only about 50% of its normal pseudouridine content. The other modified nucleoside from this region, ribothymidine, is reduced by only about 10%. Studies showed that 2-thiouracil is incoproated into the RNA of E. coli during growth in the presence of the analog. This incorporation appears to result from the replacement of uracil, occur in a random manner, and involve all RNA species. The extent of incorporation varies from 1 to 3 mol %, depending upon the preparation and RNA species examined. Electrophoresis on polyacrylamide gels and chromatography on Sephadex G-75 and reverse phase (Systen 5) columns of normal and 2-thiouracil-containing tRNAs revealed no profile differences. No accumulation of any precursor tRNA in the thiopyrimidine-treated cells is found. A partial recovery of the pseudouridine content of 2-thiouracil-containing tRNAs can be achieved in vivo by removal of the 2-thiouracil from the culture media. These transfer RNAs have also been used as substrates to study the properties of a partially purified preparation of pseudouridine synthetase II invitro and should be useful as substrates in the further purification of this enzyme.