Transport of tetracyclines through the bacterial cell membrane assayed by fluorescence: a study with susceptible and resistant strains of Staphylococcus aureus and Escherichia coli.

The fluorescence of 12 tetracyclines in buffered solutions was measured by excitation at 400 nm and emission at 520 nm. The fluorescence varied markedly for different tetracyclines at equivalent concentrations. Demethylchlortetracycline exhibited more fluorescence than both chlortetracycline and demthyltetracycline; minocycline was virtually non-fluorescent at the conditions of the study. Fluorescence was highly dependent on the polarity of the solvent; when the buffer solution in water was replaced by a solvent containing 50% methanol, fluorescence increased significantly, but to various degrees for different tetracyclines. The most striking influence of the addition of methanol was observed for doxycycline (5-oxy 6-deoxy-tetracycline), whereas the influence on anhydrotetracycline was negligible. When suspensions of a susceptible strain of Staphylococcus aureus were added to solutions of tetracyclines, membrane permeation of the drugs could be monitored by an increase in fluorescence. This increase varied strikingly with the different drugs and could not be correlated with the concentrations for 50% growth inhibition (Ki). This might be due to the quantitative variations in the intracellular level corresponding to a certain external concentration of the respective drug. When tetracycline-resistant strains of S. aureus and Escherichia coli were exposed to tetracycline, the intensity of fluorescence observed was less than for the corresponding susceptible strains; in spite of this, the quantitative differences of fluorescence exhibited by the susceptible and resistant strains seemed slight in relation to the differences in susceptibility. It was demonstrated that minocycline inhibits the membrane transport of tetracycline in S. aureus and E. coli. This inhibition seems to be competitive for S. aureus, but not for E. coli.


[tRNA-methylase study of the extreme thermophile, Thermus flavus].

tRNA methylases were studied in the extreme thermophilic culture of Thermus flavus, strain 71. Like E. coli, the culture contained only those tRNA methylases which catalysed the formation of m1A and m7G. Mg2+, Ca2+ and Na+ ions activated tRNA methylases of Thermus flavus in the series Mg greater than Ca greater than Na while Mn2+ ions inhibited the enzyme. The activity of tRNA methylases was higher in T. flavus than in E. coli, and required less protein and time for exhaustive methylation of tRNA preparations. The overall activity of methylases in T. flavus at 70 degrees C was 5-6 times higher than at 40 degrees C; the elevation of temperature had different effect on various methylases: the activity of m1A methylase increased 13-fold whereas that of m7G methylase only twofold.


[Capsule formation in saprophytic yeasts].

Among ten carbon sources and five nitrogen sources tested, D-mannose (3%) and peptone (0.2%) were found to be the best ones for stimulating capsule formation by the yeast Cryptococcus magnus. Addition of yeast extract (0.1%) to the medium favoured formation of larger capsules. Elevated concentrations of mannose and peptone inhibited capsule formation. Comparison of the results obtained with the data about the effect of cultivation conditions on capsule formation by the pathogenic yeast revealed no considerable differences between the latter and the saprophytic yeast.


A clinical trial with Alice/R-75 strain, live attenuated serum inhibitor-resistant intranasal bivalent influenza A/B vaccine.

A clinical trial was conducted with Alice/R-75 strain live attenuated intranasal influenza A/B vaccine. With double blind control 88 adult volunteers were administered 2 doses of Alice/R-75 vaccine, 93 volunteers received one dose of Alice/R-75 vaccine and one dose placebo solution and 94 subjects were administered 2 doses of placebo solution. Twenty-three other subjects received Alice strain monovalent influenza A vaccine. For comparison, data from 21 subjects who received monovalent intranasal R-75 strain influenza B in two doses is included. The vaccine was generally well tolerated. Four-fold serum hemagglutination-inhibiting (HAI) antibody titer rises to A/England/42/72 occurred in 39% of the monovalent Alice strain vaccines; in contrast 18% of those given 2 doses of bivalent Alice/R-75 vaccine and 11% of those given 1 dose of bivalent vaccine had similar four-fold HAI antibody titer rises. HAI antibody titer rises to influenza B/Hong Kong/72 occurred in 38% of R-75 strain monovalent vaccinees, 14% of Alice/R-75 2-dose vaccinees and 11% of Alice/R-75 one dose vaccinees. An epidemic of influenza at onset of the study made evaluation of the efficacy of the vaccine impossible.


Evaluation of a modified dye pour-plate auxanographic method for the rapid identification of clinically significant yeasts. Comparison with two commercial systems.

A modified dye pour-plate auxanographic (DPPA) method for the presumptive identification of medically important yeasts was evaluated, in a comparative study with a conventional procedure, the API 20C clinical yeast system (Analytab Products Inc.), and the Uni-Yeast-Tek (UYT) system. The 174 coded clinical isolates were members of the genera Candida, Cryptococcus, Rhodotorula, Saccharomyces, Torulopsis, and Trichosporon. The identification accuracies with DPPA, API, and UYT were 95%, 93%, and 99% respectively. DPPA and API required more time to inoculate but gave rapid identification profiles. UYT was simple to inoculate and both UYT and DPPA were easy to read. Cost analysis of the three rapid methods demonstrated DPPA to be the most economical making it a feasible alternative for small clinical laboratories as well as large laboratories possessing the facilities to make their own media.


[Modified method for determining water pollution by using a Serratia marcescens culture].

The method for determining the degree of water pollution with the aid of a bacterial tracer has been improved by using: (1) a technique for maintaining the culture of Serratia marcescens; (2) a medium whose composition provided for a high level of prodigiosin in the bacterial culture; and (3) an express method for assaying the degree of pigmentation during growth of Ser. marcescens on solid media.


[Morphological changes in the cells of Escherichia coli damaged by nitrous yperite].

Nitrous yperite supressed division of E. coli cells; as a result, filaments and giant abnormal cells appeared. Apparently, giant forms were caused by defects in the cell wall induced by nitrous ypertie due to a mutation in the mon gene of the strain. These cells gave rise to normal cells as well as to small bacteria resembling mini-cell.


[Role of metabolic energy in the regulation of chlortetracycline transport in Escherichia coli].

The uptake of chlorotetracycline by the E. coli K-12 strain sensitive to this antibiotic and by its resistant mutant E. coli E-6-X is regulated by the antibiotic transport being coupled and uncoupled with the metabolic energy, this being determined by the concentration of chlorotetracycline in the medium. If the level of the antibiotic is lower than the subbacteriostatic concentration, its accumulation by both strains requires the metabolic energy and can be suppressed by formaldehyde or 2,4-dinitrophenol. As the concentration of chlorotetracycline increases, it is no more absorbed by the cells of the sensitive strain, via the active transport and its accumulation does not change in the presence of 2,4-dinitrophenol or glucose. In the resistant strain, the mechanism of the energy-dependent transport of chlorotetracycline from the cells starts to function: the accumulation of the antibiotic decreases in the presence of glucose and increases in the presence of 2,4-dinitrophenol.


Fungal peritonitis and malignancy: report of two patients and review of the literature.

Two patients developed isolated Candida albicans peritonitis in association with intraabdominal malignancy. Although additional factors predisposing to the development of fungal peritonitis were present, we postulate that tumor-related local factors permitted fungi to cross the gut wall and to enter the peritoneum, where the host immune status determined whether the infection spread. These two cases are the sixth and seventh reported cancer patients who developed fungal peritonitis, but the first two who had the fungal infection localized to the peritoneum; and this is the first report known to us specifically associating intraabdominal malignancy and fungal peritonitis. Patients who develop fungal peritonitis may have a primary or metastatic intraabdominal malignancy, and fungi should be considered as a cause of peritonitis in cancer patients.


Combination chemotherapy for non-Hodgkin lymphomas: a ten year follow-up study.

In 1968 the Cancer and Acute Leukemia Group B (CALGB) demonstrated optimal control of disseminated non-Hodgkin lymphomas (NHL) with vincristine-prednisone induction followed by cyclophosphamide maintenance. A study was then begun to determine whether four drugs in combination or sequence could achieve greater control. NHL patients at each participating CALGB institution were randomly assigned to one of three regimens:I) Cyclic vincristine-streptonigrin alternating every 2 weeks with cyclophosphamide-prednisone up to 155 days; II) Sequential treatment with the same 4 drugs taken singly up to 182 days; and III) Vincristine-prednisone induction for 6 weeks followed by cyclophosphamide maintenance. Results are now reported after a 10 year follow-up period. The 203 evaluable patients are those on whom Rappaport histopathologic classification was available. Frequency of complete-response did not differ significantly among the three regimens: I) 38%; II) 30%; and III) 45%. Remission durations were significantly longer among patients receiving maintenance therapy. After ten years, two patients from Regimen I, one from Regimen II, and five from Regimen III remain alive and well. It was concluded that neither of the four-drug regimens conferred a significant advantage in terms of response rate or survival time over the standard treatment.


A minimum-interrupt digital interface for the Coulter S blood analyzer and IBM 1800 computer.

A computer interface for the fully automatic transfer of data from a Coulter model S blood analyzer to an IBM 1800 data acquisition and control system is described. The design objective was to eliminate manual handling of hematology data in order to enhance the reporting effectiveness of a computer-supported clinical pathology laboratory. Using integrated circuits and optical isolation techniques, the interface requires no modification of either the Coulter analyzer or the IBM computer. Successful operation of the interface has been demonstrated for over 4 years in two different hospitals.


Organization, replication and modification of the human genome: temporal order of synthesis and methylation of two classes of HeLa nDNA separated in Ag+--Cs2-SO4 gradients.

During the HeLa S-phase, DNA was methylated, at 1-hr intervals in isolated nuclei and fractionated in Ag+-Cs2SO4 gradients providing a heavy GC-rich peak and a main light AT-rich peak. Both size and specific methylation of these peaks changed during the nDNA duplicative phase. Replication of the heavy GC-rich nDNA fraction, which contains genes for ribosomal RNA, occurred in early S; in contrast, replication of the main AT-rich nDNA fraction was maximal in late S. Concomitantly, specific methylation of the GC-rich nDNA was maximal in the first part of S, while that of the AT-rich nDNA was maximal in the second part of S. This suggested that genes are replicated and methylated with order during the S-phase.


Origins and control of stereotyped movements.

A large number of retarded individuals exhibit repetitive and rhythmical movements of a highly stereotyped character. Common forms of these behaviors include body rocking, head nodding, hand waving, head banging and so on. Such behaviors are distinguished by their apparently maladaptive and excessive nature. The frequency of these bizarre responses in negatively correlated with measured intelligence. Stereotyped movements usually have their onsets early in the life of the individual and, in many cases, appear to have become functionally autonous. A variety of theories have been proposed to explain the onset and maintenance of stereotyped movements. None can be accepted to the exclusion of others and all have some empirical support. Problems of measurement are particularly difficult ones in this area. Of the various methods devised to suppress stereotyped movements, those based on principles of learning seem to be the most popular. However, generalization and durability of suppression effects generally have not been dealt with adequately. Much less attention has been paid to ecological analyses of the natural environments in which individuals exhibits stereotypies. A case can be made that our knowledge about the initiating conditions for these behaviors and subsequent effects on the natural environment is far less than perfect. A number of research directions are suggested.


[Isolation and properties of DNA-cytosine methyltransferase from Escherichia coli C].

The method of isolation and partial purification of DNA-cytosine-methyltransferase (DC-methylase) from E. coli C is described. The enzyme underwent approximately 100-fold purification. The obtained preparation of DC-methylase can be additionally considerably purified by sedimentation in sucrose gradient. Native molecular weight of DC-methylase from E. coli C. is 70,000. The activity of enzyme does not depend on the Mg2+ ions. DC-methylase E. coli C provides DNA of lambda phage in vitro with full resistance against restriction endonuclease EcoRII. In DNA methylated by DC-methylase the modified cytosine, mainly in C-MC and C-MC-T sequences, corresponds to the pyrimidine sequences of specific site EcoRII. DNA of lambda.B phage contains approximately 80 sites for modification by DC-methylase E. coli C. The results obtained point to the same specificity in vitro of DNA-cytosine-methylase E. coli C and DNA-methylase EcoRII.


[Modification of the RNA-polymerase of Escherichia coli by diethylpyrocarbonate].

E. coli DNA dependent RNA polymerase was modified by diethylpyrocarbonate. Optical and kinetic properties of the reaction were studied. More than 90% of RNA polymerase activity is inhibited by introduction of 9--11 ethoxyformyl groups per enzyme molecule without loss of its ability to bind DNA template. Furthermore the modified enzyme is able to form tight complexes with DNA and to compete with native enzyme for the formation of rifampicin-resistant complex. The ratio of the complex formation constants for the native and modified enzyme was determined to be equal to 10. The enzyme modified to such extent loses the activity in DNA dependent RNA as well as pppApU synthesis. Vmax value rather than Km value for both ATP and UTP decreases following the modification reaction. Incubation of the enzyme modified to the 10% of residual activity with 0.2 M hydroxylamine for 2 hours results in restoration of RNA polymerase activity. Most but not all of the modified histidyl residues restore their native structure. Two of 13 histidyl residues were modified irreversibly due to Bamberger's cleavage reaction, but these two residues were found to be unessential for RNA polymerase activity. Reaction with higher concentration of the diethylpyrocarbonate induces modification of more than 15--20 histidyl residues and leads to irreversible inactivation of the enzyme. Nevertheless the modification of the additional histidyl redidues was reversible as well as the modification of the first 11 residues. RNA polymerase modified to such extent loses the ability to bind DNA. Preformation of the initiated ternary complex of RNA polymerase with template and product fails to protect the enzyme from reversible inactivation at a low reagent concentration, but markedly decreases the rate of the irreversible and unspecific modification of sulfhydryl or amino groups of the enzyme. Reaction with the ternary complex results in reversible inactivation of the enzyme with respect to elongation of RNA chains as well as the pyrophosphate exchange reaction. The complex itself was, however, completely stable under the reaction conditions and the enzyme subunit structure was also conserved after the reaction. Evidently, the mild modification of the histidyl residues with diethylpyrocarbonate selectively inhibits RNA chain elongation.


[Hydration of a preparation of total yeast tRNA].

The interaction of water molecules from the vapour phase with the total backer's yeast tRNA preparation was studied by the dynamic aquametric method. The primary hydration sites for processes of sorption and desorption of water molecules was evaluated by means of multilayer adsorption BET-equation. It was shown that the primary hydration sites are the oxygen atoms in the ribose-phosphate backbone of the tRNA molecule. The structure of surfaces of globular proteins and tRNA molecules were compared from the point of view of their ability to interact with water molecules. The higher degree of maximal hydration (under saturated water vapour or in aqueous solution) was considered as a result of regular arrangement of the most part of tRNA primary hydration sites.


Characterization of DNA adenine methylation mutants of Escherichia coli K12.

The phenotypic traits of 7 independently isolated dam mutants of Escherichia coli have been examined. The mutant strains differ from the wildtype in the following respects: (1) decreased DNA adenine methylase activity in vivo and in vitro; (2) a 14--85-fold increase in spontaneous mutability; (3) decreased survival after ultraviolet irradiation; (4) a 10--21-fold increase in spontaneous induction of lambda phage from lysogens; (5) a 3--17-fold increase in the level of recombination; and (6) inviability of double mutants containing dam- and recB- or recC-. Unmethylated fd phage chromosomes are able to replicate normally in dam- mutants. A mutant strain in which the dcm gene is deleted is viable, showing that the dcm gene product is dispensible for growth.


The ability of ionizing radiations of different LET to induce chromosomal deletions in Aspergillus nidulans.

Conidia, derived from a strain of Aspergillus nidulans known to carry a specific chromosomal duplication, were irradiated. The duplicated segment had genetic markers, which, when eliminated from the genome, allowed the easy detection of deletion mutants. Survival curves derived following 15 MeV electron and gamma-ray irradiation were characterised by the presence of an appreciable shoulder, whilst 50 kvp X-rays gave a much smaller shoulder. Irradiation with beta-particles and alpha-particles gave rise to exponential survival curves. The RBE values for these radiations, based on the D37 value were for gamma-rays, 1.0, 15 MeV electrons 1.0, 50 kvp X-rays 1.9, beta-particles 2.1 and alpha-particles 3.4. With the exception of gamma-rays the radiations described were compared with respect to their ability to induce chromosomal deletions. When the number of deletants amongst survivors was plotted against dose, a linear relationship was found for electrons, X-rays and beta-particles. The response recorded for alpha-particles was essentially linear but with a biphasic component. The RBE values for the radiations, based on a value of unity for 15 MeV electrons were as follows: X-rays 1.3, beta-particles 0.8, alpha-particles above 7.5 krad 2.3 and below 7.5 krad 3.5. When these same data were re-plotted with number of deletants amongst survivors against log survival, electrons appeared the most efficient radiation at producing deletants amongst survivors, with an "m value" of 283 X 10(-5). Tritiated water was least efficient, the corresponding value being 182 X 10(-5). The number of deletants per 10(4) conidia plated, when plotted against dose yielded a curve which increased to a peak and then decreased linearly for all radiations. The peaks for electrons, X-rays and alpha-particles each had a value of about 14 deletants per 10(4) conidia plated and the peaks roughly corresponded with the point at which the survival curve became exponential and was clearly indicative of the accumulation of sub-lethal damage. However, for beta-particles the peak had a value of 7 deletants per 10(4) conidia plated. A non-DNA target has been implicated for cellular death following beta-particle irradiation.


Development of a liquid-holding technique for the study of DNA-repair in human diploid fibroblasts.

Liquid-holding conditions can be obtained for human diploid skin fibroblasts by keeping confluent cultures stationary over periods of 7 days or longer by means of conditioned medium. Under this condition recovery of radiation damage induced by ultraviolet light or X-rays is observed as an increase in cloning efficiency. The amount of recovery when expressed in a dose-modifying-factor appears higher than in bacteria and yeast. The repair-deficient human cell strains XP25Ro and XP7Be (xeroderma pigmentosum from complementation groups A and D respectively) exhibit less but still discernible recovery after UV-irradiation and the same was observed for AT5Bi (ataxia telangiectasia) after X-irradiation. Experiments on mutation induction indicated that the repair which takes place during liquid holding of UV-irradiated XP7Be cells reduces the mutant frequency considerably while after liquid holding of UV-irradiated wild-type cells the same or lower mutant frequencies were found for the lower exposures and the same or higher mutant frequencies for the higher exposures.


Induction of gene conversion in diploid yeast by chemicals: correlation with mutagenic action and its relevance in genotoxicity screening.

More than 200 chemicals, comprising drugs, pesticides, herbicides, industrial chemicals, food additives, etc. tested by various workers for their ability to induce gene conversion in diploid yeast, have been reviewed. This review shows that  a strong correlation exists between the convertogenic and mutagenic abilities of these chemicals. This conclusion confirms the earlier one by Zimmermann which was based on fewer mutagenic chemicals. Published data have further been analysed to confirm quantitatively Zimmermann's other important conclusion that gene conversion is not mutagen-specific. This is a positive advantage in using gene conversion in preliminary screening of chemicals because it reduces the chances of false negatives.


A combined testing protocol approach for mutagenicity testing.

The antischistosomal agent, hycanthone methanesulfonate (HMS), was employed to illustrate the utility of carrying out several mutagenicity tests in a single concurrent animal experiment. Several commonly used procedures that were successfully integrated into a multiple testing protocol included (1) metaphase analysis in bone marrow, (2) micronucleus test in bone marrow, (3) analysis of the urine for mutagenic constituents, and (4) the host-mediated assay using Salmonella typhimurium. In addition to these animal studies, in vitro mutagenicity testing with and without activation was carried out using S. typhimurium. HMS produced positive, dose--response effects in in vitro tests, metaphase analysis, micronucleus test, and urine analysis, but not in the host-mediated assay. The results of these integrated techniques suggest that such a protocol may be a benefit to those concerned with mutagenicity testing of chemicals.


The mutagenic action of nitroimidazoles. IV. A comparison of the mutagenic action of several nitroimidazoles and some imidazoles.

The mutagenic action of 51 imidazoles was investigated. The fluctuation test of Luria and Delbrück was used, with Klebsiella pneumoniae as test organism. 8 compounds, including 5 with a weak mutagenic action in the fluctuation test, were also investigated by the Ames test in which Salmonella typhimurium TA100 was used. Of the 51 imidazoles examined, 33 were nitroimidazoles. 31 of the latter appeared to be mutagenic, whereas out of the 18 other imidazoles without a nitro group only 2 were mutagenic. Several of the substances tested for mutagenicity showed an antimicrobial activity. No direct relationship between antimicrobial action, growth inhibition and mutagenicity was established. With methyl-nitroimidazoles a relationship was found between the chemical structure and mutagenic action. However, when the nitroimidazoles had a more complex chemical structure, a relationship between this structure and mutagenicity could not be established.


Mutagenicity of plant flavonols in the Salmonella/mammalian microsome test: activation of flavonol glycosides by mixed glycosidases from rat cecal bacteria and other sources.

Over 70 naturally occurring and synthetic flavonoids were screened for mutagenicity with 5 tester strains in the Salmonella/mammalian microsome assay: TA1535, TA100, TA1537, TA1538 and TA98. Frameshift mutagenicity was confined to the flavonols (flavon-3-ols) in strain TA98, TA1537 and TA100. The two most mutagenic falvonols, namely, quercetin (3,3',4',5,7-pentahydroxyflavone) and kaempferol (3,4',5,7-tetrahydroxyflavone), exhibiting 12 and 7 revertants/nmol in TA98 respectively, are also the most common flavonols occurring in plants. Other flavonols exhibited less activity (revertants/nmol): galangin (2.0), rhamnetin (0.45), kaempferide (0.24), fisetin (0.14), myricetin (0.12), robinetin (0.06) and morin (0.05). All of these flavonols apparently exhibited significant activation by Aroclor 1254 induced rat-liver microsome preparations (S9). However, subsequent study revealed that only those flavonols either lacking or possessing one B ring hydroxyl group had an absolute requirement for microsomal activation. Alternatively, quercetin with two B-ring OH groups is not activated by microsomal enzymes, but by soluble (S100) enzymes from liver which are apparently constitutive and not subject to the usual chemical induction. 3 flavonol glycosides, namely, quercetrin (quercetin-3-O-rhamnoside), rutin (quercetin-3-O-rutinoside) and robinin (kaempferol-3-O-galactosido-rhamnoside-7-O-rhamnoside), were found to be nonmutagenic. They could, however, be activated by a variety of mixed glycosidases incorporated in the usual pour plate procedure. The most effective enzyme mixtures were obtained from rat cecal bacteria and from the snail Helix pomatia.


Detection of mutagens in the urine of rats following topical application of hair dyes.

Mutagens were detected in the urine of rats following topical application of two commercial oxidative-type hair dye preparations. The test system used was induction of back mutation with the bacterial tester strain TA1538, a histidine-dependent mutant of Salmonella typhimurium. Various quantities of dye were applied to the shortened hair on the backs of the test animals. The dye was allowed to remain on the hair for 20 min after application and was then removed by shampooing and thorough rinsing. Maximal levels of mutagenic activity occurred with urine collected during first 24 h following dye application, and a dose--response was observed when increasing volumes of mutagenic urine were tested. Mutagens were detected in rat urine after intraperitoneal injection, and also after topical application of 4-nitro-o-phenylenediamine, one of the constituents of the hair-dye preparations.


Hydrazines as mutagens in a histidine-requiring auxotroph of Salmonella typhimurium.

Hydrazines have been found naturally in tobacco and mushrooms. Other hydrazines are used in industry, medicine, and agriculture. Although about 38 hydrazines are carcinogenic, few, if any, have been tested successfully in rapid bacterial mutagenesis assays. We have utilized a tester strain of Salmonella typhimurium (TA1530) in order to determine the mutagenic activity of a number of hydrazines and related compounds. This strain is thus shown to be effective as a tester organism for the facile detection of hydrazines as mutagens.


Comparison of the induction by cigarette smoke condensates of sister-chromatid exchanges in Chinese hamster cells and of mutations in Salmonella typhimurium.

Three cigarette smoke condensates were tested for the induction of sister-chromatid exchanges in ovary cells of the Chinese hamster and for mutations in Salmonella typhimurium. In the sister-chromatid exchange test an effect was obtained that was not enhanced by the inclusion of a system for metabolic activation. In the Salmonella test, an effect was only obtained by including rat-liver homogenates derived from rats treated with inducers of the enzyme systems necessary for metabolic activation. It appears that the SCE test and the Salmonella test are sensitive to different components of cigarette smoke condensates.


Chromosomal aberration tests on 29 chemicals combined with S9 mix in vitro.

A metabolic activation system with rat-liver microsome fraction plus cofactors (S9 mix) was applied to chromosomal aberration tests in vitro for the screening of chemical mutagens or carcinogens in the environment. Dialkylnitrosamines only induced chromosomal aberrations in Chinese hamster cells (CHL) when treated with S9 mix. The incidence of chromosomal aberrations in CHL varied with experimental conditions, e.g. incubation time, recovery time, components of S9 mic and inducers used for preparation of S9. For dimethylnitrosamine (DMN), the maximal incidence was obtained when the cells were incubated with S9 mix for 3 h and harvested 24 h after treatment. Therefore, this system (3 h incubation and 24 h recovery) was routinely applied to further screening of other chemicals with S9 prepared from PCB-pretreated rats. 10 carcinogens (e.g. 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene, quinoline, etc.) out of 16 induced aberrations when they were treated with S9 mix, whereas the remaining 6 carcinogens (e.g., 3-methyl-cholanthrene, 4-o-tolylazo-o-toluidine, etc.) induced few or no aberrations even after activation. Two insecticides, allethrin and diazinon, were strongly positive at relatively low doses only when they were activated with the S9 mix. Medical drugs, such as ethenzamide, methyl p-hydroxybenzoate and nitrofurazone, and a food additive, sodium hypochlorite, were positive on activation. Chemicals used for industry, such as styrene monomer and tris-dichloropropylphosphate, were also positive in our activation system.


Superiority of nabilone over prochlorperazine as an antiemetic in patients receiving cancer chemotherapy.

Two double-blind, crossover trials comparing the antiemetic effectiveness of nabilone, a new synthetic cannabinoid, with that of prochlorperazine were conducted in patients with severe nausea and vomiting associated with anticancer chemotherapy. Of 113 patients evaluated, 90 (80 per cent) responded to nabilone therapy, whereas only 36 (32 per cent) responded to prochlorperazine (P less than 0.001). Complete relief of symptoms was infrequent, occurring only in nine patients (8 per cent) given nabilone. When both drugs were compared, both nausea (P less than 0.01) and vomiting episodes (P less than 0.001) were significantly lower in patients given nabilone. Moreover, patients clearly favored nabilone for continued use (P less than 0.001). Predominant side effects noted by patients were similar for both agents and included somnolence, dry mouth and dizziness but were about twice as frequent and more often severe in patients receiving nabilone. In addition, four patients (3 per cent) taking nabilone had side effects (hallucinations in three, hypotension in one) that required medical attention. Euphoria associated with nabilone was infrequent (16 per cent) and mild.


Autoantibodies to gastrin-producing cells in antral (type B) chronic gastritis.

Autoantibodies that react exclusively with the gastrin-secreting cell of human antrum have been detected by immunofluorescence in eight of 106 patients with histologic evidence of chronic atrophic gastritis, Type B, involving mainly the antrum. These antibodies were of the IgG class and of low titer. However, follow-up studies one to two years later showed persistently positive reactions, despite symptomatic treatment. These data support the concept of an autoimmune variant of chronic "antral" gastritis, Type B.


[In vitro toxigenesis of Aspergillus fumigatus strain. Production of fumitoxins].

A new class of mycotoxins has been characterized from a strain of Aspergillus fumigatus: the fumitoxins A, B, C and D. The in vitro production of these metabolites is studied. Fumitoxins are common in cultures extracts of most strains of A. fumigatus. They are not detected from A. fischeri. Variations of the levels of these products during the incubation of cultures, and also by using different media, are noted. At all events, the toxicity of crude extracts of the mould, for the chick embryo, is equal to the one of the fumitoxins.


Pulmonary nocardiosis report of the first two Brazilian cases.

The first two Brazilian cases of human pulmonary nocardiosis are reported. Diagnosis was established on material obtained by direct lung puncture. A review of selected aspects on pulmonary nocardiosis in Latin American countries is presented.


Aspergillus fumigatus and Aspergillus niger in two potted ornamental plants, cactus (Epiphyllum truncatum) and clivia (Clivia miniata). Biological and epidemiological aspects.

The presence and growth of Aspergillus fumigatus and Aspergillus niger in the soil of ornamental plants have been demonstrated. The ecological conditions in the soil of such plants as influenced by temperature, humidity, desiccation, fertilization and ventilation obviously influence such fungal growth. The epidemiological significance of these findings is of interest with a view to the present efforts to control aspergillosis in the environment of susceptible persons. Observations of a preferential growth of certain Aspergillus species in the soil of defined plants under defined conditions raise problems of soil microbiology.


Actin-containing microprocesses in the fusion of cultured chick myoblasts.

Scanning electron microscopic studies of myoblasts from 11- to 13-day-old chick embroyonic breast muscle cultured on collagen-coated glass coverslips showed six stages of development into multinucleated myotubes: (1) growth of flattened, spread-out cells for 20-30 hr following initiation of monolayer cultures; (2) extension of microprocesses (1-150 microM) from cells that have become spindle shaped; (3) contact and adherence of microprocesses from adjacent cells; (4) thickening of fused processes; (5) approximation of the cells; and (6) coalescence of the cells to form a spindle-shaped myotube. When the calcium-ion concentration in the growth medium was lowered--either by increasing the concentration of ethylene-glycol-bis(aminoethyl ether)N,N'-tetraacetate (EGTA)or by decreasing the cconcentration of free calciumion used--the number of microprocesses present on the cells was reduced. Presumably, however, these microprocesses could still fuse together, provided that the calciumion concentration was greater than 160 microM. Indirect immunofluorescence assay with actin-specific antibody indicated that actin is a major component of the myoblasts' microprocesses. Cytochalasin B (5 microgram/ml) caused the microprocesses to retract within 15 min and the myoblasts to round up and detach from the glass substrate. This was presumably caused by the action of the drug on actin filaments.


A seven-day volumetric spore trap for use within buildings.

The Burkard Volumetric spore trap, designed to operate for seven days continuously in the field, was modified to sample still air within buildings. The efficiency with which spores of Lycopodium clavatum and Agaricus bisporus were trapped at two rates of suction was determined. Spore distribution within traces and deposition on surfaces not beneath the orifice were assessed. In an appendix catches of four spore types by the Hirst and Burkard (field model) spore traps operating over mown grass were compared.


Systemic candidiasis in mice treated with prednisolone and amphotericin B. 1. Morbidity, mortality and inflammatory reaction.

Prednisolone potentiated candidiasis in mice when given as dosages of 1 mg, s.c. at-1 and + 24 h in relation to the time of inoculation, i.p. with any of 4 isolates of C. albicans which differed in degree of pathogenicity. Enhancement was shown by increased intra-abdominal colonisation, haematogenous dissemination and percentage mortality. There was at least a seven-fold increase in the mean area occupied by fungal colonies in median, longitudinal sections of kidneys after prednisolone treatment. Compared with those of fungal controls, renal lesions were deficient in inflammatory cells, only a few polymorphonuclear leukocytes occurring peripherally. Amphotericin B (AmB) at a non-toxic, total dosage of 0.5 mg given in two injections of 0.25 mg, i.p. at intervals of 24 h rendered infections non-lethal, or significantly reduced their severity, when started 24 or 48 h after inoculation. When antifungal treatment was delayed until 72 h, however, early deaths occurred despite the fact that the mean renal section area occupied by pseudomycelium was little more than that of controls and fibrosis of lesions was characteristic. Early mortalities increased even further when AmB, commencing at 72 h, was combined with prednisolone. This effect was thought to be due to the release of some toxic factor(s) following the leaching of cells by AmB combined with depleted antibody levels. Ascitic fluids from infected mice given Ehrlich ascites tumour cells showed marked increases in the concentrations of alpha2 and gamma globulins. Concentrations were almost reduced to normal levels in animals treated with prednisolone and/or AmB.


Systemic candidiasis in mice treated with prednisolone and amphotericin B. 2. Ultrastructure and evidence for fungal toxin.

The principal ultrastructural changes in cells of Candida albicans treated with amphotericin B (AmB), either in vitro or in vivo, and in the presence or absence of prednisolone included plasmolysis, vacuolation and destruction of organelles. Lamination of the cell wall, although discernible after 4 h antibiotic treatment in vitro, was conspicuous in vivo, especially in prednisolone-treated mice given AmB 72 h after inoculation and was seen in both phagocytosed cells and those free in inflammatory exudates. Somatic extracts from control cells and somatic extracts and leachates from  AmB-treated cells showed the presence of low-grade toxic components when given i.p. to mice receiving antinomycin D(AMD) s.c. Culture filtrates were negative. Eighteen hour cultures were more toxic than those grown for 3 days and no toxicity was shown for cultures after 8 or 14 days. The behaviour of toxic materials during electrophoresis in polyacrylamide gels suggested that they were proteins of relatively high molecular weight.


Dermatophytes in a population of bank voles and woodmice.

A population of bank voles (Clethrionomys glareolus) and wood mice (Apodemus sylvaticus) inhabiting an oak wood in Somerset was examined for dermatophytes at monthly intervals for 2 years. The marked animals were frequently retrapped, allowing a study of host fungus relationship over a period of time. Microsporum persicolor (Sabouraud) Guiart er Grigorakis and Trichophyton mentagrophytes (Robin) Blanchard were isolated from both animal species, but M. persicolor predominated in bank voles and T. mentagrophytes in wood mice. In their most favoured host, both dermatophyte species often persisted for several months, but in the less favoured host they were never isolated at more than one sampling. Males of both animal species were infected more often than females. The existing evidence for the geophilic nature of both fungi is reviewed and shown to be very weak, especially for M. persicolor. This is the first report from Great Britain of T. mentagrophytes infection of wood mice not in contact with human habitation.


A new fluorescent viability test for fungi cells.

The fluorescein diacetate-ethidium bromide (FDA-EB) fluorescence method, primarily used to determine viability of mammalian cells, was applied to several fungi species. Living fungi cells produced fluorochromasia, i.e., an intracellular accumulation of fluorescein which could be easily visualized as a green color under the U.V. microscope. Dead cells showed a red bright color due to ethidium bromide penetration. The FDA-EB test can be successfully employed to assay yeast and yeast like cells viability since a good correlation was observed between this assay and the colony count technique. The main advantages of FDA-EB test are its speed, high sensitivity and simplicity.


Enzyme-linked immunosorbent assay of antigens from Candida albicans circulating in infected mice and rabbits: the role of mannan.

Various antisera raised either to antigens of Candida albicans or to sub-lethal infections of blastospores (convalescent sera) were tested for their efficacy in diagnosing systemic disease in artifically infected animals. Globulin from convalescent serum, when conjugated with alkaline phosphatase and used in enzyme-linked immunosorbent assays (ELISA), was the only antiserum type which detected circulating Candida-related antigen in the serum of infected animals. Conjugates made from anti-mannan, anti-blastospore or antimycelial globulin did not detect antigen. Mannan did not appear to be related to an antigen produced in sera of experimentally infected mice. The significance of these results in the diagnosis of systemic candidosis is discussed.


Kallikrein, kininogen and kinins in control of blood pressure.

Plasma kallikrein releases bradykinin when activated by gram-negative septicemia or irreversible hemorrhagic shock. Pancreatitis releases glandular kallikrein causing hypotension and increased vascular permeability. Bradykinin in the brain produces hypertension. Renal kallikrein is released by high arterial pressure, vasodilators, low doses of noradrenaline, angiotensin II, mineralocorticoids and rapid volume expansion. It has a biphasic relation to sodium excretion. In essential hypertension, kallikrein release into the blood and urine is low and facilitates hypertension. High renin in Bartter's syndrome is balanced by high PGE and kallikrein without hypertension.


On the control of renin release.

The role of carotid, aortic and cardiopulmonary receptors and of prostaglandins in the control of renin release was reviewed. It is suggested that the receptors in the low- and high-pressure circuits of the cardiovascular system control renin release through the renal sympathetic nerves and adrenal medulla. It is suggested that prostaglandins play a role in the control of renin release under certain pathophysiological conditions.


Isolation of granules and vesicles rich in LHRH and catecholamines from the hypothalamus of the female rabbit.

In the female rabbit hypothalamus (HYP) the luteinizing hormone-releasing hormone (LHRH) and catecholamines (CAT) are mostly present in the granules and vesicles. A fraction of granules containing LHRH and vesicles containing CAT was obtained with 70% purity by ultracentrifugation in sucrose gradient. This fraction was devoid of synaptosomes.


Local changes in immunoreactive gonadotropin releasing hormone in the rat median eminence during the estrous cycle. Correlation with the pituitary luteinizing hormone.

The immunocytochemical approach was used to explore the possibility of local changes in immunoreactive gonadotropin releasing hormone (GnRH) of the median eminence (ME) during the estrous cycle in rats, and an attempt was made to correlate these changes with immunoreactive luteinizing hormone (LH) content in gonadotropic cells. On the day of proestrus, between 9:00 and 18:00 h, a severe depletion of GnRH from the medial palisade zone (MPZ) of the rostral part of the ME was observed whenever any marked changes in the density of GnRH present in the lateral palisade zone and in organum vasculosum of the lamina terminalis (OVLT) were visible. The depletion of GnRH from the MPZ was accompanied by a degranulation of LH-cells (stained with anti-pLH-beta) in the pituitary which occurred over the same period. It is postulated that the MPZ of the rostral part of the ME contains the majority of nerve endings originating from the perikaya responsible for the cyclic surge of LH.


Enzyme kinetic studies and inhibition by oligopeptides of LH-RH degradation in rat hypothalamus and pituitary.

The enzyme kinetic parameters of the degradation of luteinizing hormone-releasing hormone (LH-RH) and L-cystine-bis-(4-nitroanilide) (Cys-NA) by rat hypothalamic (HYP) and pituitary (PIT) extracts and the effect of various oligopeptides on the rate of LH-RH inactivation were investigated in vitro. The 105,000 x g supernatant of 1 rat HYP inactivated 57 microgram LH-RH during a 30 min incubation (Km = 12.4 microM, V max = 2.33 microgram LH-RH/mg protein/min), and of one rat anterior PIT, 48 microgram LH-RH during 30 min of incubation (Km = 12.2 microM, V max = 8.0 microgram LH-RH/mg protein/min). The synthetic substrate Cys-NA competitively inhibited LH-RH degradation with a Ki of 8.5 microM in the HYP and 6 microM in the PIT enzyme preparation. Vice versa, LH-RH also competitively inhibited the cleavage of Cys-NA with inhibition constants of 14 microM (HYP) and 15 microM (PIT) indicating that the 2 substrates are probably cleaved by the same enzyme. The most effective inhibitors of LH-RH degradation were found to be angiotensin-related peptides, neurotensin, bradykinin, and bacitracin. A relatively weak effect was obtained with oxytocin, enkephalin and puromycin. It is concluded that endogenous oligopeptides such as angiotensins, neurotensin, bradykinin, etc., may possibly influence H-RH degradation in the PIT and the HYP. The synthetic substrate Cys-NA may be an appropriate substrate for measuring the activity of an LH-RH-degrading peptidase, which therefore could be classified as arylamidase.


Catecholaminergic drugs in chronic schizophrenia.

The effect of dopaminergic-related and stimulatory drugs have been studied in chronic hebephrenic schizophrenics untreated with neuroleptic drugs. 6 patients received therapy of 2 g L-dopa + 200 mg carbodopa per day orally for 30 days, then placebo for 30 days. Following that 3 of the same patients received therapy of 2 g L-dopa + 200 mg carbodopa + 300 mg imipramine orally for 30 days, then placebo for 30 days. Following that the same 3 patients received 1 mg apomorphine s.c. for 15 days, then placebo for 15 days, then 1 mg apomorphine s.c. + 2 g L-dopa + 200 mg carbodopa per os daily for 15 days, then placebo for 15 days. The patients were examined psychologically by the Wittenborn Rating Scale, the Weigl Object-Sorting Test, and tests for verbal learning and verbal association, before and after each therapeutic trial. Levels of FSH, LH, testosterone and GH were assayed radioimmunologically before, in the middle of and after each course of therapy. 2 patients showed improvement in the affective-behavioural symptomatology during therapy, while the other 4, who had a more severe degree of mental deterioration and destruction, were unchanged. FSH and LH levels, very low under basal conditions, did not change under therapy. Testosterone was very low before therapy and increased in only 1 subject. Normal basal GH levels increased during therapy in some of the patients, but not constantly. The results obtained are discussed in relation to the catecholamine hypotheses of schizophrenia.


Guidelines for supervised wound care by emergency nurse practitioners.

Nearly 10 million patients with traumatic lacerations are treated annually in Emergency Departments in the United States. Since these wounds do not usually pose a threat to the patient's life, they assume a lower level of priority than emergent conditions. Consequently, treatment of patients with lacerations is often delayed until the emergent patient is resuscitated. In the event that wound care is inadvertently delayed, bacteria may proliferate to levels that result in infection. In selected patients, supervised wound care by an emergency nurse practitioner (ENP) can safely eliminate this delay in treatment. During their graduate training in the University of Virginia Nursing School, the ENPs are instructed to treat traumatic wounds with physician supervision. The criteria for patient selection and care are outlined in comprehensive guidelines reported herein. The bases for these treatment guidelines were clinical and experimental studies conducted at our medical center which examined the influence of various therapeutic decisions on the ultimate fate of the wound.


Familial malignant melanoma of the female genitalia: a case report and review.

An additional case of familial occurrence of malignant melanoma is presented, involving mother and son. This is the fourth case of familial occurrence of malignant melanoma in the female genitalia and the first occurring in the vagina reported in the literature. We present a review of the literature concerning the hereditary property of malignant melanoma, including a list of all 92 families and 240 patients reported. The importance of a family history of malignant melanoma for the early diagnosis of this disease in relatives at risk is stressed.


Ascending infection following cervicovaginal exposure to herpesvirus hominis type II.

An animal model is presented that demonstrates that following cervicovaginal inoculation, infective doses of herpesvirus hominis type II induce a form of herpetic encephalitis. Immunofluorescent results indicate that there is both neural and hematogenous spread of the herpes infection. The possible role of a latent viral infection in the genesis of an acute disease of the central nervous system is investigated in this experimental system.


Prophylactic antibiotics in gynecologic surgery.

A 2-year prospective double-blind study of prophylactic antibiotics in 317 patients undergoing elective total abdominal or vaginal hysterectomy was conducted. Patients randomly received placebo, penicillin, or cefazolin 30 minutes prior to surgery and at 6-hour intervals thereafter for 48 hours. Rigid criteria for postoperative morbidity were established. Vaginal hysterectomy patients given either penicillin or cefazolin prophylaxis had fewer postoperative infections (P less than 0.01) compared to those given placebo. A similar trend was noted among women undergoing abdominal hysterectomy; however, this trend was not statistically significant (P greater than 0.05). Despite continuous surveillance, no change in nosocomial flora or antibiotic sensitivity of bacterial isolates was noted. Adverse drug effects and antibiotic-resistant secondary infections were encountered with similar frequency in all treatment groups.


Therapeutic trial of the combination in an eye lotion of adrenaline (0.5 or 1%) and guanethidine (3 or 5%) in cases of open-angle glaucoma.

Treatment of open-angle glaucoma has always been problematical, either because of the lack of efficacy, the inconvenience of multiple medication (handled by the patient with difficulty), or due to the high incidence of side effects. This study showed that an association of adrenaline and guanethidine in drop form, used in cases of open-angle glaucoma, achieved a rapid and lasting stabilisation of intraocular pressure. The main advantage of this form is the combination of two active ingredients for instillation twice daily. In addition, the pH and the nature and concentration of the ingredients are such that side effects, if they occur, do so at the beginning of treatment and are of short duration.


Method of production of and antibody response to an oil adjuvant Bacteroides nodosus bacterin.

A procedure is described for the production of a highly concentrated Bacteroides nodosus bacterin. An oil adjuvant bacterin which elicited the best antibody response in rabbits produced a response in sheep which lasted for at least 6 months.


Eikenella corrodens: a new pathogen.

Oral infections that result from trauma or dental manipulation are often thought to be of minor importance to the practicing clinician. The majority of oral infections respond well to treathis form of simple therapy is no longer as efficacious as it has been though to be. Soment with the common antibiotics without the need for laboratory culture and sensitivity. There are instances, becoming increasingly frequent, when this form of simple therapy is no longer as efficacious as it has been thought to be. Some exotic infections are frequently recalcitrant to treatment. These infections are usually caused by ubiquitious organisms, such as Eikenella corrodens. We are presenting several of our cases that were caused by this organism, as well as a review of some aspects of its microbiology, in order to attract the attention and interest of our colleagues in dental practice to this increasingly frequent problem.


Evaluation of sedative/analgesic combination for postoperative pain.

Current pain concepts suggest a dual role in pain perception. Both motivational, effective, and somatosensory components are noted. Pain affects the patient's psyche and, conversely, the patient's state of mind modifies pain perception. Consequently, it might be expected that a sedative/analgesic combination might be more effective than either the sedative or the analgesic alone in controlling pain. A clinical study was carried out to compare a promethazine-A.P.C. combination with promethazine and A.P.C. alone. One hundred forty-nine patients undergoing third molar removal were studied, with each patient serving as his own control. The results indicated a significant difference between the promethazine-A.P.C. combination and prometazine alone or Phenergan alone. No significant difference was noted between the promethazine-A.P.C. combination and A.P.C. alone.


Muscular form of glycogenosis type II (Pompe's disease).

An 11-year-old boy who was previously thought to have progressive muscular dystrophy was studied clinically, biochemically, and histologically. He was seen initially with an amyotonic syndrome with no clinical evidence of heart disease. Light and histochemical examination showed vacuolar degeneration and abnormal accumulation of glycogen in the muscular fibers. Electron microscopy showed aggregates of glycogen granules surrounded by a well-defined membrane, as in previously reported cases of type II glycogenosis. Enzymatic study disclosed that acid alpha-glucosidase was deficient in muscle, liver, and heart tissue, although neutral alpha-glucosidase was present within normal ranges. Measurement of acid and neutral alpha-glucosidase activity in muscle from the patient and his sisters and in urine from them and their parents indicated that his sisters are heterozygotes and his parents probably are heterozygotes. The disease was transmitted as an autosomal-recessive trait.


Preferential distribution of lobar emphysema and atelectasis in bronchopulmonary dysplasia.

The chest roentgenograms of 142 neonates who survived mechanical ventilation for respiratory distress syndrome (N = 99) and prolonged apnea (N = 43) were reviewed. Thirty-seven infants had bronchopulmonary dysplasia (BPD) and 17 of these developed lobar hyperinflation of the right lower lobe and collapse of the right upper lobe. Regional lung function was measured with a xenon 133 technique in three of these infants and in five other patients who either died or were lost to follow-up. All had BPD with right lower lobe overinflation. Ventilation was less in the lower regions than the upper regions bilaterally (P less than .001), indicating that the hyperinflation of the lower lobes was not compensatory for upper lobe collapse but was due to emphysema. Mean regional perfusion was equal in the upper and lower regions of the chest. This preferential distribution of lobaremphysema and ipsilateral atelectasis in BPD tended to present and regress simultaneously, but in many infants it lasted as long as eight weeks. Only one infant with persistent atelectasis developed pneumonia. The best mode of therapy appears to be supportive.


Successful treatment of juvenile chronic granulocytic leukemia with marrow transplantation.

A 46-month-old boy with juvenile chronic granulocytic leukemia was treated intensively with hydroxyurea, dimethyl myleran, cyclophosphamide, and total body irradiation. He then received a marrow transplant from an HL-A matched brother. Thirty-two months after the transplantation, he is hematologically normal and remains disease free on no-maintenance therapy. The successful outcome of this case suggests that a bone marrow transplant for any patient with a suitable histocompatible donor should be considered in the treatment of this disease.


Risk of respiratory distress syndrome after prenatal dexamethasone treatment.

A prospective double-blind randomized clinical trial was carried out to determine whether glucocorticoid treatment reduces the risk of respiratory distress syndrome (RDS) in prematurely born infants. There were 127 infants born to 122 mothers who received either steroid (dexamethasone phosphate) or placebo. No differences between groups occurred in risk factors for RDS (eg, prepartum asphyxia, male sex, cesarean section). When those who received a full course of dexamethasone therapy were compared with those who received placebo, a significant reduction was found in risk, severity, and deaths due to RDS. An increased incidence of infection in mothers treated with steroid was evident, particularly after premature rupture of membranes. We conclude that steroids are effective in reducing risk of RDS, but safer and more efficacious approaches for the prevention of RDS should be sought.


The antenatal use of betamethasone in the prevention of respiratory distress syndrome: a controlled double-blind study.

One hundred forty-six pregnant women were enrolled in a prospective double-blind study to assess the effectiveness and side-effects of antenatal administration of betamethasone in the prevention of respiratory distress syndrome (RDS) in potentially premature infants. On admission to the study, the women were given, at random, either 12 mg of betamethasone or placebo. The same dose was repeated 24 hours later and then weekly up to 34 weeks of gestation. Gestational age of the infants ranged from 25 to 34 weeks, and birth weights ranged between 730 and 2,650 gm. Statistically significant differences in favor of the infants in the betamethasone group were found in the incidence of RDS, 20.7% in the betamethasone group compared with 59.5% in the control group (P less than .005); in the severity of RDS (P less than .05); and in the death rate (P less than .05). A higher incidence of hypoglycemia was found among infants in the betamethasone group (P less than .05). Prolonged rupture of the membranes played no protective role against RDS, and the incidence of infection was similar in both groups.


The chemiluminescence response and bactericidal activity of polymorphonuclear neutrophils from newborns and their mothers.

Chemiluminescence (CL) was measured in the polymorphonuclear neutrophils (PMN) of 18 normal term infants, their mothers, and controls during phagocytosis of opsonized zymosan particles. Chemiluminescence was significantly lower in the PMN of newborns in comparison with the PMN of their mothers and of the controls. Depressed bactericidal activity was demonstrated in newborn PMN, in comparison with the activity of the PMN of their mothers and controls, when challenged with Escherichia coli at large bacteria-PMN ratios. Uptake of radio-labeled bacteria by PMN was identical in newborns, mothers, and controls, which indicates that reduced CL was not a result of impaired ingestion. Therefore, PMN of normal term infants have both depressed oxidative metabolic responsiveness as measured by CL and depressed bactericidal capacity.


Localization and therapy of urinary tract infections of childhood.

One hundred four patients with 124 episodes of urinary tract infection were studied. Serum C-reactive protein (CRP) was determined on diagnosis of each patient. Children with a CRP equal to or greater than 30 micrograms/ml (CRP-pos) differed significantly from those with values less than 30 micrograms/ml (CRP-neg) in age, clinical presentation, K type of Escherichia coli causing disease, frequency or radiographic abnormalities, and presence of antibody coating of bacteria in the urinary sediment. E. coli K1 strains caused disease significantly more often in CRP-pos than in CRP-neg patients, and children with K1 infections were younger than those with non-K1 infections. The antibody-coated bacteria test was neither sensitive nor specific for localization of infection in pediatric patients. Determination of K1 antibody concentrations in serum and urine of E. coli K1-infected children provided data supporting the measurement of CRP as one means of localizing urinary tract infections. Patients with CRP-neg infections were treated as successfully with four days of antimicrobial therapy as with ten days.


Shigella vaginitis: report of 38 patients and review of the literature.

Thirty-eight cases of Shigella vaginitis were identified in a retrospective review of records kept over the past 14 years. The cases of vaginitis were due to three subgroups of Shigella. Vaginitis varied in severity and duration, persisting for several months in some instances. In 47% of the cases, there was associated bloody vaginal discharge. Only two children had diarrhea temporally associated with vaginitis, but six others had had diarrhea. Many treatment modalities were used. Systemic antibiotic therapy appeared more effective than topical antimicrobials in the few patients who could be evaluated. Review of the literature revealed 32 additional cases, including four in adult women.


Effect of heparinization of fluids infused through an umbilical artery catheter on catheter patency and frequency of complications.

Heparinization of fluids (1 unit/ml) infused through an umbilical artery catheter (UAC) was efficacious in prolonging catheter patency in a double-blind, randomized, controlled clinical study. On the basis of life-table analysis, the half-life of catheter function was seven days in the heparinized group as compared with just over two days in the nonheparinized group (P less than .01). UAC occlusion occurred in 4 of 32 patients in the heparinized and 19 of 30 in the nonheparinized group (chi 2 = 17.6, P less than .01). Blood transfusions, number of arterial blood gases drawn through the UACs, and fluid infusion rates were not related to catheter occlusion. Heparinization of the UAC infusion did not alter the partial thromboplastin time or the incidence of catheter-related thromboembolic phenomena in the extremities. Heparinization of fluids infused through a UAC appears to be useful in the care of critically ill neonates.


Immunofluorescence in group B streptococcal infection and idiopathic respiratory distress syndrome.

Immunofluorescence was performed on lung tissue obtained at necropsy from 18 newborn infants, including five with group B streptococcal (GBS) sepsis, seven with idiopathic respiratory distress syndrome (IRDS), and six control infants who died from other causes. Deposits of C3, IgG, and fibrin were found within hyaline membranes of infants who died with GBS sepsis or IRDS within 48 hours after birth. In some cases C4, factor B, and IgM were also observed. In five infants with IRDS who died more than five days after birth, immunofluorescent lung findings were less common and less intense. Hyaline membranes, attributed to mechanical ventilators and oxygen therapy in two infants who did not have GBS infection or IRDS, were negative for complement and immunoglobulins although fibrin was detected in one specimen. These data suggest that immunologic processes may contribute to the pathogenesis of certain types of acute lung injury, particularly in infants who die from GBS infection or IRDS during the early neonatal period.


Effect of storage processes on the bacterial growth-inhibiting activity of human breast milk.

Human breast milk samples were collected from lactating mothers, and aliquots were maintained at room temperature, frozen, and pasteurized. Samples were inoculated with 10 to 50 colony-forming units per milliliter (cfu/ml) of Escherichia coli or group B streptococcus, and incubated at 37 C. Quantitative growth was measured at eight and 24 hours. No inhibitory activity was demonstrated by control broth, commercial formula, and pasteurized breast milk, which had a rapid logarithmic growth to a maximum of 10(8) to 10(9) cfu/ml at 24 hours. Compared with these controls, fresh breast milk, fresh frozen breast milk, and breast milk frozen for 21 days demonstrated a significant inhibition of bacteria growth. A trend toward gradual loss of inhibiting activity was noted with prolonged freezing of breast milk. Although freezing may quantitativly decrease the amount of some breast milk host-defense factors, it cannot be assumed that comparable functional reductions will necessarily result.


[Cis-diammino-dichloro-platinum therapy of cancers; phase II therapeutic trial].

We have conducted a phase II trial of cisdiammino-dichloro platinum (CDDP) which demonstrates its remarkable activity in testis embryonic carcinoma, in ovary carcinoma and in epidermoid cancers, especially head and neck and uterus cervix carcinoma. Its toxicity in mainly digestive and renal. This compound is now indicated in combinations in the case of the above mentioned tumors.


[Production of O antigens and antibodies during urinary tract infections due to Escherichia coli (author's transl)].

In 138 adults with monomicrobial E. coli urinary tract infections, IgG coated bacteriuria (ACB) are found in 9/10 acute pyelonephritis, 21/24 chronic pyelonephritis, 2/5 acute prostatitis, and in only 6/99 lower UTI. These urinary antibodies are synthetised localy because IgA-S are found in 75%, in the kidney because humoral IgG antibodies are detected in only 40% of the ACB + patients. O6, O18, O22, O75, are the four most frequent O antigens (47,5%) and no difference were found in the distribution of O serotypes according to ACB production or clinical signs. But rough strains are significantly more frequent in pyelonephritis, suggesting a modification of the bacterial wall. The use of ACB test and O serotyping allows a better follow-up of patients, but relapses with ACB- and reinfection with ACB+ show the complexity of the relation host-E. coli.


[Physiopathological and anatomical classification of small bowel diseases (author's transl)].

The first small bowel diseases have been known for three centuries, but essential breakthroughs have occured in the last twenty years. A new classification may be proposed according to, the physiopathology, the anatomo-pathology, the etiology. From the physiopathological point of view one may separate enterocytic, pre-enterocytic or post-enterocytic malabsorptions. Small bowel injuries described by the pathologist include small bowel resections, villous atrophy, lesion of the lamina propria, or transmural lesions, the vascular pathology and the ulceration of the small bowel. A new syndrome is proposed: the rupture of the cellular continuity or intercellular syndrome.


[Morphology and deformability of erythrocytes in muscular dystrophy].

In recent years, the presence of red cell morphological abnormalities in patients with Muscular Dystrophy has made the object of numerous, often contradictory reports. A possible source of such confusion may lie in the fact that human erythrocytes are extremely sensitive to morphologic transformations resulting from various manipulations or environmental conditions in vitro. We have examined the morphology and deformability of erythrocytes from 7 patients with Duchenne and 9 patients with Steinert (myotonic) Muscular Dystrophy. To avoid preparation artifacts, fresh, unwashed red cells suspended in their own plasma were examined under phase contrast microscopy for the presence of either echinocytes and stomatocytes. Deformability was measured by filtration of dilute cell suspensions at constant flow rate through nucleopore membranes (nominal pore diameter = 3 micrometer). No significant difference was found between the patients' cells and those of 22 healthy volunteer controls. We conclude that previously reported abnormalities may have been the result of preparation artifacts. It appears possible, however, that erythrocytes from Muscular Dystrophy patients may be more sensitive than normal ones to certain stimuli originating from red cell manipulations in vitro.


Addition of oligonucleotides to the 5'-terminus of DNA by T4 RNA ligase.

Bacteriophage T4-induced RNA ligase catalyzes the controlled template-independent addition of RNA to the 5'-phosphoryl end of large DNA molecules. Restriction enzyme-generated fragments of Co1E1 DNA with completely basepaired or cohesive ends and linear single-stranded øX174 viral DNA were all good substrates. DNA molecules from 10 to 6000 nucleotides long were quantitatively joined in an hour to a number of different RNA homopolymers. The most efficient of these was A(pA)5; I(pI)5 and C(pC)5 were also utilized while U(pU)5 was not. The optimum ribohomopolymer length was six nucleotides. Joining of ribohomopolymers between 10 and 20 nucleotides long occurred at approximately 1/2 the maximal rate and a trimer was the shortest substrate. Thus RNA ligase provides a method for generating extensions of predetermined length and base composition at the 5'-end of large DNA molecules that complements the available procedures for extending the 3'-hydroxyl terminus of DNA.


2'-Deoxy-2'-fluorouridine-5'-triphosphates: a possible substrate for E. coli RNA polymerase.

dUflTP was tested as substrate in the E. coli RNA polymerase system using poly(dAT) as template. dUflTP could replace UTP when Mn++ was utilized as divalent cation instead of Mg++. The level of transcription with the fluoro analog was then 55% of that with UTP.


Nucleotide sequence of the transposable DNA-element IS2.

The complete sequence of the transposable DNA element IS2 in gal OP-308:: IS2 (I) has been determined. This element is 1.327 bp long. The integrated element is flanked by a five base pair long sequence duplication. The termini of IS2 are not perfect inverted repeats, but a close approximation.


In vitro methylation of yeast tRNAAsp by rat brain cortical tRNA-(adenine-1) methyltransferase.

Rat brain cortices from young animals contain large amounts of tRNA (adenine-1)methyltransferase(s). The enzyme(s) can methylate E. coli tRNA and to a lower degree yeast tRNA. Among yeast tRNA species which can be methylated we have selected tRNAAsp as a substrate for the brain enzyme. The digestions of in vitro methylated [Me-3H]-tRNAAsp with pancreatic and/or T1 ribonucleases followed by chromatographies on DEAE-cellulose, 7 M urea, suggested that the methylation of tRNAAsp occurred at a single position within the D-loop. Further digestion of the radioactive oligonucleotide recovered after DEAE-cellulose chromatography by phosphomonoesterase and snake venom phosphodiesterase enzymes followed by bidimensional thin layer chromatography enabled us to determine the location of the adenine residue which becomes methylated by the brain enzyme. This one resulted to be the adenine 14 in the D-loop of yeast tRNAAsp.


Photosensitized formation of thymine dimers in DNA by tyramine, tyrosine and tyrosine-containing peptides.

The formation of Thy-Thy in DNA in the presence of tyramine, tyrosine and tyrosine-containing peptides such as Lys-Tyr and Lys-Tyr-Lys was studied with monochromatic UV irradiation. The formation of Thy-Thy by UV irradiation was enhanced in the presence of these compounds. The action spectrum of the photosensitization has a peak near 280 nm corresponding to the absorption spectrum of tyrosine. The triplet quencher reduced the sensitization substantially. The sensitization in native DNA was more than six times larger than that in denatured DNA. increasing the concentration of salts suppressed the sensitization. The nature of the interaction between DNA and the sensitizer is discussed.


Cloning, mapping and expression of the genetic determinant that encodes for the K88ab antigen.

The K88 antigen, a plasmid-specified virulence factor of E. coli involved in porcine neonatal diarrhoea, is often found to be associated with the ability to metabolize raffinose (Raf). Plasmid pRI8801 (51 megadalton) was used to clone the determinants of K88 and Raf with the vector pBR322. K88 was found to be encoded by a 7.7 megadalton HindIII fragment. The expression was highly dependent on the orientation of the HindIII fragment within pBR322. By in vitro generation of deletions, the HindIII fragment was reduced in size to 4.3 megadalton. The expression of K88 by pRI8801 and the recombinant plasmids was studied using an enzyme-linked immunosorbent assay. Raf was found to be located on a 4.0 megadalton SalI fragment. A physical map of pRI8801 was constructed. The K88 antigen and Raf genes are not closely linked but separated by a stretch of DNA of about 20 megadalton.


Nucleotide sequence of the cro-cII-oop region of bacteriophage 434 DNA.

The nucleotide sequence of a 869 bp segment of phage 434 DNA including the regulatory genes cro and cII is presented and compared with the corresponding part of the phage lambda DNA sequence. The 434 cro protein as deduced from the DNA sequence is a highly basic protein of 71 amino acid residues with a calculated molecular weight of 8089. While the cro gene sequences of phage 434 and lambda DNA are very different, the nuleotide sequences to the right of the lambda imm434 boundary show differences only at 11 out of 512 positions. Nucleotide substitutions in the cII gene occur with one exception in the third positions of the respective codons and only one out of several DNA regulatory signals located in this region of the phage genomes is affected by these nucleotide substitutions.


Chemical modification study of aminoacyl-tRNA conformation.

Chemical reactivity of cytosines in 32P-labeled E. coli tRNA1Leu, E. coli tRNAPhe and yeast tRNAPhe before and after aminoacylation was examined by use of a cytosine-specific reagent, semicarbazide-bisulfite mixture. In all the three tRNA species examined, the cytosine residues that were susceptible to the modification were the same in the aminoacylated tRNA and the unacylated tRNA. Only a limited number of the cytosine residues were modifiable: those that occur in the anticodon, the 3'-CCA terminus, the D-loop, and the extra loop. The sites accessible by the reagent are in good agreement with the general three-dimensional structure of tRNA proposed in literature. These results indicate that the gross conformation of these tRNAs does not change on aminoacylation, and consequently favor the view that the T psi C(G) sequence could become exposed in later steps of protein synthesis in order to achieve the binding of aminoacyl tRNA to ribosomes.


Construction and analysis of recombinant DNAs containing a structural gene for rat prolactin.

Poly A-containing RNA enriched in prolactin-coding sequences was isolated from female rate pituitaries after induction with diethylstilbesterol. Double stranded cDNA was synthesized from this RNA and inserted into plasmid pBR322 at the Pst I site via the poly(dG):polyy(dC) tailing method. E. coli was transformed with this DNA and the recombinant plasmid in one of the transformants characterized in detail. About half of its 900 base pair cDNA insert was sequenced. The DNA sequence is consistent with most of the reported amino acid sequence of rat preprolactin. In addition, the recombinant plasmids in two of the other transformants appear to contain growth hormone coding sequences.


Antibacterial properties of sodium nalidixate against avian Salmonellae in liquid and on solid media.

The in vitro inhibitory effect of sodium nalidixate (NaNA) for 206 avian salmonella cultures of varying serotypes was established in both liquid and on solid media. Salmonella cultures were much more resistant to the lethal effects of NaNA in veal infusion broth than on a solid medium such as MacConkey agar. The inhibitory effects of NaNA varied in liquid media, depending on the nutritive support afforded by the medium. Growth-suppressive effects were enhanced by less nutritive media. Reasons are suggested as to the possible differences in the inhibitory effect of NaNA on avian salmonella cultures grown in liquid and on solid media.


Preparation and study of a fluorescent sugar analog competitive inhibitor of yeast hexokinase.

The preparation of 2-deoxy-2-amino-N-(5-dimethylamino-1-naphthalene sulfonyl)-glucose (III) designed as a fluorescent competitive inhibitor of hexokinase was achieved after reacting 2-deoxy-2-aminoglucose and 1-dimethylamino-5-naphthalene sulfonyl chloride. (III) showed fluorescence excitation and emission maxima in water at 330 and 507 nm, respectively. (III) was found to competitively inhibit hexokinase and a value of Ki = 3.0 x 10(-3)M was obtained for the system hexokinase B + Mg.ATP + glucose at pH 8.4.


Isolation of Trypanosoma cruzi from the blood of infected mice by column chromatography.

Trypomastogotes of three strains of Trypanosoma cruzi were isolated from the blood of infected mice employing lymphoprep for separation of the red blood cells and a column of DEAE cellulose for removal of white cells and platelets. An average recovery of 45 to 58 percent of actively motile, infective organisms, free of contaminating blood cells was obtained. Protein and carbohydrate assays of the separated organisms revealed significant differences between the Tulahuen, a reticulotropic strain, and the House 510 and House 11, two myotropic strains of this parasitic species. The present procedure should provide sufficient parasites for physiological and biochemical studies; it has also served to indicate particular strain characteristics which may aid in a taxonomic classification of these organisms.


High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules.

A set of vector DNAs (Y vectors) useful for the cloning of DNA fragments in Saccharomyces cerevisiae (yeast) and in Escherichia coli are characterized. With these vectors, three modes of yeast transformation are defined. (i) Vectors containing yeast chromosomal DNA sequences (YIp1, YIp5) transform yeast cells at low frequency (1--10 colonies per microgram) and integrate into the genome by homologous recombination; this recombination is reversible. (ii) Hybrids containing endogenous yeast plasmid DNA sequences (YEp2, YEp6) transform yeast cells at much higher frequency (5000--20,000 colonies per microgram). Such molecules replicate autonomously with an average copy number of 5--10 covalently closed circles per yeast cell and also replicate as a chromosomally integrated structure. This DNA may be physically isolated in intact form from either yeast or E. coli and used to transform either organism at high frequency. (iii) Vectors containing a 1.4-kilobase yeast DNA fragment that includes the centromere linked trp1 gene (YRp7) transform yeast with an efficiency of 500--5000 colonies per microgram; such molecules behave as minichromosomes because they replicate autonomously but do not integrate into the genome. The uses of Y vectors for the following genetic manipulations in yeast are discussed: isolation of genes; construction of haploid strains that are merodiploid for a particular DNA sequence; and directed alterations of the yeast genome. General methods for the selection and the analysis of these events are presented.


Interaction of Escherichia coli ribosomal protein S1 with ribosomes.

The binding affinity of Escherichia coli ribosomal protein S1 for 30S ribosomal particles has been determined by a sucrose gradient band sedimentation technique; the association constant (K) for the binding of one S1 protein per active 30S ribosomal subunit is approximately 2 X 10(8) M-1. The involvement of the two polynucleotide binding sites of S1 protein (site I binding single-stranded DNA or RNA, and site II binding single-stranded RNA only) in the S1--ribosomal interaction have been examined by competition experiments with polynucleotides of known affinity for the two sites. We find that site I does not contribute to the interaction; site II binding appears to provide a major part of the binding free energy, presumably by interaction of S1 with the 16S rRNA of the 30S particle. The remaining binding free energy is probably derived from the interaction of S1 protein with other proteins of the 30S subunit. The affinity of S1 for 70S ribosomes is about the same as that for the 30S subunit; the affinity of S1 for 50S subunits is much less. Binding affinities and stoichiometries of S1 protein with "inactive" 30S ribosomal subunits have also been examined.


Localization of the decoding region on the 30S Escherichia coli ribosomal subunit by affinity immunoelectron microscopy.

The decoding region of the Escherichia coli ribosome has been localized by affinity immunoelectron microscopy. Valyl-tRNA1Val, derivatized at the alpha-amino end with the dinitrophenyl group, was bound to the ribosomal P site of 70S ribosomes and crosslinked specifically to 16S RNA by 310- to 325-nm irradiation. Previous work has shown that crosslink occurs between the 5' anticodon base of the tRNA and a pyrimidine in the 3' one-third of the 16S RNA. By reaction of the dinitrophenyl group with antibody, dimers of the 30S tRNA covalent complexes were prepared containing one covalently attached tRNA per 30S subunit. Electron microscopic visualization of the antibody attached to the dinitrophenyl group located the position of the 3' end of the tRNA. Several sites, with a strong preference for the large protrusion or cleft region in the upper one-third of the subunit, were found. The multiplicity of sites is likely due to the freedom of orientation of the 3' end of the tRNA which is approximately 80 A from the site of crosslinking. By extrapolating this distance from each of the antigenic sites, we concluded that the anticodon of tRNA when in the P site is probably located in the cleft region of the 30S subunit.


Biochemical assay designed to detect formation of recombination intermediates in vitro.

A biochemical assay that is designed to detect recombination intermediates formed in vitro is described. The assay measures the fusion of two essentially homologous plasmids, one of which is radioactively labeled and the other of which carries several copies of the lac operator. The fusion product is radioactive and can be bound to a nitrocellulose filter by lac repressor. This assay for genome fusion is rapid and readily applicable to the many fractions that result during enzyme purification. The fused product is not destroyed in the assay and may be recovered from the filter for further analysis by electron microscopy. The product is then seen to consist of figure 8 structures that can be cleaved by the restriction enzyme EcoRI to give chi forms, structures similar to those recovered from recombination-proficient cells. It is expected that this assay will be useful in the purification of the "recombinase-type" activity detected in crude cell lysates. To demonstrate this point, the assay was applied to the protein fractions recovered from a molecular sieve column. The results indicate that the fusion activity has an apparent molecular weight of 50,000--100,000.


Enzymatic insertion of purine bases into depurinated DNA in vitro.

An enzymatic activity that inserts purines into depurinated DNA was found in a soluble enzyme extract of Escherichia coli. This activity brings about the insertion of adenine and guanine into the appropriate apurinic sites in double-stranded DNA by using the corresponding deoxyribonucleoside triphosphates as the purine donors. Magnesium ions are required for this activity, it is inhibited by caffeine, and it does not act on depurinated single-stranded DNA. The insertion activity described here may represent a step in a repair mechanism, "base-insertion repair," whereby apurinic sites (which may occur in double-stranded DNA either due to the removal of damaged purines with specific glycosylases or by spontaneous depurination) are directly filled with the correct missing purine base.


Mutations reducing the activity of c17, a promoter of phage lambda formed by a tandem duplication.

We report the isolation of four independently selected mutations (scs) in the c17 promoter of phage lambda that reduce or eliminate the promoter activity. The c17 promoter is not normally present in lambda, and has been shown to be generated by a tandem duplication which creates a "Pribnow Box," a heptamer sequence implicated in promoter activity. This sequence is located upstream from the site of transcription initiation and is present, with some variation, in all promoters whose sequences have been determined. Analysis of the c17 duplications carrying the scs mutations reveals that three of these mutants carry single base-pair changes in the most highly conserved base pairs of the Pribnow Box and that the other mutation is a reversion to the wild type sequence in this region (i.e., a loss of the duplicated base pairs).


Nucleotide sequence of the region of an origin of replication of the antibiotic resistance plasmid R6K.

A 2.1-kilobase segment of the antibiotic resistance plasmid R6K carries sufficient information to replicate as a plasmid in Escherichia coli. This segment contains a functional origin of replication and a structural gene for a protein, designated pi, that is required for the initiation of R6K replication. The nucleotide sequence of a 520-base-pair portion of this 2.1-kilobase segment that includes the functional origin of replication and the region adjacent to the start of the pi structural gene was determined. A striking feature of the sequence is the presence of seven 22-base-pair direct repeats joined in tandem in the region adjacent to the start of the pi gene. A possible role of the tandem repeats in the regulation of expression of the pi protein and the control of initiation of replication of the plasmid R6K is discussed.


Bacteriophage f1 infection of Escherichia coli: identification and possible processing of f1-specific mRNAs in vivo.

[3H]Uracil-pulse-labeled RNA from Escherichia coli infected with f1 bacteriophage was fractionated on polyacrylamide gels containing urea. Eight phage-specific RNA species were present with approximate lengths ranging from 2100 to 400 nucleotides. The amount of the seven largest species was increased when the infected bacteria were incubated at 41 degrees C. When the RNA was isolated and used as message in an in vitro protein-synthesizing system, most of the RNA species appeared to direct the synthesis of the phage gene VIII protein. The six largest species also directed the synthesis of the phage gene V protein. Some of the labeled smaller RNA species increased in amount after addition to rifampicin, suggesting that they may have resulted from cleavage of larger RNA species. These particular smaller RNA species also were present in infected bacteria containing a mutant RNase III. The data are discussed in terms of the regulation of synthesis of the phage-specific proteins.


Soluble precursor of an integral membrane protein: synthesis of procoat protein in Escherichia coli infected with bacteriophage M13.

Prior to virus assembly, the major coat protein of coliphage M13 is an integral protein of the host cytoplasmic membrane. Coat protein synthesized in vitro is initially made with an NH2-terminal "leader peptide" of 23 amino acids and is termed "procoat." We now report that procoat is a biosynthetic precursor of coat protein in vivo. Conversion of procoat to coat occurs within 30 sec in cells infected with wild-type virus. This proteolytic processing is delayed in cells infected by M13 mutants (in genes 1, 5, or 7) that are defective in virus assembly. Pulse--chase experiments in combination with subcellular fractionation show that procoat is synthesized in a soluble form in the cytoplasm and is then incorporated into the cytoplasmic membrane, where it is converted to coat protein. This finding is supported by the observation that procoat is synthesized exclusively by polysomes that are not membrane bound. These results are interpreted in terms of the "membrane-triggered folding" hypothesis of membrane protein assembly.


Requirements of acetyl phosphate for the binding protein-dependent transport systems in Escherichia coli.

In Escherichia coli, acetyl phosphate can be formed from acetyl-CoA via the phosphotransacetylase (phosphate acetyltransferase; acetyl-CoA:orthophosphate acetyltransferase, EC 2.3.1.8) reaction and from acetate (plus ATP) via the acetate kinase (ATP:acetate phosphotransferase, EC 2.7.2.1) reaction. By restricting acetyl phosphate formation to the phosphotransacetylase reaction alone, through the use of metabolic inhibitors, we were able to show that, with pyruvate as a source of energy, mutants defective in phosphotransacetylase are unable to transport glutamine, histidine, and methionine. However, with the same energy source, mutants defective in acetate kinase are normal in the transport of these amino acids. The inability of the phosphotransacetylase mutants to transport is due to their presumed inability to form acetyl phosphate, because pyruvate is found to be metabolized to acetyl-CoA in these mutants. Thus acetyl phosphate has been implicated in active transport. Evidence is also presented that neither the protonmotive force nor the ecf gene product is required for the shock-sensitive transport systems.


Replication in vitro of the 2-micrometer DNA plasmid of yeast.

Cell-free extracts prepared from growing cells of the budding yeast Saccharomyces stimulated DNA synthesis directed by the supercoiled 2-micrometer yeast DNA plasmid. The major products of the reaction were open-circular daughter molecules possessing newly synthesized full-length linear DNA strands. Some of these were ligated and supertwisted by the extracts to yield a supercoiled DNA product. Both of the complementary DNA strands of the template were replicated. In addition, the extracts induced the appearance of theta-forms of the plasmid DNA, which are presumed to be replicative intermediates. The results of experiments utilizing BrdUTP incorporation indicated that DNA repair did not contribute significantly to the overall reaction. Extracts prepared from the cell division cycle mutants cdc7 and cdc8, held in culture at the nonpermissive temperature, possessed diminished activity. Because these mutants define a dependent sequence of events leading from the start of the cell cycle through G1 to S phase, this result suggests that the activity that stimulates 2-micrometer DNA replication in vitro is subject to control in the yeast cell cycle.


Membrane biogenesis: cotranslational integration of the bacteriophage f1 coat protein into an Escherichia coli membrane fraction.

The coat protein (CP) of bacteriophage f1 is integrated into an Escherichia coli plasma membrane fraction consisting of inverted vesicles when it is synthesized in a cell-free, coupled transcription--translation system supplemented with the inverted vesicles. By using proteolytic enzymes as probes, we found by subsequent peptide mapping and determination of the sequence of the proteolytic products that CP was inserted into the inverted vesicles in an orientation indistinguishable from that in inverted vesicles prepared from infected E. coli: only a COOH-terminal portion of approximately 10 residues was accessible to proteolysis, whereas the remainder of CP (CP') was entirely protected. Protection of CP' was dependent on the integrity of the vesicle membrane, because it was abolished when proteolysis was done in the presence of nonionic detergents. Insertion was observed when the inverted vesicles were present during translation in the cell-free system, not when they were added after translation. Thus, the asymmetric insertion of this type of integral membrane protein is strictly coupled to translation. These findings are discussed with respect to prokaryotic membrane biogenesis and are related to bacteriophage f1 assembly and infection.


Differentiation of a rat mammary cell line in vitro.

We have studied the development of fusiform (probably related to myoepithelial) cells in Rama 25 cultures [Bennett, D. C. Peachey, L. A., Durbin, H. & Rudland, P. S. (1978) Cell 15, 283--298]; we show that they are generated from special differentiated structures (projections) that contain a rapidly differentiating cell type (F-precursor cells). Clonal sublines isolated from projections develop in several directions under both environmental and genetic control. Some types of differentiation are reversible; others are irreversible. The various cell occurring in vitro may correspond to specific cell types in vivo.


Simultaneous localization of type II collagen and core protein of chondroitin sulfate proteoglycan in individual chondrocytes.

In order to investigate the coordinated synthesis of matrix components by individual chondrocytes, specific antibodies to type I collagen, type II collagen, and chondroitin sulfate proteoglycan core protein were used in simultaneous double immunofluorescence reactions. Extensive accumulation of core protein surrounding chondrocytes and the intracellular accumulation of type II collagen were observed. Extracellular core protein immunofluorescence obscured the intracellular reaction product, but the extracellular immunoreactive material could be removed by digestion with purified testicular hyaluronidase prior to fixation. Subsequent to digestion, core protein and type II collagen were observed in the same chondrocytes within discrete, sometimes identical, cytoplasmic regions, thus demonstrating the simultaneous localization of these two products characteristic of differentiating cartilage.


Human melanoma cells have both nerve growth factor and nerve growth factor-specific receptors on their cell surfaces.

Human melanoma cells were examined in an indirect membrane immunofluorescence assay for surface nerve growth factor (NGF) and NGF receptors. This assay revealed that human melanoma cells have various levels of NGF and NGF receptors on the plasma membrane, whereas a variety of human sarcoma and carcinoma tumor cells and normal human fibroblasts are negative. Surface NGF could be detected on melanoma cells with a rabbit antiserum directed to NGF at titers as high as 1:64; prior adsorption of this antibody with mouse 2.5S NGF resulted in a loss of fluorescence. The melanoma cells were positive whether or not they were grown in the presence of fetal calf serum. NGF production by human melanomas is a previously unrecognized property of this differentiated cell type. Although other cells in culture have been shown to produce NGF, the association of NGF production with the presence of NGF receptors on the cell surface is rare among tumor cells, and may represent an opportunity for "autostimulation" of melanoma cells by this growth factor.


Aflatoxin B1 mutagenesis, DNA binding, and adduct formation in Salmonella typhimurium.

Salmonella typhimurium strain TM677 was mutagenized with aflatoxin B1 (AFB1) in liquid suspension culture in the presence of a rat liver postmitochondrial supernatant. Forward mutation to 8-azaguanine resistance was measured in the treated cultures and was found to increase linearly with AFB1 concentration. DNA purified from mutagenized cells was analyzed for AFB1 adduct formation by high-pressure liquid chromatography after adduct liberation. AFB1 exposures at 0.16 and 0.32 micrometer for 35 min produced 15 and 22 AFB1--DNA adducts per genome, respectively, and induced 8-azaguanine-resistant fractions of 4.9 X 10(-4) and 9.6 X 10(-4). Approximately 70% of the AFB1 bound to DNA was chromatographically identical to 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 at the two AFB1 levels used.


Strand exchange in site-specific recombination.

The site-specific recombination system of phage lambda promotes crossovers at its attachment site (att). In this report we show that when phage are crossed in conditions where only the site-specific recombination system is active, a low frequency of crossovers can also be detected in a region that is close to but does not contain att. These crossovers require the phage int gene, the host hip gene, and the integrity of att. They are not detected if one of the parents carries a substitution of a heterologous attachment site (attB instead of attP). To explain these findings we suggest that site-specific recombination can proceed by exchange of single strands between the participating chromosomes at att and migration of the resulting junction outside of att.


Bradykinin-like immunoreactive neuronal systems localized histochemically in rat brain.

Bradykinin-like immunoreactive structures were localized in rat brain by the indirect immunofluorescence method. Specificity of staining was demonstrated by: (i) the absence of fluorescence when preimmune serum was used, (ii) the disappearance of fluorescence when sera were preadsorbed with bradykinin, and (iii) the presence of identical staining with two different antisera. Immunoreactive neuronal cells are observed only in the hypothalamus, with especially dense clusters overlying the periventricular and dorsomedial nuclei. Fibers and varicose processes are observed in the periaqueductal gray matter, hypothalamus, perirhinal and cingulate cortices, the ventral portion of caudate-putamen, and the lateral septal area.


Myelin-associated glycoprotein demonstrated immunocytochemically in myelin and myelin-forming cells of developing rat.

The unlabeled peroxidase-antiperoxidase method has been used with antiserum against "myelin-associated glycoprotein" to establish the presence of the glycoprotein in myelin and myelin-forming cells of the developing rat nervous system. Myelin-associated glycoprotein is found in oligodendroglial cytoplasm before the beginning of myelination. Staining intensity of oligodendroglia increases during early development and slowly declines during the period of rapid myelination. Myelin staining is confined to the periaxonal region of the myelin sheath and does not increase as large, compact sheaths are formed. Antiserum to central nervous system myelin-associated glycoprotein also stains Schwann cells in developing trigeminal ganglia and the periaxonal region of peripheral myelin sheaths.