Biological and physicochemical characteristics of four serotypes of Salmonella enteritidis.

Four serotypes of Salmonella enteritidis, Anatum ATCC 9270, Newbrunswick ATCC 1608, Oranienburg 200 E, and Pullorum RM, were studied to determine biological, chemical, or physical differences which might explain variations in Salmonella virulence as previously reported by McCullough and Eisele (J. Infect. Dis. 88:278-289, 1951; 89:259-265, 1951). These investigators found that serotype Pullorum was significantly less virulent than serotypes Newport, Derby, Barielly, Meleagridis and Anatum when fed to healthy humans. Results of our own experiments showed that serotype Pullorum RM had a generation time approximately twice that of serotype Anatum 9270. The volume of serotype Pullorum was approximately one-half the volume of the other serotypes used (Anatum 9270, Newbrunswick 1608, Oranienburg 200 E, Cubana 12007, and Meleagridis DR). The number of cells required to yield 1 g dry weight was substantially higher for serotype Pullorum RM than for serotypes Anatum 9270, Newbrunswick 1608, and Oranienburg 200 E. The yield of endotoxin per gram dry weight for serotype Pullorum RM averaged 22 mg/g, whereas yields of endotoxin for serotypes Anatum 9270, Newbrunswick 1608, and Oranienburg 200 E averaged 32 to 35 mg/g. The relative abundance of the four major fatty acids (measured by gas chromatography) also showed distinct differences among the serotypes. Pullorum RM contained less lauric and 3-hydroxymyristic acids and more myristic and palmitic acids than the other three serotypes. The identity of 3-hydroxymyristate was confirmed by mass spectroscopy. Serotype Pullorum RM required 10 times more lipopolysaccharides (endotoxin) to obtain a 50% lethal dose in mice than the other three serotypes. When the lipid part was separated from the polysaccharide and solubilized with bovine serum, the 50% lethal dose of serotype Pullorum RM was equal to that of the other three.


Binding of glucosyltransferase and glucan synthesis by Streptococcus mutans and other bacteria.

Lyophilized and heat-treated cells from the seven serotypes of Streptococcus mutans were examined for their ability to bind added insoluble-product glucosyl-transferase (GTase) and to synthesize cell-associated glucan from [(14)C]sucrose. Lyophilized cells of serotypes a and g did not synthesize any more additional glucan than did the controls after exposure to GTase. These cells, however, synthesized four- to eightfold-greater quantities of glucan than did the cells of the remaining serotypes. Lyophilized cells of serotypes b, c, d, e, and f synthesized two- to threefold-greater quantities of glucan after exposure to GTase than did the controls without added enzyme. Lyophilized cells of serotypes a and g synthesized 6- to 10-fold-greater quantities of glucan than did heat-treated cells of the same strain after binding of GTase. Lyophilized cells of the remaining serotypes synthesized only 1.6- to 3.3-fold-greater quantities of glucan than did the heat-treated cells. These results demonstrate that heat treatment to inactivate cell-associated GTase does not create additional GTase binding sites in S. mutans and that serotypes a and g are considerably more active in cell-associated glucan synthesis than cells of the other five serotypes. Ten species of gram-positive and gram-negative bacteria from five genera which do not produce in vitro plaque synthesized 10- to 100-fold-less glucan than did the S. mutans strains after exposure to GTase. Of these species, S. sanguis, Actinomyces viscosus, and A. naeslundii synthesized the largest quantities of glucan. Three mutant strains of S. mutans which possess a reduced ability for in vitro adherence but do agglutinate with glucan or dextran synthesized only one-third as much glucan after binding of GTase as the control. These results are discussed in relation to in vitro and in vivo plaque development and the agglutination of S. mutans. The results support earlier findings which indicate that the presence of bacterial species other than S. mutans in smooth-surface dental plaque is due in part to contact of the cells with glucan in the developing plaque and not to the binding of cell-free GTase and the in situ synthesis of glucan. The results obtained with these representative strains of the seven serotypes of S. mutans may not apply to the same extent to other strains within the serotypes.


Escherichia coli pili as possible mediators of attachment to human urinary tract epithelial cells.

Presence of pili of fimbriae on Escherichia coli bacteria isolated from the urine of patients with urinary tract infection was related to the ability of the bacteria to attach to human uroepithelial cells. Piliated E. coli strains agglutinated guinea pig erythrocytes. D-Mannose and alpha-methyl-D-mannopyranoside inhibited this agglutination with all but one of the 12 strains tested. D-Mannose, D-galactose, alpha-methyl-D-mannopyranoside, and L-fucose did not afect attachment of piliated strains to uroepithelial cells. Heating as well as washing of piliated strains caused a parallel decrease of piliation and adhesive ability. Growth in glucose-enriched medium increased capsule formation but decreased piliation and adhesion. Capsulated strains retained their adhesive ability provided that pili extended outside the capsule.


Immunization of suckling pigs against enteric enterotoxigenic Escherichia coli infection by vaccinating dams with purified pili.

Pregnant swine (gilts) were vaccinated parenterally with a suspension of purified pili from the porcine enterotoxigenic Escherichia coli strain 987 (09:K103::NM). Gilts injected with placebo served as controls. Suckling pigs born to gilts in both groups were challenged intragastrically with virulent strain 987. The percentage of deaths, incidence and duration of diarrhea, numbers of E. coli in the ilea, and E. coli attachment to the villous epithelia were significantly less in suckling pigs of vaccinated gilts than in those of controls. These results are consistent with the hypothesis that pili of some enterotoxigenic E. coli facilitate adhesion to intestinal epithelia. Vaccination of dams with pili appears to be a means of immunizing against diarrheal disease caused by enterotoxigenic E. coli in suckling neonates. This work confirms the role of somatic pili as colonization and virulence factors and provides another example of safe and effective purified pilus vaccines.


Immunofluorescence and electron microscopy of the attachment of Mycoplasma synoviae to chicken embryo fibroblasts.

The techniques of immunofluorescence and of transmission and scanning electron microscopy were used to demonstrate the attachment of Mycoplasma synoviae to chicken embryo fibroblasts. Although the organisms colonized the surface of many of the cells, there was marked variation in the number of organisms associated with individual cells.


Growth of salmonellae in orally infected germfree mice.

Germfree mice were infected intragastrically, intravenously, or intraperitoneally with 10(3) to 10(9) viable Salmonella typhi Ty2, S. gallinarum 9240, or S. enteritidis 5694. The 50% lethal doses were compared with those for conventionally raised mice. Substantial growth of the salmonellae occurred in the intestinal tract of the germfree mice but, despite the presence of more than 10(9) viable S. typhi or S. gallinarum in the lumen, the liver and spleen cultures remained Salmonella-free, and all of the mice survived the oral challenge. The ileal and cecal Peyer's patches and the mesenteric lymph nodes of these mice contained 10(3) to 10(4) viable salmonellae within 24 h of introduction of the inoculum into the stomach. Despite this local involvement, the infection did not spread systemically even when host resistance was reduced by means of sublethal, whole-body gamma irradiation before oral challenge. Germfree mice infected orally with as few as 10 mouse-virulent S. enteritidis quickly developed severe diarrhea and died within 5 to 8 days as a result of a spreading systemic disease.


Immunoglobulin G and immunoglobulin M enzyme-linked immunosorbent assays and defined toxoplasmosis serological patterns.

Enzyme-linked immunosorbent assay (ELISA) for toxoplasmosis was evaluated in serum samples presenting defined toxoplasmosis serological patterns, as determined by results in immunoglobulin (Ig)G and IgM immunofluorescence (IgG-IF, IgM-IF), hemagglutination, and complement fixation tests. ELISA was carried out with alkaline phosphatase-labeled anti-IgG and anti-IgM antibodies. Serum titer was expressed as the serum dilution end point determined by mere observation of color development in the test. A straight agreement was found between IgG-ELISA and IgG-IF titers, both in group A sera of ancient infections (patterns II and III) and group B sera of recent infections (pattern I). A similar agreement was found between IgG-ELISA and hemagglutination titers in group A sera, for which coincident IgG-IF and hemagglutination titers are also frequent. However, in group B sera, in spite of the same toxoplasma extract being used to sensitize both plastic surfaces and erythrocytes, IgG-ELISA titers were much higher than hemagglutination titers, in a way similar to that observed for IgG-IF titers. IgM-ELISA was positive in every group B serum, with higher titers than corresponding IgM-IF titers. Occasional low-titered positive IgM-ELISA results were seen for group A sera, sometimes due to IgM-antiglobulin antibodies. An easy test to perform, ELISA seems to be an adequate substitute for toxoplasmosis IF tests for routine purposes.


Intraintestinal toxin in infant mice challenged intragastrically with Clostridium botulinum spores.

Conventionally raised suckling mice were injected intragastrically with 10(5) spores of a Clostridium botulinum type A culture. Botulism was not observed, but 80% or more of mice challenged when 8 to 11 days old had botulinum toxin in the large intestine 3 days later. Mice younger than 7 days or older than 15 days were resistant to the challenge. When in vivo toxin production was started by spores given to 9-day-old mice, toxin was present in the intestine at 1 through 7 days postchallenge but with greatest consistency between 1 and 4 days. Total toxin in an intestine ranged up to 1,920 50% lethal doses as titrated intraperitoneally in adult mice. The dose infecting 50% of a group of 9-day-old mice was 700 (95% confidence limits of 170 to 3,000) spores per animal. Toxin was formed in the lumen of the large intestine; it was not associated with the ileum. Injection of 10(5) spores intraperitoneally into 9-day-old mice resulted in toxin production in the large intestines of 30% of the test animals.


Adherence of Candida albicans to human buccal epithelial cells.

The adherence of Candida albicans to human buccal epithelial cells after 2 h at 37 degrees C was significantly greater in human saliva than in phosphate-buffered saline. in saliva, viable fungi adhered much better than did nonviable fungi, and this adherence was greater at 37 than at 25 degrees C. Viable yeasts, preincubated in saliva for 90 min at 37 degrees C before being washed and mixed with epithelial cells in phosphate-buffered saline, adhered better than nonviable yeasts or yeasts preincubated in phosphate-buffered saline. Enhanced adherence in saliva appeared to be associated with germination of the yeast cells. Conditions permitting germination (growth in tissue culture medium 199 at 37 degrees C but not at 25 degrees C) also supported enhanced adherence. After germination had occurred, the fungi could be killed with Formalin without interfering with their rapid and efficient adherence to epithelial cells. These data indicate that the enhanced adherence of C. albicans observed after incubation in saliva is related to changes in the fungi, rather than to a requirement for prolonged interaction between fungi and epithelial cells.


Immunotherapy of a guinea pig hepatoma with mycobacterial vaccines: comparison of BCG cell walls and cell wall skeletons.

BCG cell wall skeletons (SK) derived from BCG cell walls (CW) by treatment with proteolytic enzymes and organic solvents were tested for their potency to cause regression of a transplanted guinea pig hepatoma. On a weight basic, SK were as effective as CW in causing tumor regression, and they, as well as purified protein derivative of mycobacteria, provoked delayed cutaneous hypersensitivity reactions in animals immunized with CW or with SK. On a weight basis, CW were more active than SK in eliciting delayed cutaneous hypersensitivity in sensitized guinea pigs whether the animals were immunized with CW or with SK. In unimmunized animals the inflammatory response to intradermally administered CW was greater than that evoked by SK. CW and SK provoked delayed cutaneous hypersensitivity reactions of similar strength in animals immunized with living BCG. This study provided no compelling reasons for using SK instead of CW in clinical trials of cancer treatment by mycobacterial vaccines.


Immunological cross-reactivity between a heat-labile enterotoxin(s) of Escherichia coli and subunits of Vibrio cholerae enterotoxin.

Heat-labile enterotoxin from Escherichia coli is immunologically related to both subunits, A and B, of cholera enterotoxin as demonstrated by neutralization and immunodiffusion.


Opposite effects of BCG on spleen and lymph node cells: lymphocyte proliferation and immunoglobulin synthesis.

C57BL/6 mice were immunized intravenously (i.v.), intraperitoneally (i.p.), or subcutaneously with one dose of Bacillus Calmette-Guérin (BCG). At various time intervals after injection, the lymphocyte response, as measured by thymidine incorporation into DNA, and the number of immunoglobulin-secreting cells were determined in vitro before and after mitogenic stimulation with phytohemagglutinin, concanavalin A, or lipopolysaccharide. In unstimulated cultures, the spontaneous thymidine incorporation and immunoglobulin synthesis of spleen cells were increased to some extent in mice infected i.p. or i.v. with BCG, as compared with noninfected mice. In contrast, after mitogenic stimulation, a marked depression of the proliferative response of spleen cells to both T- and B-cell mitogens and a marked inhibition of LPS-induced immunoglobulin secretion were observed in mice infected i.v. and to a lesser extent in those infected i.p. The depression of lymphoblastogenesis in spleens was fully established 15 days after infection and persisted for a long period of time. When unfractionated or plastic-adherent spleen cells from BCG-infected mice were cultured with normal spleen cells, a strong depression of their reactivity to phytohemagglutinin, concanavalin A, and lipopolysaccharide was observed. After the removal of cells adherent to plastic, the response was partially restored in the nonadherent population from mice infected i.p., but not in that from mice infected i.v. After mitogenic stimulation, lymph node cells of mice inoculated subcutaneously showed a response to mitogen higher than that of normal cells. These results thus demonstrate that, depending on the route of administration, BCG exerts very different effects.


Phagocytosis of Cryptococcus neoformans by normal and thioglycolate-activated macrophages.

Phagocytosis of Cryptococcus neoformans by normal and thioglycolate-activated mouse peritoneal macrophages was studied. Thioglycolate-activated macrophages exhibited a lower percent phagocytosis than did normal macrophages. Differences in phagocytosis could not be attributed to differences in macrophage viability, minor variations in the concentration of adherent macrophages, or a general depression in activated macrophage phagocytosis. Thioglycolate-activated macrophages required heat-labile opsonins for optimal phagocytosis of non-encapsulated cryptococci, whereas nonactivated macrophages did not require heat-labile opsonins for phagocytosis of the yeast. Both types of macrophages exhibited similar sensitivity to the phagocytosis-inhibiting properties of cryptococcal polysaccharide. The results show that depletion of heat-labile opsonins from serum or inactivation of yeast-bound, heat-labile opsonins by polysaccharide cannot account for the phagocytosis-inhibiting properties of cryptococcal polysaccharide.


Association of elevated levels of extracellular neuraminidase with clinical isolates of type III group B streptococci.

The level of total extracellular neuraminidase produced by 74 clinical isolates of group B streptococci isolated from diseased or asymptomatically colonized infants was assayed. Extracellular neuraminidase was obtained from concentrated filtrates of exponentially growing cultures of group B streptococci grown in a chemically defined medium (FMC) containing supplemental protein. The total activity of extracellular enzyme produced by these clinical isolates ranged from less than 10 to 360 nmol of sialic acid released per min per mg of cell dry weight. Strains were arbitrarily classified as either nonproducers (less than 10 nmol/min per mg of cell dry weight), low producers (greater than 10 to less than or equal 140 nmol/min per mg of cell dry weight), or high producers (greater than 140 to 360 nmol/min per mg of cell dry weight). Type III isolates from diseased infants were significantly more often classified as high producers than strains of group B streptococci of other serotypes from diseased infants (P less than  0.001). Furthermore, the serotype III strains isolated from neonatal infections were more often high producers than those of the same serotype from asymptomatically colonized infants (P less than 0.025). These results suggest that the ability to produce elevated levels of neuraminidase may be related to the frequent association of type III strains with disease among neonates.


Group A streptococcal pyrogenic exotoxin: pyrogenicity, alteration of blood-brain barrier, and separation of sites for pyrogenicity and enhancement of lethal endotoxin shock.

Group A streptococcal pyrogenic exotoxin type C (SPE C) was shown to produce fever by crossing the blood-brain barrier. The toxin directly stimulated the hypothalamic fever response control center, thus bypassing a requirement for endogenous pyrogen release. SPE C was detected in the cerebrospinal fluids of toxin-treated rabbits by pyrogen tests and a hemagglutination inhibition assay. The toxin altered the permeability of the blood-brain barrier to endotoxin, Streptococcus pneumoniae, and Haemophilus influenzae as well as to itself. SPE C did not alter the in vivo differential and total counts of peripheral blood leukocytes and did not elicit endogenous pyrogen release from leukocytes in vitro. In vivo, peripheral blood platelet counts remained unchanged after SPE treatment. Cycloheximide pretreatment of rabbits did not inhibit fever production by SP C. In contrast to the hypothermia observed in mice treated with endotoxin intravenously susceptibility to lethal endotoxin shock. The abilities of SPE C to produce fever and enhance lethal shock were shown to be separate functions of the molecule; fever results from stimulation of the hypothalamus, and enhancement appears not to involve the central nervous system.


Immunological relationship of different preparations of coliform enterotoxins.

Antisera raised in rabbits to ultrafiltrate toxin preparations containing either the heat-labile (LT) toxin form obtained from whole cell lysates or broth filtrates or the heat-stable (ST) toxin form prepared from broth filtrates from nontoxigenic and toxigenic strains of Escherichia coli and Klebsiella were examined for their ability to neutralize the secretory effect on water transport of these toxins in the rat jejunum as determined by the in vivo marker perfusion technique. Antisera to the heat-labile toxin derived from whole cell lysate preparations from nontoxigenic strains had no neutralizing effect. Antisera to both types of LT preparation from both toxigenic strains neutralized, with several exceptions, all of the homologous and heterologous LT toxins as well as a heat-labile toxin preparation derived from sequential ultrafiltration of cell-free whole cell lysates which had a defined molecular weight of between 30,000 and 100,000. These antisera also neutralized homologous and heterologous ST preparations obtained from broth filtrates, but they had no neutralizing effect on low-molecular-weight, ST toxin material obtained during the sequential ultrafiltration of cell lysates. Antisera to ST prepared from broth filtrates had no neutralizing capacity against either LT or ST toxin preparations. These observations (i) indicate that the immunological relationship of E. coli and Klebsiella LT and ST toxins extends to antisera raised against LT prepared by several different methods, (ii) raise the possibility that, based on the response to antisera to LT, there may be several immunologically heterogeneous forms of low-molecular-weight ST toxin, and (c) confirm the lack of immunogenicity of ST.


Surface structures of Escherichia coli that produce diarrhea by a variety of enteropathic mechanisms.

Strains of Escherichia coli, mostly of human origin, were obtained from several different investigators who had isolated them from patients with diarrhea from many different parts of the world. The mechanisms by which these E. coli were thought to have caused diarrhea included: (i) synthesis of labile, stable, or Shigella dysenteriae-like enterotoxins; (ii) invasion of the intestinal mucosa; and (iii) unknown. Each strain was carefully examined for pili or flagella to correlate the presence or absence of such surface structures with a particular mechanism of diarrhea. The presence of pili was determined by colony morphology on minimal media, pellicle formation in static broth culture, and transmission electron microscopy. The pili were categorized as type 1 if the bacteria fermented rhamnose and if pellicle formation was inhibited by alpha-methyl-d-mannoside. The presence of flagella was confirmed in motility media and by transmission electron microscopy. Six invasive E. coli strains lacked pili and flagella. Ten E. coli strains that synthesized enterotoxins or produced diarrhea by an unknown mechanism were piliated (seven with type 1 pili), and all but one had flagella. Pili and flagella seem to be associated with strains of E. coli that produce diarrhea by enterotoxin synthesis or unknown mechanisms. Strains that produce diarrhea by mucosal invasion lack both types of surface structures.


Delayed-type hypersensitivity to rabies virus in mice: assay of active or passive sensitization by the footpad test.

With a purified beta-propiolactone-inactivated rabies virus, a significant increase in footpad swelling was elicited in normal or in BCG-pretreated mice after immunization with varying doses of rabies vaccine. These footpad reactions were shown to peak at 24 h and to be associated with an infiltration of newly formed blood monocytes demonstrated by histology and [125I]deoxyuridine labeling. A relationship between the lymphoproliferation and the degree of sensitization is described, and the susceptibility to cyclophosphamide treatment is also examined. Adoptive transfer of specific reactivity to normal recipient mice with immune lymphoid cells, but not with immune serum, was demonstrated, and the results represent another argument for a cell-mediated immunological mechanism.


Characterization of group A streptococcal T-12 protein purified by ion-exchange column chromatography.

The aim of the present study was to describe the physicochemical characteristics of streptococcal T antigen. T protein isolated from Streptococcus pyogenes type 12 (R53/1077, Colindale) and purified by ion-exchange column chromatography resulted in a preparation that was homogeneous when tested electrophoretically (in two systems, in presence and in absence of sodium dodecyl sulfate) and by gel filtration on Sephadex G-100. The purified T antigen was resistant to enzymatic degradation by trypsin and pepsin. It formed a single precipitin line with standard T-12 antiserum and was not contaminated with group A carbohydrate and M protein. The molecular weight of protein T, determined by means of polyacrylamide gel electrophoresis and calculated from its amino acid composition, was about 39,000. The molecular weight of this protein, determined by means of high-speed sedimentation equilibrium, ranged between 80,000 and 120,000. Glutamic and asparatic acids, lysine, alanine, and leucine were the predominant amino acids.


Adhesive bonding of various materials to hard tooth tissues--solubility of dentinal smear layer in dilute acid buffers.

While the acid etch technique has largely solved the problem of bonding restorative materials to enamel, attempts to improve adhesive bonding to dentin have so far had only limited success. One aspect to which insufficient attention has been paid is the presence of a smear layer on cut surfaces and of pellicle on surfaces exposed to saliva, both of which would reduce or prevent durable adhesive bonds. The present study attempts to evaluate the usefulness of various acidic solutions in removing these surface layers while causing minimal damage to the vital contents of the dentinal tubules. A series of twelve 0.16 M buffer solutions were applied to cut dentin surfaces which were then washed, dried and sputter-coated with gold/palladium in preparation for examination by scanning electron microscopy. It is concluded that the smear layer on cut dentin surfaces can be substantially removed in vitro by a 30 second exposure to an isotonic solution of buffered monobasic acids having pKa values between 3.8 and 2.5. Since these solutions did not always remove material from grooves in the prepared surface clinical techniques should aim at producing a smooth cut surface. The response of the vital pulp to this treatment still awaits investigation.


Is there a role for serotonin (5-hydroxytryptamine) in feeding?

A review of evidence indicates that experimentally induced changes in the activity of serotonin (5-hydroxytryptamine) systems are associated with pronounced changes in feeding behaviour. In general, treatments and procedures believed to lead to an increased availability of 5-HT in the synaptic cleft or which directly activate 5-HT receptors reduce food consumption, while procedures which either directly or indirectly decrease 5-HT receptor activation bring about the opposite effect. Interpretation of findings is hindered by methodological difficulties involved in the experimental manipulation of serotonin metabolism, by the lack of precise behavioural measures of feeding, and by the presence of large stores of serotonin outside the brain. However, available data favour the idea that serotonin systems play an inhibitory role in feeding, possibly in the mediation of satiety. This proposal has implications for further experimental investigations of the control of food intake, and for the aetiology and treatment of obesity.


Distribution of fenfluramine in normal and obese mice.

Concentrations of fenfluramine and its metabolite, norfenfluramine, were measured in blood, brain and parametrial adipose tissues 15, 30, 60 and 120 min after intraperitoneal (i.p.) administration of fenfluramine hydrochloride (10 mg/kg) to normal and gold-thioglucose obese mice. Concentrations of fenfluramine were higher in the brain of obese mice than in normal mice, while there was no difference in norfenfluramine levels in the two groups. This difference may be related to higher blood levels of fenfluramine but not of norfenfluramine in obese animals. The drug was presented in about the same concentrations in the adipose tissue of the two groups. These results may explain previous findings that fenfluramine was more effective as an anorectic agent in obese than in lean mice.


Conformation of disulfide bridges in peptides with pepsin partial sequences.

On the basis of Raman spectra investigation of two model heterodetic cyclic peptides, containing partial sequences of pepsin fragments 45--50 and 206--210 of the chain, it was concluded that the disulfide bridge conformation in pepsin is determined not only by the size and conformation of the peptide loops created by disulfide bridges, but also by the peptide fragments located outside these loops.


On the nature of damage involved in liquid-holding recovery in diploid yeast after gamma- and alpha-irradiation.

Cells surviving after liquid-holding recovery following gamma- and alpha-irradiations are found to be slightly more sensitive to a second series of radiation doses. Further, the shoulder on the gamma survival curve of the pre-irradiated and liquid-held cells disappears. The shoulder and sensitivity are restored only when these cells are grown in broth before the second series of doses. In addition to this, liquid-holding recovery reduces progressively if the cells after irradiation are incubated in broth for different periods of time before holding. These observations suggest that: (1) the so-called potentially lethal damage may consitute that part of the sub-lethal damage which interact with one another to form lethal damage; (2) during liquid-holding, the interaction among sub-lethal damage transforming them to the status of lethal damage is inhibited; (3) the 'recovered' cells are saturated with sub-lethal damage, the repair of which will be completed only when the cells are placed in a nutrient medium. The inhibitory process is not a passive one, but requires energy metabolism.


Medical and surgical treatment of the dry eye.

An overview of the dry eye syndrome and its medical and surgical management has been presented. It is apparent that any single treatment is generally ineffective and that a combination of therapeutic modalities is required for success. The Experimental Slow Release Artificial Tear Insert (Ocusert) shows the most promise for a future solution.


Reflex control of the circulation by afferents from skeletal muscle.

Functionally, the pressor reflex, rather than the depressor reflex, appears to be the normal physiologic response to exercise. Available data indicate that chemoreceptors, probably in the form of free nerve endings, are stimulated by local factors, e.g., hypoxia or hyperkalemia. Impulses from these receptors are mediated by group IV afferent nerves to the central nervous system. Then, through efferent sympathetic and parasympathetic nerves, systemic blood pressure, cardiac output, heart rate, and myocardial contractility all increase. The baroreceptors tend to modify the reflex increase in heart rate. The potential significance of local control of the systemic circulation by neural reflexes from skeletal muscle is apparent when one considers that during exercise local control allows both rapid and precise control of the circulation from the metabolically active region of the body. However, the exact role of neural reflexes from skeletal muscle during voluntary exercise still remains to be defined. Clearly, more studies are necessary to elucidate fully their function in the overall control of the circulation in conscious animals and man.


The effect of intramural injection of immunotherapeutic agents on bladder histology and systemic humoral response.

Bacillus Calmette-Guerín (BCG), Corynebacterium parvum, and various mycobacterium fragments were injected intramurally into mouse bladders and the resulting systemic humoral stimulation and local histologic reactions were evaluated. C. parvum and methanol extracted residue of BCG elicited significant immunostimulation without producing irreversible bladder damage. The severe histologic disruption caused by BCG could be reduced by the antituberculus drug isoniazed without abrogating the humoral stimulation. Intramural bladder injection of BCG produced greater humoral stimulation than subcutaneous administration.


Autoantibodies to acrosomal antigens of spermatozoa in vasectomized mice.

A total of 102 mice from seven different inbred strains were vasectomized or sham vasectomized. Sera from these animals were collected before vasectomy and at 7 and 10 months after the operation. Indirect immunofluorescence on sperm smears showed that 26 per cent of vasectomized mice had antibodies to arosomal antigens of spermatozoa. This response was restricted to three of seven strains. None of the sera obtained from animals before vasectomy or from sham-vasectomized control mice contained similar antibodies.


Effect of immunization of bacterial adherence to urothelium.

In order for a urinary tract infection to begin, the pathogenic bacteria must first adhere to the urothelial surface. An in vivo method for quantitation of bacterial adherence to urothelium has been developed. Immunization by instillation of bacterial antigens into the rat bladder decreased bacterial adherence. Bladder immunization was more effective than subcutaneous immunization. Preinfection by Escherichia coli decreased subsequent adherence of Klebsiella pneumoniae. Increasing antiadherence factors may offer a new approach to the treatment of recurrent urinary tract infections.


Chronic pyelonephritis. Electron microscopic study. II. Persistence of variant bacterial forms.

Ascending nonobstructive pyelonephritis was produced in nonhuman primates by ureteral catheterization which delivered Escherichia coli (04:H1) to the renal pelvis while creating intrarenal reflux. Female rhesus monkeys (Macaca mulatta) were immunosuppressed by cyclophosphamide before infection and weekly thereafter and compared to those with infection only. Kidney tissue was examined by electron microscopy in an effort to compare the development of the infection in the two groups of monkeys, i.e., immunocompetent and immunosuppressed. Reorganization of bacterial cytoplasm into small dense bodies (averaging 250 A in diameter) was seen in two of the suppressed animals. These particles were within bacteria that were either free in the medullary interstitium or in macrophages. Clusters of electron-dense bodies of the same size and morphology were also seen within subendothelial spaces of glomerular capillaries. Protoplast-like forms were observed within the medullary interstitium. One cell wall-less form contained particles (as previously described) within a large peripheral vesical. Gross pyelonephritic scarring occurred in all immunosuppressed animals. This study has shown morphologically that classical bacterial organisms placed into the intact kidneys of partially immunoincompetent nonhuman primates will cause pyelonephritis and continue to exist for 18 days. These observations of the futile efforts by suppressed populations of leukocytes to clear intrarenal bacteria raise interesting questions about the host-paradise relationship in chronic renal infection.


Quantitative determination of prostatic epithelial and stromal hyperplasia by a new technique. Biomorphometrics.

Recent studies of neonatal and pathologic growth of the prostate have postulated the possible importance of stromal-epithelial interaction in the control of prostatic growth. To allow further analysis of the possible importance of this interaction, a new technique has been developed that permits quantification of the total number of stromal and epithelial cells in the prostate gland. This technique has been termed biomorphometrics and is a combination of standard morphometric and biochemical methodology. This method has been applied to analyze the hormonal response of the stromal and epithelial cells of the rat ventral prostate.


The multicomponent genome of a different cytoplasmic polyhedrosis virus isolated from Heliothis armigera.

The genome of a new cytoplasmic polyhedrosis virus (CPV) isolated from Heliothis armigera has been shown to consist of 10 double-stranded RNA segments, as in other CPVs, but the genome profile on PAGE bears no resemblance to previously described CPVs isolated from H.armigera.


[Skin incisions in inguinal lymph-node excision].

Wound complications in the postoperative period of the inguinal lymph node dissection are frequent events. The main factor in wound complications is due to dissection of the vascular architecture. In most of the cases the impaired wound healing occurs distal to Poupart's ligament. Suggestion for its prevention is the use of an oblique, subinquinal, wide spindle-S-shaped skin excision crossing over Scarpa's triangle with an extension to the anterior superior iliac spine from its lateral and a vertical prolongation onto the thigh from its medial end. The wide spindle-S-shaped excision minimizes the later devascularisation of skin flaps. The anatomic outline of the inguinal lymphatic basin to be removed can easily be reached by the extend of these two incision lines. Besides an undisturbed healing the technique offers two further possibilities. If malignant skin tumors are in anatomical positions which lend themselves to incontinuity dissection, removing primary tumor, intervening lymphatics and regional lymph nodes en bloc can be combined with the incision lines. Furthermore the excised groin skin can be used for a free flap to cover the excision wound of the primary tumor. The method is described in ten cases.


[Reconstructive surgery of the thorax and breast].

Various possibilities of breast plasty after tumor surgery or for the correction of combined anomalies of the thorax and the breasts are discussed. In the augmentive procedures either local flaps or mammary prostheses as well as silicone rubber were used. The authors show a personal method where the remaining healthy breast is used for reconstruction.


[Mediastinoscopy or thoracotomy? A prospective randomized study].

A prospective, controlled, randomized trial on the value of mediastinoscopy was performed during a 12-month period (1969-1970) on 130 patients with a bronchogenic carcinoma. Patients to be admitted to the study had to meet the criteria of being clinically operable. 126 patients could be followed up for the following 5 years. Of these patients 62 had a mediastinoscopy previous to the thoracotomy (Group A) and 64 had directly a thoracotomy (Group B). A curable resection was possible in Group A in 56.4% and in 73.5% in Group B. The perioperative mortality (30 days) is 8% for Group A and 16% for Group B. The mean survival rate is for patients with a positive mediastinoscopy which did not underwent thoracotomy with 6.8 months better than for patients which underwent directly an exploratory thoracotomy (2.7 months). The 5-year survival rate is despite the high perioperative mortality for Group B (16%)higher than for Group A(10%). Mediastinoscopy is with its low mortality and its better short surviving rate of great value for high risk patients and inoperable patients with bronchogenic carcinomas, but reduces the overall resectability and the 5-year survival rate.


[Breast wound reconstruction with plastic mesh].

A combined network plasty is presented which allows covering of thoracic wall defects without difficulties. The technique consists in closing the defect with a nylon netting in order to reestablish stability, and in air-tight covering of this netting with the greater omentum. This wound is covered after 10 to 14 days with split skin grafts. The method was applied to 7 patients with local recurrences of a breast cancer, two of them infected. The follow-up after 4 to 13 months reveals well-healed wounds in every case. The stomach is not altered, neither clinicallly nor in X-ray controls.


[Swiss physicians and their contribution to surgery of the hand].

The origins of five important Swiss contributions to plastic and hand surgery since the last third of the 19th century are described: free grafting of the epidermis (J.L. Reverdin), a pollicization (C. Roux), de Quervains's stenosing tendovaginitis, Secrétan's oedema and the first voluntary movable artificial hand (S. Stodola and F. Sauerbruch). These contributions are related to the development of general surgery. Their history illustrates the advantages which may result from prolonged contact and exchange between members of different cultures.


[Surgical and apparative treatment of patients with meningomyelocele with special reference to their hip problems].

Children with thoracic lesions should have hip surgery done only if this is necessary for the child to be able to walk with braces. In lumbar lesions hip surgery and especially SHARRARD'S procedure of iliopsoas transfer is done to establish muscular balance. The amount of braces for walking can thus be reduced. In sacral lesions there is no place for hip surgery, as functional gain would not be expected.


[Treatment of knee joint deformities in spina bifida].

182 cases of surgical treatment of knee deformities in children with spina bifida between 1968 and 1977 are reviewed. The most number of cases had very good or good success. 138 supracondylar osteotomies produced very good results in 18 cases; reosteotomy was necessary in 10 cases. EGGERs procedure in 26 cases made very good results and good results in 6 cases. Quadriceps-Z-plasty with anterior capsulotomy produced very good results in 11 cases, only one bad result here was produced by skin-breakdown.


[Effect of n-dipropyl-acetate on the elimination of urinary amino acids].

N-dipropyl-acetate (Depakine) induces a hyperglycinuria and simultaneously an increase of urinary excretion of alanine and a decrease of serine, glutamate and/or glutamine, histidine and/or ornithine. The mechanism of this side effect of n-dipropyl-acetate is discussed.


Light and electron microscopic studies of intramitochondrial bodies in bovine adrenocortical cells by proteolytic digestion.

The nature of the intramitochondrial bodies in bovine adrenocortical cells was investigated both light and electron microscopically, by applying enzymatic digestion on paraffin and epon sections. The result that these bodies were extracted completely either by pepsin or by trypsin strengthened the validity of the previous conclusion that their nature is proteinaceous.


The size of the fibre populations in rabbit skeletal muscles as revealed by indirect immunofluorescence with anti-myosin sera.

Antibodies against myosin of the fast long. Dorsi and the slow soleus muscle of rabbits were induced in guinea pigs. With the aid of a new technique, the gel-electrophoresis-derived-enzyme-linked-immunosorbent assay (GEDELISA) it could be shown that they are directed against the heavy and the light chains of fast (M. long. dorsi) and slow (M. soleus) myosin. In the indirect immunofluorescence test each antiserum only stained one population of fibres in five different muscles tested. The single fibres were observed to react only with one of the two types of antisera. The following percentage of fibres showed a positive reaction with the anti-fast myosin serum: M. long. dorsi, 95%; M. psoa maior, 95%, M. psoa minor, 92%; M. tibialis ant., 90%; M. soleus, 15%.


The histochemical demonstration of sialic acid residues in pancreatic islets.

Using a modified colloidal iron reaction in connection with neuraminidase extraction test 3 different sialic acid-containing components have been demonstrated in pancreatic islets comprising golgi region and glycocalyx layer of islet cells and intrainsular capillary walls. The colloidal iron positive cationophilia increased markedly after treatment with alkali; an effect which might be due to deesterification, thus exposing additional free carbonyl groups of sialic acid residues.


Action of heparin on protein fractions of isolated nuclei and on their DNA content.

Isolated nuclei of rat hepatocytes were incubated with 0.05% sodium heparinate for 2 to 10 min. Alterations in the nuclei were controlled biochemically, determining the amounts of DNA and histones, and by cytophotometric methods determining the amounts of total and nonhistone proteins and DNA. Under the selected experimental conditions 95% of histones are bound already after 5-min incubation with heparin; nonhistone proteins of cell nuclei remain unchanged. The blockade of histones is followed by DNA diffusion into the incubation medium. Experiments with nuclear staining with alcian blue proved the specificity of heparin binding with histones and showed that heparin-histone complex remains in the nuclei, and its histones lose their extractability with 0.25 n HCl.


Volume, flow, and timing of each breath during positive-pressure breathing in man.

This study is a breath-by-breath analysis of the effects of 5, 10, and 15 cmH2O positive-pressure breathing (PPB) on man's steady-state breathing pattern. Inspiratory (TI), expiratory (TE), and cycle (TT) durations, tidal volumes (VT), minute ventilation (VE), mean inspiratory flow rate (VT/TI), and mean expiratory flow rate (VT/TE) were determined from pneumotachograph and Wedge spirometer recordings before and during steady states on PPB. End-tidal CO2 was continuously recorded. Seventeen adults, seated in a full body-box, breathed quietly for 8 min through a mouthpiece on a bag-in-box. Pressure in the body-box was lowered to the desired level prior to 4 min of stress. On all pressure levels, end-expiratory volume, VT, VE, VT/TI, and VT/TE increased; end-tidal CO2, TE, and TT decreased with no consistent change in TI. Calculated alveolar ventilations indicated that the increases in VE were true hyperventilations. Each individual increased VE by using a unique combination of VT, TI, and TE. End-expiratory volume increased less and expiratory flow increased more than would occur passively. Hence, it is concluded that active reflexes account for the resistance of the systems to the passive distention, the facilitation of expiratory flow, and the shortening of TE.


The inactivation of gentamicin and netilmicin by carbenicillin: its effect on Serratia marcescens.

Ten clinical isolates of Serratia marcescens were tested on Mueller-Hinton agar containing gentamicin or netilmicin with carbenicillin. The isolates grew on plates where inactivation occurred, at higher antibiotic concentrations, but failed at lower concentrations. This growth response was individualistic and not closely related to the minimum inhibitory concentrations.


Role of the Escherichia coli aromatic amino acid aminotransferase in leucine biosynthesis.

Strains of Escherichia coli that lack the branched-chain amino acid amino-transferase because of mutations in the ilvE gene had no growth requirement for leucine when the cells contained the aromatic amino acid aminotransferase that is the product of the tyrB gene. The presence of leucine increased the generation time of these cells and decreased the specific activity of the aromatic amino acid aminotransferase. It is concluded that this enzyme functions efficiently in leucine biosynthesis and can be repressed by leucine as well as by tyrosine.


Suppressors of a genetic regulatory mutation affecting isoleucine-valine biosynthesis in Escherichia coli K-12.

Escherichia coli K-12 mutant PS187 carries a mutation, ilvA538, in the structural gene for the biosynthetic L-threonine deaminase that leads to a leucine-sensitive growth phenotype, an isoleucine- and leucine-hypersensitive L-threonine deaminase, and pleiotropic effects resulting in abnormally low and invariant expression of some of the isoleucine-valine biosynthetic enzymes. Fifty-eight derivatives of strain PS187 were isolated as resistant to growth inhibition by leucine, by valine, or by valine plus glycly-valine and were biochemically, genetically, and physiologically characterized. All of these derivatives produced the feedback-hypersensitive L-threonine deaminase, and thus presumably possess the ilvA538 allele of the parent strain. Elevated synthesis of L-threonine deaminase was observed in 41 of the 58 isolates. Among 18 strains analyzed genetically, only those with mutations linked to the ilv gene clusters at 83 min produced elevated levels of L-threonine deaminase. One of the strains, MSR91, isolated as resistant to valine plus glycyl-valine, was chosen for more detailed study. The locus in strain MSR91 conferring resistance was located in four factor crosses between ilvE and rbs, and is in or near the ilvO gene postulated to be a site controlling the expression of the ilvEDA genes. Synthesis of the ilvEDA gene products in strain MSR91 is constitutive and derepressed approximately 200-fold relative to the parent strain, indicating that the genetic regulatory effects of the ilvA538 allele have been suppressed. Strain MSR91 should be suitable for use in purification of the ilvA538 gene product, since enzyme synthesis is fully derepressed and the suppressor mutation is clearly not located within the ilvA gene.


Reduction of postreplication DNA repair in two Escherichia coli mutants with temperature-sensitive polymerase III activity: implications for the postreplication repair pathway.

Daughter strand gaps are secondary lesions caused by interrupted DNA synthesis in the proximity of UV-induced pyrimidine dimers. The relative roles of DNA recombination and de novo DNA synthesis in filling such gaps have not been clarified, although both are required for complete closure. In this study, the Escherichia coli E486 and E511 dnaE(Ts) mutants, in which DNA polymerase I but not DNA polymerase III is active at 43 degrees C, were examined. Both mutants demonstrated reduced gap closure in comparison with the progenitor strain at the nonpermissive temperature. These results and those of previous studies support the hypothesis that both DNA polymerase I and DNA polymerase III contribute to gap closure, suggesting a cooperative effort in the repair of each gap. Benzoylated, naphthoylated diethylaminoethyl-cellulose chromatography analysis for persistence of single-strand DNA in the absence of DNA polymerase III activity suggested that de novo DNA synthesis initiates the filling of daughter strand gaps.


Evidence for an intermediate in quinolinate biosynthesis in Escherichia coli.

Evidence for the formation of an unstable intermediate in the synthesis of quinolinate from aspartate and dihydroxyacetone phosphate by Escherichia coli was obtained using toluenized cells of nadA and nadB mutants of this organism and partially purified A and B proteins in dialysis and membrane cone experiments. The results of these experiments indicate that the nadB gene product forms an unstable compound from aspartate in the presence of flavine adenine dinucleotide, and that this compound is then condensed with dihydroxyacetone phosphate to form quinolinate in a reaction catalyzed by the nadA gene product.


Isolation and characterization of an actinomycin D-sensitive mutant of Saccharomyces cerevisiae.

A single mutation in Saccharomyces cerevisiae conferred sensitivity to low concentrations of actinomycin D. Treatment with actinomycin D preferentially inhibited synthesis of rRNA's. Residual rRNA synthesized was processed normally. Total protein synthesis and inducibility of the enzyme maltase were relatively unaffected at concentrations of actinomycin D which severely inhibited rRNA synthesis.


Genetic separation of high- and low-affinity transport systems for branched-chain amino acids in Escherichia coli K-12.

The Escherichia coli K-12 mutant strain AE4107 (livH::Mu) is defective in the high-affinity binding protein-mediated uptake system for L-leucine, L-valine, and L-isoleucine (LIV-I). We have used this strain to produce mutations in the residual LIV-II membrane-bound branched-chain amino acid uptake system. Mutants selected for their inability to utilize exogenous L-leucine were found to be defective in the LIV-II system and fell into two classes. One class, represented by strain AE410709 (livP9), showed a complete loss of saturable uptake for L-leucine, L-valine, and L-isoleucine up to 50 muM, and a second class, represented by strain AE4017012 (liv-12), showed a residual component of saturable leucine uptake with increased Km. These mutations, livP9 and liv-12, were closely linked and mapped in the 74 to 78 min region of the E. coli genetic map. Strains constructed so that they lacked both LIV-I and LIV-II transport systems excreted leucine. Strains of the genotype livH+ livP were found to have normal high-affinity binding protein-mediated transport (LIV-I and leucine specific), whereas the low-affinity (LIV-II) transport was completely missing. We concluded from these studies that the high-affinity binding protein-mediated transport systems (LIV-I and leucine specific) can operate independently of the membrane-bound LIV-II system.


Chromosome replication during the division cycle in slowly growing, steady-state cultures of three Escherichia coli B/r strains.

The period of DNA synthesis C during the cell cycle was determined over a broad range of generation times in slowly growing, steady-state batch cultures in the exponential phase and in chemostat cultures of three strains of Escherichia coli, strains B/r A, B/r K, and B/r TT, utilizing measurements of average amounts of DNA per cell and cell survival after radioactive decay of 125I incorporated into the DNA of synthesizing cells. At each growth rate, values for cell survival and for C periods were the same within experimental errors for the three strains. The length of the DNA synthesis period increased linearly with generation (doubling) time T of the culture and approached a limiting value of C = 0.36T at very long generation times. In very slowly growing cultures, DNA replication was limited almost entirely to the final third of the cell cycle. D periods, between termination of DNA replication and cell division, were found to be relatively short at all growth rates for each strain. Average amounts of DNA per cell measured in slowly growing cultures of strains B/r A and B/r TT were indistinguishable from results for strain B/r K at the same growth rates. Amounts of DNA per cell calculated from the cell survival values alone are completely consistent with the measured DNA per cell.


Purification and characterization of a tRNA methylase from Salmonella typhimurium.

A tRNA methylase, in which supK strains of Salmonella typhimurium are deficient, was purified from strain LT2 and characterized. Column chromatography of protein extracts from wild-type cells on phosphocellulose, diethylaminoethyl-Sephadex A-50, and hydroxlapatite resulted in an enzyme that was estimated to be about 50% pure. tRNA from S. typhimurium which had been incubated at pH 9.0 served as a substrate for this methylase. The enzyme has a molecular weight of about 50,000 as estimated by gel chromatography and by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. The optimal assay conditions, as well as the kinetics and stability of the enzyme, were studied. As with other tRNA-methylating enzymes, S-adenosylhomocysteine is a potent inhibitor.


Effect of tryptophan analogs on derepression of the Escherichia coli tryptophan operon by indole-3-propionic acid.

The abilities of 14 tryptophan analogs to repress the tryptophan (trp) operon have been studied in Escherichia coli cells derepressed by incubation with 0.25 mM indole-3-propionic acid (IPA). trp operon expression was monitored by measuring the specific activities of anthranilate synthase (EC 4.1.3.27) and the tryptophan synthase (EC 4.2.1.20) beta subunit. Analogs characterized by modification or removal of the alpha-amino group or the alpha-carboxyl group did not repress the trp operon. The only analogs among this group that appeared to interact with the trp aporepressor were IPA, which derepressed the trp operon, and d-tryptophan. Analogs with modifications of the indole ring repressed the trp operon to various degrees. 7-Methyl-tryptophan inhibited anthranilate synthase activity and consequently derepressed the trp operon. Additionally, 7-methyltryptophan prevented IPA-mediated derepression but, unlike tryptophan, did so in a non-coordinate manner, with the later enzymes of the operon being relatively more repressed than the early enzymes. The effect of 7-methyltryptophan on IPA-mediated derepression was likely not due to the interaction of IPA with the allosteric site of anthranilate synthase, even though feedback-resistant mutants of anthranilate synthase were partially resistant to derepression by IPA. The effect of 7-methyltryptophan on derepression by IPA was probably due to the effect of the analog-aporepressor complex on trp operon expression.


Biosynthesis of uridine diphosphate N-acetylmannosaminuronic acid in rff mutants of Salmonella tryphimurium.

In Salmonella typhimurium, three groups of genes located in rfb, rfe, and rff clusters are known to be involved in the biosynthesis of the enterobacterial common antigen. We found that enzymatic synthesis of uridine diphosphate N-acetylmannosaminouric acid, the activated form of a constituent sugar of the common antigen, followed the pathway previously described in Escherichia coli (N. Ichihara, N. Ishimoto, and E. Ito, FEBS Lett. 39:46--48, 1974). All of the six rff mutants tested, which fail to synthesize the common antigen, were deficient in one or both of the two enzymes needed for the synthesis of this sugar nucleotide from uridine diphosphate N-acetylglucosamine; these results established the physiological role of the pathway studied for the biosynthesis of N-acetylmannosaminuronic acid residues. The levels of these enzymes were not reduced in rfe mutants or rfb deletion mutants, although they produced no or only traces of the common antigen.


Control of vacuole permeability and protein degradation by the cell cycle arrest signal in Saccharomyces cerevisiae.

Saccharomyces cerevisiae responds to deperivation of nutrients by arresting cell division at the unbudded G1 stage. Cells situated outside of G1 at the time of deperivation complete the cell cycle before arresting. This prompted an investigation of the source of nutrients used by these cells to complete division and the mechanisms controlling their availability. We found a close correlation between accumulation of unbudded cells and loss of previously formed allophanate hydrolase activity after nutrient starvation. These losses were not specific to the allantoin, system since they have been observed for a number of other enzymes and also when cellular protein levels were monitored with [3H]leucine. Loss of hydrolase activity was also observed when protein synthesis was inhibited either by addition of inhibitors or loss of the prtl gene product. We found that onset of nutrient starvation brought about release of large quantities of arginine and allantoin normally sequestered in the cell vacuole. Treatment of a cells with alpha-factor resulted in both the release of allantoin and arginine from the cell vacuole and the onset of intracellular protein degradation. These effects were not observed when either alpha cells or a/alpha diploid strains were treated with alpha-factor. These data suggest that release of vacuolar constitutents and protein turnover may be regulated by the G1 arrest signal.


6-cyanopurine, a color indicator useful for isolating mutations in the nif (nitrogen fixation) genes of Klebsiella pneumoniae.

6-Cyanopurine (6-CP) can be used as a color indicator for certain classes of nif (N2 fixation) mutations in Klebsiella pneumoniae. Under N2-fixing conditions, Nif+ colonies and most Nif- colonies are purple on media containing 6-CP. Twenty-two Nif- mutants with altered color on medium containing 6-CP were isolated. All white mutants contained mutations in the regulatory genes, nifAA-nifL. Mutants which were more darkly colored than the wild type had mutations distributed among six nif genes. Medium with 6-CP was used to isolate Nif- mutants with deletions internal to the nif genes, and 6-CP was used to identify strains depressed for nitrogenase synthesis in the presence of NH4+.


Fine-structure mapping and complementation analysis of nif (nitrogen fixation) genes in Klebsiella pneumoniae.

Four hundred and eighty-nine independent Nif- strains containing 260 point, 130 millimicron-induced, and 99 deletion mutations in nif in the Klebsiella pneumoniae chromosome were isolated. Three hundred and ninety insertion and point mutations were mapped with millimicron-induced deletions carried on 44 plasmids derived from pTM4010, a recombinant R factor containing the his-nif region of K. pneumoniae. The 99 chromosomal deletions in the nif region were mapped with 69 derivatives of pTM4010 carrying insertion and point mutations in nif. Complementation analysis between 84 derivatives of pTM4010 carrying nif mutations and Rec- derivatives of the 390 Nif- mutants identified 14 genes. The nif mutations were ordered into 49 deletion groups with a gene order of his...nifQBALFMVSNEKDHJ. Complementation analysis of millimicron-induced, amber, frameshift, and deletion mutations indicates there are five polycistronic and two monocistronic operons: nifQ nifB, nifA nifL, nifF, nifM nifV nifS, nifN nifE, nifK nifD nifH, and nifJ. Transcription is from right to left in all polycistronic operons.


Regulation and characterization of protein products coded by the nif (nitrogen fixation) genes of Klebsiella pneumoniae.

Two hundred and thirty-five Nif- strains of Klebsiella pneumoniae were characterized by two-dimensional polyacrylamide gel electrophoresis. Forty-two of these strains were tested further by in vitro acetylene reduction assays. By these techniques, nine nif-coded polypeptides were identified, and eight of these were assigned to specific nif genes. Nitrogenase component I required nifK and nifD, which coded for the beta and alpha subunits, and nifB, -E, and -N were required for the iron-molybdenum cofactor, which is a part of the active site of nitrogenase. nifH coded for the structural protein of component II, and nifM and nifS products seemed to be necessary for the synthesis of an active component II. There were two genes, nifF and nifJ, that were required for N2 fixation in vivo but not for N2 fixation in vitro. There were at least two cases (nifE and nifN, nifK and nifD) of two proteins that seemed to require each other for stability in vivo. Regulation of N2 fixation is apparently complex, and this is reflected by the assignment of regulatory functions to the gene products of nifA, nifL, nifK, nifD, nifH, and NIFJ.


Cell envelope and shape of Escherichia coli: multiple mutants missing the outer membrane lipoprotein and other major outer membrane proteins.

Starting with an Escherichia coli strain missing the outer membrane lipoprotein, multiple mutants were constructed than in addition to this defect miss the outer membrane proteins II, Ia and Ib, or Ia, Ib, and II. In contrast to all single mutants or strains missing the lipoprotein and polypeptides Ia and Ib, drastic influences on the integrity of the outer membrane and cell morphology were observed in mutants without lipoprotein and protein II. Such strains exhibited spherical morphology. They required increased concentrations of electrolytes for optimal growth, and Mg2+ or Ca2+ were the most efficient. These mutants were sensitive to hydrophobic antibiotics and detergents. Electron microscopy revealed abundant blebbing of the outer membrane, and it could clearly be seen that the murein layer was no longer associated with the outer membrane.


Major outer membrane protein in Salmonella typhimurium induced by maltose.

A maltose-induced major outer membrane protein (the 44K protein) is demonstrated in Salmonella typhimurium. This protein resembles the lambda receptor of Escherichia coli in its location, induction properties, apparent molecular weight, and association with the peptidoglycan layer of the cell wall. The 44K protein is missing in certain Salmonella Mal- mutants, which are also missing a protein analogous to the maltose-binding protein of E. coli. Thus, these mutants may be defective in the control of maltose genese in Salmonella. The proteins appear to be closely related, as indicated by cross-reaction of the Salmonella protein with the antiserum raised against the lambda receptor; however, they are not identical, since the peptide patterns obtained after limited proteolysis are completely different. Bacteriophage lambda does not use the 44K protein as a receptor.


Altered nuclear pore diameters in G1-arrested cells of the yeast Saccharomyces cerevisiae.

Nuclear pores in cells of the yeast Saccharomyces cerevisiae were examined by using the freeze-fracture technique. Nuclear pore diameters in actively growing cells appear to be exclusively of the normal diameter (75 to 115 nm), whereas some pore diameters in abnormally small G1-arrested cells produced by nitrogen starvation are unusually wide (120 to 160 nm). There may be a correlation between nuclear pore size and nuclear envelope size, the larger pores tending to occur in the smaller envelopes. The finding suggests that nuclear pore diameter may not function in regulating the flow of informational molecules from nucleus to cytoplasm, but may be implicated in regulating the flow of substrates into the nucleus.


Cloned DNA fragment specifying major outer membrane protein a in Escherichia coli K-12.

Plasmid pMC44 is a recombinant plasmid that contains a 2-megadalton EcoRI fragment of Escherichia coli K-12 DNA joined to the cloning vehicle, pSC101. The polypeptides specified by plasmid pMC44 were identified and compared with those specified by pSC101 to determine those that are unique to pMC44. Three polypeptides specified by plasmid pMC44 were localized in the cell envelope fraction of minicells: a Sarkosyl-insoluble outer membrane polypeptide (designated M2), specified by the cloned 2-megadalton DNA fragment, and two Sarkosyl-soluble membrane polypeptides specified by the cloning plasmid pSC101. Bacteria containing plasmid pMC44 synthesized quantities of M2 approximately equal to the most abundant E. coli K-12 outer membrane protein. Evidence is presented that outer membrane polypeptide M2, specified by the recombinant plasmid pMC44, is the normal E. coli outer membrane protein designated protein a by Lugtenberg and 3b by Schnaitman.


Absence of DNA sequences homologous to transposable element Tn5 (Kan) in the chromosome of Escherichia coli K-12.

The DNA hybridization procedure of Southern has been used to search for homology between the transposable kanamycin resistance determinant Tn5 and sequences in the chromosome of Escherichia coli K-12. No homology was detected under conditions in which a segment homologous to 5% or more of the 5,300-base pair Tn5 element would have been seen.


Bacteriophage Mu-induced modification of DNA is dependent upon a host function.

The DNA of bacteriophage Mu, extracted from induced lysates, is partially resistant to digestion by the endonuclease BalI. This modification of DNA is controlled by the Mu modification function (mom), which acts in conjunction with the dam (DNA-adenine methylation) function of Escherichia coli. Since the BalI recognition site is apparently different from the dam recognition site, these results imply that either the specificity of the dam function is changed by the mom function or the mom function requires the dam function for its activity.


Comparison of ribosomes from Coxiella burnetii and Escherichia coli by gel electrophoresis, protein synthesis, and immunological techniques.

Ribosomes and postribiosomal supernatant fluid (S-100) were isolated from Coxiella burnetii. The ribosomes functioned in polyuridylic acid-directed polyphenylalanine synthesis in the presence of S-100 from either C. burnetii or Escherichia coli. C. burnetii S-100 promoted translation with E. coli ribosomes. Antisera against E. coli elongation factor G and ribosomal proteins L7/L12 cross-reacted with rickettsial S-100 and ribosomes, respectively. Ribosomal proteins were analyzed by two-dimensional gel electrophoresis.


Interactions between uv radiation of different energies in the inactivation of bacteria.

A strong lethal interaction was observed between various monochromatic wavelengths (254, 334, 365, and 405 nm) in the repair-proficient E. coli K-12 strain AB 1157, except in the case of preexposure to 405-nm radiation which resulted in a protection against the inactivation resulting from subsequent exposure to 365-or 254-nm radiations. The results may be tentatively explained by assuming two classes of DNA lesions and two classes of damage to repair (reversible and inrreversible) whose proportions vary according to wavelength.


Dimer excision and repair replication patch size in recL152 mutant of Escherichia coli K-12.

Dimers are excised slowly in a recL152 mutant. This observation is not an artifact of altered DNA degradation because degradation is the same in recL+ and recL strains. The repair patch size was measured by the bromodeoxyuridine-313 nm radiation photolysis technique. In the recL+ strain, the average patch size was found to be about 30 nucleotides in length, but in the recL mutant, it was about 360.


The F plasmid may cosegregrate with either DNA strand of the Escherichia coli chromosome.

A stable association exists between the plasmid Flac and one of the polynucleotide strands of the bacterial chromosome. This polynucleotide strand was isolated and tested for uniqueness by DNA-DNA hybridization analysis. The association was found to involve either bacterial DNA strand.


Enchancement of nitrosoguanidine mutagenesis by chloramphenicol in Escherichia coli K-12.

N-methyl-N'-nitro-N-nitrosoguanidine-induced mutagenesis in E. coli K-12 can be enchanced up to 50-fold by the addition of chloramphenicol with minimal effect on survival. Chloramphenicol does not produce the expected proportionate increase in closely linked double mutations.


Membrane damage associated with inositol-less death in Saccharomyces cerevisiae.

The effect of inositol deficiency was studied on fermentation, respiration, and sugar and amino acid transport. It was found that the loss of fermetnation and respiration and sugar transport activity parallel the loss of cell viability. The loss of sugar transport activity is associated with the development of cell membrane damage. It is concluded that the ultimate cause of cell death is cell membrane leakiness.


Proline transport carrier-defective mutants of Escherichia coli K-12: properties and mapping.

A series of mutants of Escherichia coli K-12 requiring a high concentration of L-proline for growth were isolated from a proline auxotroph strain, JE2133. Genetic studies of the mutants, PT19, PT21, and PT22, showed that all the mutations (proT) were point mutations, and these were mapped at 82 min on the E. coli genetic map. Intact cells and cytoplasmic membrane vesicles of these mutants were specifically defective in L-proline transport activity. Strain PT21 had no detectable activity of the L-proline transport carrier at all, and strains PT19 and PT22 had only 1/35 and 1/70, respectively, of the transport activity of the parental strain. The mutants were also shown to have a defect in proline-binding function of the carrier by measuring specific binding of proline to sonically disrupted membranes. These results indicate that the gene proT determines the function of proline carrier in the cytoplasmic membrane.


Effect of mutation in the aromatic amino acid pathway on sporulation of Saccharomyces cerevisiae.

Mutations in ARO1 and ARO2 genes coding for enzymes involved in the common part of the aromatic amino acid pathway completely block the sporulation of Saccharomyces cerevisiae when in a homozygous state, whereas mutations in all the other genes of the same pathway do not. This effect is not due to the lack of any intermediate metabolite but rather to the accumulation of a metabolite preceding chorismic acid. Shikimic acid or one of its precursors was identified as the possible inhibitor. The presence of the three aromatic amino acids in the sporulation medium restores the ability to undergo meiosis. This seems not to be due to a feedback inhibition of the first enzymes of the pathway but rather to a competition between aromatic amino acids and the inhibitor on a site specific for the meiotic process. The inhibition of sporulation seems to occur at a very early step in meiosis, as indicated by the lack of premeiotic DNA synthesis in aro1 and aro2 mutants.


Relationship between phosphate content and immunochemical properties of subfractions of bakers' yeast mannan.

The mannan of bakers' yeast (Saccharomyces cerevisiae) was fractionated on a column of diethylaminoethyl-Sephadex into five subfractions. Phosphate content of these mannan subfractions was proportional to the concentration of NaCl solutions used in the chromatographic separation. Quantitative precipitin reactions showed that the serological reactivities of the subfractions were proportional to the content of phosphate. The result of acetolysis study showed that the amounts of mannotetraose and phosphate-containing oligosaccharide fractions increased proportionally to the acidity, whereas the amount of mannose decreased inversely. The results from quantitative precipitin reaction tests and acetolysis study demonstrated that both phosphate contents and multiplicity of branching moieties of mannan subfractions increased proportionally, i.e., micro-heterogeneity concerning the acidity comprised in the parent bulk mannan is not attributable merely to the coexistence of molecular species containing different amounts of phosphate but also to the presence of more of the branching moieties.


Stimulation of deletions in the Escherichia coli chromosome by partially induced Mucts62 prophages.

Deletion of bacterial DNA fragments is stimulated in induced Mucts62 lysogens. The host genes located proximally to the prophage are more frequently lost than those which are unlinked to the Mu genome. Genes located on either side of a Mu genome are deleted in the same manner. Like the other Mu-induced rearrangements, this process is recA independent and requires the participation of Mu DNA, as indicated by the fact that a phage genome always replaces the deleted genes. Data are presented which strongly suggest that both ends of the Mu genome are involved in deletion formation.


Chromosomal location of a gene (nmpA) involved in expression of a major outer membrane protein in Escherichia coli.

The phenotypic expression of protein E, a recently described major outer membrane protein, is associated with a mutation at a locus on the Escherichia coli chromosome that we call nmpA. nmpA is located between rbsK and uncA at 82.7 min on the E. coli linkage map. The nmpA locus is also the site of the mutations which lead to the formation of major outer membrane proteins Ic or e. It is likely proteins E, Ic, and e are closely related or identical. The mutant nmpA allele is dominant.


Evolution of propanediol utilization in Escherichia coli: mutant with improved substrate-scavenging power.

Wild-type strains of Escherichia coli are unable to use L-1,2-propanediol as a carbon and energy source. A series of mutants, able to grow on this compound at progressively faster rates, had been isolated by repeated transfers to a medium containing 20 mM L-1,2-propanediol. These strains synthesize at high constitutive levels a propanediolmicotinamide adenine dinucleotide oxidoreductase, an enzyme serving as a lactaldehyde during L-fucose fermentation by wild type cells. In this study, a mutant that can grow rapidly on the novel carbon source was subjected to further selection in a medium containing L-1,2-propanediol never exceeding 0.5 mM to obtain a derivative that has an increased power to extract the substrate from the medium. The emerging mutant exhibited four changes at the enzymatic level: (i) fuculose 1-phosphate aldolase activity is lost; (ii) the constitutive propanediol oxidoreductase activity is increased in its level; (iii) lactaldehyde dehydrogenase becomes constitutive and shows an elevated specific activity in crude extracts; and (iv) at low concentrations of propanediol, the facilitated diffusion across the cell membrane is enhanced. Changes two to four seem to act in concert in the trapping of propanediol by hastening its rate of entry and conversion to an ionized metabolite, lactate.


Metabolic interconversion of free sterols and steryl esters in Saccharomyces cerevisiae.

The interconversion of free and esterified sterols was followed radioisotopically with [U-14C]acetate and [methyl-14C]methionine. In pulse-chase experiments, radioactivity first appeared mainly in unesterified sterols in exponential-phase cells. Within one generation time, the label equilibrated between the free and esterified sterol pools and subsequently accumulated in steryl esters in stationary-phase cells. When the sterol pools were prelabeled by growing cells aerobically to the stationary phase and the cells were diluted into unlabeled medium, the prelabeled steryl esters returned to the free sterol form under several conditions. (i) During aerobic growth, the prelabeled sterols decreased from 80% to 45% esters in the early exponential phase and then returned to 80% esters as the culture reached the stationary phase. (ii) Under anaerobic conditions, the percentage of prelabeled steryl esters declined continuously. When growth stopped, only 15% of the sterols remained esterified. (iii) In the presence of an inhibitor of sterol biosynthesis, which causes accumulation of a precursor to ergosterol, prelabeled sterols decreased to 40% steryl esters while the precursor was found preferentially in the esterified form. These results indicate that the bulk of the free sterol and steryl ester pools are freely interconvertible, with the steryl esters serving as a supply of free sterols. Furthermore, there is an active cellular control over what types of sterol are found in the free and esterified sterol pools.


Repair of cross-linked DNA and survival of Escherichia coli treated with psoralen and light: effects of mutations influencing genetic recombination and DNA metabolism.

Repair of cross-linked DNA was studied in Escherichia coli strains carrying mutations affecting DNA metabolism. In wild-type cells, DNA strands cut during cross-link removal were rejoined during a subsequent incubation into high-molecular-weight molecules. This rejoining was dependent on gene products involved in genetic recombination. A close correlation was found relating recombination proficiency, the rate of strand rejoining, and formation of viable progeny after DNA cross-linking by treatment with psoralen and light. Wild-type cells and other mutants which were Rec+ (sbcB, recL, recL sbcB, recB recC sbcA, recB recC sbcB, xthA1, and xthA11) rejoined cut DNA strands at a rate of 0.8 +/- 0.1 min -1 at 37 degrees C and survived 53 to 71 cross-links per chromosome. recB, recC, recB recC, recF, or polA strains showed reduced rates of strand rejoining and survived 4 to 13 cross-links per chromosome. Recombination-deficient strains (recA, recB recC sbcB recF, recB recL) and lexA failed to rejoin DNA strands after crosslink removal and were unable to form colonies after treatments producing as few as one to two cross-links per chromosome. Strand rejoining occurred normally in cells with mutations affecting DNA replication (dnaA, danB, dnaG, and dnaE) under both permissive and nonpermissive conditions for chromosome replication. In a polA polB dnaE strain strand rejoining occurred at 32 degree C but not at 42 degree C, indicating that some DNA synthesis was required for formation of intact recombinant molecules.


Cell division during nutritional upshifts of Escherichia coli.

Nutritional shifts of Escherichia coli B/r to richer media have been analyzed is synchronously growing and exponential-hase populations. Early perturbations in the timing of cell division were observed. At the slow growth, division progressed at a rate equal to or less than the preshift rate for about 1 h. At intermediate growth, both delays and acceleration in division were observed. The extent of the perturbation depended upon the age of the cells at the time of the shift and the composition of the preshift and postshift media. The perturbation was different in the two substrains of E. coli B/r used in this study.


Insertion of bacteriophage lambda into the deo operon of Escherichia coli K-12 and isolation of plaque-forming lambdadeo+ transducing bacteriophages.

A procedure has been devised to isolate plaque-forming lambda cI857S7 transducing bacteriophage which carry the internal promoter, P3, of the deo operon of Escherichia coli and the deoB and deoD genes, while lacking the deoP and cytP promoters of the same operon, in order to study, specifically, regulation at the P3 site. This has been accomplished by selecting for the insertion of bacteriophage lambda into the deoA gene in a strain deleted for the normal lambda attachment site (delta att lambda) and isolating from this lysogen lambda spi- and lambda EDTAr phage. Among these, lambda pdeoB+D+ phage were identified by their transducing abilities. From in vivo enzyme induction experiments performed on a delta deo strain lysogenized with such phage, they were shown to carry the P3 promoter while lacking the deoP and cytP promoters. A lambdapdeo B+D+ phage phage was used to lysogenize a deo+ delta att lambda strain, integration of lambda occurring within the region of homology, and, from a heat-induced lysate of this strain, a plaque-forming lambda+ phage carrying the complete deo operon was obtained. Phage lambda was also inserted into the deoB and deoD genes and into the tdk gene. By isolating lambdaspi- and lambdaEDTAr phage from the deo::(lambda) mutants and determining which bacterial genes they carried and whether they retained the int gene of lambda, it was found that lambda had inserted into deoD with the same orientation as lambda inserted into attlambda, whereas lambda inserted into deoA and deoB had the opposite orientation. Deletions extending from the site of lambda insertion into the bacterial chromosome were isolated by selecting for heat-resistant revertants. These confirmed the order of markers to be deo-serB-trpR-thr and also placed a locus, msp, determining sensitivity or resistance of male strains to male-specific phages, between trpR and thr. For some reason unknown, but which may be related to the orientation of the lambda prophages, short deletions rendering the bacterium Ser- Thr+ were of much lower frequency from the deoD::(lambda) lysogen than from the other two lysogens. From an examination of the residual deoD enzyme levels in deoB::(lambda) mutants, it was deduced that there may be two promoter sites within the deoB::(lambda) mutants, it was deduced that there may be two promoter sites within the deoB gene, transcription from one of these being sufficient to account for the noncoordinate nature of the induction of deoB and deoD gene products.


Role of lipopolysaccharide and outer membrane protein of Escherichia coli K-12 in the receptor activity for bacteriophage T4.

Lipopolysaccharide isolated from Escherichia coli K-12 did not inactivate phage T4, although the cell envelopes with 1% sodium deoxycholate resulted in the release of cytoplasmic membrane proteins, 70% of the lipopolysaccharide, and almost all of the phospholipid. The reconstitution of phage receptor activity was achieved from deoxycholate-soluble and -insoluble fractions by dialysis against a solution of magnesium chloride. Lipopolysaccharide was the only essential component in the deoxycholate-soluble fraction. PhageT4-resistant mutants YA21-6 and YA21-82, having defects in the deoxycholate-soluble and -insoluble fractions, respectively, were isolated. The deoxycholate-soluble fraction of YA21-6 possessed heptoseless lipopolysaccharide, and this defect was responsible for the phage resistance. The deoxycholate-insoluble fraction of YA21-82 lacked outer membrane protein O-8. The addition of O-8 to this fraction together with the wild-type lipopolysaccharide resulted in the appearance of the receptor activity. Furthermore, the reconstitution was successfully achieved with only O-8 and the wild-type lipopolysaccharide, indicating that O-8 was an essential component in the deoxycholate-insoluble fraction.


Mechanism of colicin E3 production in strains harboring wild-type or mutant plasmids.

As previously reported by others, more than 90% of the colicin E3 synthesized soon after induction of colicinogenic bacteria was found to be cell bound, about half of it being in a salt-extractable state at the cell surface. Evidence is presented that the colicin molecules remain preferentially bound to the cell which produced them, rather than being secreted and randomly distributed in the cell population. Secretion of colicin E3 may in fact never occur, all or most of the colicin found free in the medium perhaps being released during lysis of the producing cells long after induction. Among 19 mutant plasmids selected on the basis of their inability to produce an active colicin, only 3 were found to code for a protein which although it lacked any bactericidal activity, had the same molecular weight as wild-type colicin E3 and displayed a reaction of immunological identity with it. These three inactive colicins fail to be exported to the cell surface and seem to be blocked at some intermediate stage in the export process.


Genetic mapping of tyramine oxidase and arylsulfatase genes and their regulation in intergeneric hybrids of enteric bacteria.

The genes for arylsulfatase (atsA) and tyramine oxidase (tynA) have been mapped in Klebsiella aerogenes by P1 transduction. They are linked to gdhD and trp in the order atsA-tynA-gdhD-trp-pyrF. Complementation analysis using F' episomes from Escherichia coli suggested an analogous location of these genes in E. coli, although arylsulfatase activity was not detected in E. coli. P1 phage and F' episomes were used to create intergeneric hybrid strains of enteric bacteria by transfer of the ats and tyn genes between K. aerogenes, E. coli, and Salmonella typhimurium. Intergeneric transduction of the tynK gene from K. aerogenes to an E. coli restrictionless strain was one to two orders less frequent than that of the leuK gene. The tyramine oxidase of E. coli and S. typhimurium in regulatory activity resemble very closely the enzyme of K. aerogenes. The atsE gene from E. coli was expressed, and latent arylsulfatase protein was formed in K. aerogenes and S typhimurium. The results of tyramine oxidase and arylsulfatase synthesis in intergeneric hybrids of enteric bacteria suggest that the system for regulation of enzyme synthesis is conserved more than the structure or function of enzyme protein during evolution.


Arrangement of glycan chains in the sacculus of Escherichia coli.

A novel of Escherichia coli endopeptidase was used for a selective partial hydrolysis of the peptide bridges which interlink the glycan chains in E. coli sacculi. The loosening of the murein network revealed, in the electron microscope, a preferential orientation of the glycan chains, more or less perpendicular to the length axis of the cell. Control incubations with E. coli transglycosylase or egg-white lysozyme did not leave ordered structures behind.


Analysis of sequences transposed by complementation of two classes of transposition-deficient mutants of Tn3.

The Tn1 and Tn3 elements are closely related transposons which carry the structural gene for ampicillin resistance. Two classes of deletion mutants of the plasmid pMB8::Tn3 (RSF1050) are unable to transpose ampicillin resistance but can be complemented in trans by a coresident Tn1 or Tn3 element. The analysis of the sequences transposed upon complementation of one class of mutants (type I) showed that the mutant element had undergone bona fide transposition. Complementation of the type II mutants led to the transposition of a sequence analogous to bacteriophage mu-promoted integration of non-mu DNA. The transposed sequence consisted of two Tn3 elements which flanked a single copy of the pMB8 portion of the RSF1050 genome. Complementation data indicated that the type II mutants are defective in at least one trans-acting function which must be supplied for transposition to occur. The nature of sequence transposed from the type II mutant is the consequence of a defective cis-acting function (or site). In addition, the type II mutants were defective in a trans-acting function which regulated the frequency of transposition.


Pesticin-dependent generation of somotically stable spheroplast-like structures.

Homogenous pesticin, a bacteriocin produced by Yersinia pestis, promoted rapid dose-dependent killing of Escherichia coli phi but permitted residual generation of cell mass. Both growing cells and those blocked in net synthesis of nucleic acids or protein were converted by pesticin to osmotically stable spheroplast-like forms. Morphology and viability of cells starved for fermentable carbohydrate were not affected by pesticin. Similar spheroplast-like structures were formed from sensitive cells of Yersinia pseudotuberculosis, Yersinia enterocolitica, and Y. pestis.


Trypsin peptide patterns of tryptophan synthase beta2 protein among four species of the Enterobacteriaceae.

The tryptophan synthase beta 2 protein (EC 4.2.1.20) of Escherichia coli, Enterobacter aerogenes, Serratia marcescens, and Erwinia carotovora was purified and compared. Two-dimensional total peptide patterns for each of the four beta2 proteins obtained after digestion with trypsin showed that approximately three quarters of the total peptides are common to all four peptides. Examination of only arginine-containing peptides showed that approximately half of these peptides are common. From a comparative standpoint, the data provide evidence that the primary structure of beta 2 proteins is relatively similar, indicating that the trpB cistron is evolutionarily conserved in the enteric bacteria group.


Plasmids of incompatibility group P code for the capacity to propagate bacteriophage IKe.

Seven of eight plasmids of incompatibility group P were found to code for the capacity to propagate bacteriophage IKe in Escherichia coli. Six of the seven plasmids allowed propagation of IKe by one bacterial host (RG172) but not by another (RG176); the other plasmid allowed IKe propagation by both hosts. IKe propagation by a number of E. coli K-12 strains was quite variable. IKeh, an extended host range mutant of IKe, was found to plaque specifically on N+ and P+ strains.


Orotic acid excretion in some wild-type strains of Escherichia coli K-12.

During rapid growth,  the excretion of pyrimidines, predominantly uracil, is a common phenomenon in procaryotes and eucaryotes. In Escherichia coli, some K-12 strains excrete orotic acid and not uracil. This is caused by a mutation in the pyrF gene.


A guide to the use of psychotropic medication in children and adolescents.

There are a number of clinical entities in which medication can be a useful adjunct to the overall therapeutic approach to a child or adolescent. This guide will outline the most common medications used and some of the important problems encountered with the medication.


Construction of an L-arginine-producing mutant in Serratia marcescens. Use of the wide substrate specificity of acetylornithinase.

L-Arginine biosynthesis in Serratia marcescens Sr41 was found to be controlled by (a) feedback inhibition of N-acetylglutamate synthetase and (b) repression of some L-arginine biosynthetic enzymes, and an L-arginine-degrading system was found to exist. Accordingly, an L-arginine-producing mutant (aru argR argA) of S. marcescens Sr41 was constructed as follows. A mutant incapable of L-arginine utilization (aru) was obtained from the wild strain. Subsequently, from the lysine auxotroph (lysA) of aru mutant, a mutant having derepressed L-arginine biosynthetic enzymes (argR) was isolated by screening for colonies that could utilize Nalpha-acetyl-L-lysine in the presence of L-arginine. This selection was based on the finding that acetylornithinase of S. marcescens hydrolyzed Nalpha-acetyl-L-lysine. On the other hand, to obtain a mutant with feedback-resistant N-acetylglutamate synthetase (argA), the proAB argD argR triple mutant was isolated from the indirectly suppressed revertant (proAB argD) of the proline auxotroph (proAB). Next, the argA mutant was isolated from the triple mutant by selection for resistance to 3,4-dehydro-DL-proline in the presence of L-arginine. The argA mutation was introduced into the aru lysA argR strain by PS20-mediated cotransduction with lysA+. The aru argR argA lysA+ transductant produced 25 mg/ml of L-arginine in the medium.


Local migration of myosin in F-actin plus ATP solution on the boundary of a diffusion cell.

The diffusion phenomena of myosin (myosin A, H-meromyosin or subfragment-1) in F-actin plus ATP solutions were investigated. The upper part of the diffusion cell was filled with F-actin plus ATP, and the lower part was filled with F-actin, ATP, and myosin, then both parts were brought into contact so that a boundary of the two solutions was formed and the diffusion of myosin in F-actin plus ATP solutions started. The diffusion pattern was observed with a schlieren lens system. When almost all the ATP in the lower part of the cell had been consumed by actomyosin, a hyper-sharp schlieren pattern appeared near the boundary. On analyzing this pattern, it was found that a local fast migration of proteins was occurring. Simple Brownian motion of myosin molecules could not explain the hyper-sharp phenomenon. This phenomenon occurred in ther pesence of Mg2+ or Ca2+, but very little in the presence of EDTA. Although it is well known that the superprecipitation of myosin B suspension occurs only at physiological ionic strength, this phenomenon occurred over a relatively wide range of ionic strengths. The molecular mechanism of this phenomenon is discussed in relation to the basic mechanism of the interaction between myosin and F-actin.


Proteolysis of the major capsid protein T4 bacteriophage polyheads limited by quaternary structure.

Bacteriophage T4 carrying an amber mutation in gene 22 plus an amber mutation in gene 21 form aberrant, tubular structures termed rough polyheads, instead of complete phage when they infect Escherichia coli B. These rough polyheads consist almost entirely of the major capsid protein in its uncleaved form (gp23). When rough polyheads are treated under mild conditions with any of the five proteases, trypsin, chymotrypsin, thermolysin, pronase, or the protease from Staphylococcus aureus V8, the gp23 is rapidly hydrolyzed at a limited number of peptide bonds. In contrast, cleaved capsid protein (gp23) in mature phage capsids is completely resistant to proteolysis under the same conditions. A major project in this laboratory requires determining the primary structure of gp23, a large protein (Mr = 58,000) quite rich in those amino acids at which cleavages are achieved by conventional means. Recovery of peptides from the complex mixtures resulting from such cleavages proved to be extremely difficult. The limited proteolysis of gp23 in rough polyheads had yielded a set of large, easily purified fragments which are greatly simplifying the task of determining the primary structure of this protein.


Phospholipid composition and membrane function in phosphatidylserine decarboxylase mutants of Escherichia coli.

Temperature-sensitive conditional lethal mutants in phosphatidylserine decarboxylase (psd) accumulate large amounts of phosphatidylserine under nonpermissive conditions (42 degrees C) prior to cell death. In addition, the ratio of cardiolipin to phosphatidylglycerol is increased. At an intermediate temperature (37 degrees C), high levels of phosphatidylserine can be maintained with little effect on cell growth or viability. Under these conditions, both the rate of induction and the function of the lactose transport system are normal. At 42 degrees C addition of Mg2+ or Ca2+ to mutant cultures produces a partial phenotypic suppression. Growth is prolonged and the filaments normally present at 42 degrees C do not form. Upon transfer to the nonpermissive temperature, there is a considerable lag before accumulation of phosphatidylserine begins and the growth rate is affected. Based on the kinetics of heat inactivation of phosphatidylserine decarboxylase activity in extracts, in intact nongrowing cells, and in growing cells, it appears that the enzyme newly synthesized at 42 degrees C is more thermolabile in vivo than enzyme molecules previously inserted into the membrane at the lower temperature. Thus, the older, stable enzymatic activity must be diluted during growth before physiological effects are observed.


The peptide chain elongation factor genes tufA and fus of Escherichia coli are intimately related physically.

Recent work from several laboratories has established the following points about the synthesis of the polypeptide chain elongation factors Tu and G in Escherichia coli. (i) Elongation factor Tu is the product of duplicate, highly conserved genes, tufA and tufB, which are widely separate parts of the chromosome. (ii) The molar  concentration of this factor is considerably higher than that of elongation factor G which is encoded by the fus gene. (iii) Although the tufA and fus genes are close together and can be co-transcribed in the direction from fus to tufA, the tufA gene product is synthesized at several times the rate of the fus gene product. In an attempt to understand what mechanism(s) could account for the differential expression of the tufA and fus genes, we sought to obtain more precise information on the physical relationship of these genes. By examining heteroduplexes between restriction endonuclease-generated fragments of DNA containing the tufA, fus, and tufB genes, we have demonstrated that the fus and tufA genes are intimately related physically in one of two possible arrangements. Either the NH2-terminal region of the tufA gene is contiguous with the COOH-terminal region of the fus gene or the beginning of the tufA gene overlaps part of the fus gene. These results mean that if the tufA gene is always co-transcribed with the fus gene, then some mechanism must allow the tufA portion of the transcript to be translated more often than the fus gene portion of the transcript.