[Electron microscopic study on the focal immune response in the choroid (author's transl)].

Pigmented rabbits were sensitized by subcutaneous injection of BCG together with Freund's complete adjuvant and heat killed tubercle bacilli together with adjuvant. After the titer of serum antibody to BGG was elevated or tuberculin skin test became positive, choroiditis was induced in these eyes by an injection of BGG or PPD directly through the posterior sclera in the vicinity of the optic nerve. The eyes were enucleated at various intervals and the choroid was studied by light and electron microscopy. In the choroid challenged with BGG, infiltrates such as polymorphonuclear leukocytes and lymphocytes were seen, in early stages, invading in the posterior segment and peripheral area near the ciliary body. These cells decreased in number within a few days, and after one week a few plasma cells were detected in the posterior uvea. In the choroid challenged with PPD, infiltration of monocytes and lymphocytes was seen in early stages, reaching its peak in 48 hours. After 4 and 5 days lymphocytes decreased in number, but monocytes showed mitotic and phagocytotic activity, and accumulated near the sclera. After one week typical epithelioid cells were detected along the suprachoroidal space. These events were restricted within the choroid. The results obtained suggest, that in the focal immune reaction of the choroid, the cellular sequence of events and the range of tissue involved are different depending upon the kinds of antigen used. It may indicate humoral immunity on one side and cell-mediate immunity on the other.


[Clinical and experimental study on hepatorenal syndrome in terms of obstructive jaundice (author's transl)].

In our department, hepatorenal syndrome was highly associated with obstructive jaundice and was seen in 9.2 percent of patients with obstructive jaundice. This syndrome developed when in underlying hepatic disease, acute renal failure supervened. Histological findings of renal failure caused by obstructive jaundice were summarized as tubular necrosis and the presence of protein-like substance in the glomerulus, which were mostly reversible and similar to those seen in shock. To investigate the pathogenesis of hepatorenal syndrome, experimentally the common duct in dog was ligated to produce obstructive jaundice and B. Klebsiella was injected into the bile duct to produce cholangiolitis. From this study, it was evident that cholangiolar infection was closely correlated with the manifestation of renal failure. In fact, without acquiring infection, renal failure did not occur. Moreover, this infection produced endotoxemia with high incidence. Therefore, it is assumed that this cholangiolar infection plays an important role in the pathogenesis of hepatorenal syndrome caused by obstructive jaundice.


Effects of serotonin, of its biosynthetic precursors and of the anti-serotonin agent metergoline on the release of glucagon and insulin from rat pancreas.

We have evaluated the effect of serotonin (5-HT) and of its biosynthetic precursors 5-Hydroxytryptophan (5-HTP) and tryptophan (TRP) on the release of immunoreactive glucagon (IRG) and insulin (IRI) from isolated islets and pieces of pancrease of the rat. In isolated islets, 5-HT inhibited the IRI response to a high glucose concentration (3.0 mg/ml), without affecting the IRG response to either a low (0.5 mg/ml) or a high glucose concentration; TRP stimulated the IRG and IRI response to the low glucose concentration, while 5-HTP was ineffective. When pieces of pancreas were used, 5-HT and 5-HTP inhibited IRG response to both glucose concentrations, while IRI release was inhibited only by 5-HT. The anti-5-HT agent metergoline enhanced the release of IRG and IRI by pieces of pancreas at both glucose concentrations. The results indicate that exogenous and endogenous 5-HT inhibit basal as well as glucose-mediated IRG and IRI release; that isolated islets are less sensitive than pieces of pancreas to the inhibitory effect of 5-HT and that TRP acts as an amino acid and not as a precursor of 5-HT.


The effect of luteinizing hormone releasing hormone (LRH) on pituitary gonadotropins in male homosexuals.

Basal serum LH and FSH values were found to be within normal limits in 9 homosexual men. The mean LH and FSH responses following the intravenous administration of 100 microgram of LRH were not significantly different from that of heterosexual controls. In addition, the mean basal plasma serum testosterone was similar in the two groups. There is thus no definite implication of endocrine factors in the genesis of male homosexuality.


The amino acid sequences of the tryptic, chymotryptic and peptic peptides from the L-2 light chain of rabbit skeletal muscle myosin.

The amino acid sequences of the tryptic peptides from the aminoethylated L-2 light chain of rabbit skeletal muscle myosin were determined by various enzymatic hydrolyses, partial hydrolysis with dilute acetic acid and Edman degradation. The amino acid sequences of the chymotryptic and peptic peptides from the carboxymethylated L-2 light chain were partially analysed in the same manner as the tryptic peptides. The primary structure of the L-2 light chain of rabbit skeletal muscle myosin was deduced from the above results.


Release of granulocyte elastase in lethal canine endotoxin shock.

The release of granulocyte elastase and its interaction with plasma protease inhibitors was studied in dogs receiving a slow infusion of a lethal dose of Escherichia coli endotoxin. During endotoxin infusion a marked decline in leucocyte counts was parallelled by a rapid increase in plasma granulocyte elastase concentrations. Maximal values were reached after 3 h, when the infusion was ended. Crossed immunoelectrophoresis with antiserum against granulocyte elastase did not reveal the presence of elastase components with the electrophoretic mobility of free elastase, but elastase-alpha1-antitrypsin complexes were detected. A gradually decreasing plasma concentration of alpha2-macroglobulin was noted during the experiments. Crossed immunoelectrophoresis, however, did not reveal any electrophoretic heterogeneity. It is concluded that the release of granulocyte proteases might be of significance for several pathophysiological changes seen in endotoxin shock.


The bone marrow in Hodgkin's disease: the non-involved marrow.

One hundred and nineteen methacrylate-embedded trephine biopsy specimens from 80 patients with Hodgkin's disease are reviewed. Although marrow involvement was found in 4% of the untreated patients, the present study was mainly concerned with the marrow unaffected by specific infiltrates and in negative specimens. The non-involved bone marrow in Hodgkin's disease invariably showed alterations of three types; stromal damage, inflammatory infiltration, and disturbed haematopoiesis. Each of these features can be found in the absence of one or both of the others. In severe examples, all three of these components usually occur simultaneously, giving histological pictures that mimic Hodgkin-specific infiltrates, sometimes in association with clinical suggestions of bone marrow involvement or replacement. But trephine biopsies of sufficiently high histological quality offer alternative explanations for the pancytopenia, for instance intramedullary phagocytosis, reactive sclerosing inflammation resembling auto-immune disorders, or a disturbance of haematopoiesis itself. This disturbance could be due to a defect inherent in the haematopoiesis associated with Hodgkin's disease, possibly predisposing for leukaemia in longterm survivors who have received chemotherapy and/or irradiation. The disturbed erythropoiesis proved to be strongly correlated with the stage of the disease at the time of biopsy. This finding could contribute to staging procedures, when laparotomy is contraindicated.


The immunofluorescence characteristics of the basement membrane in squamous carcinoma of the uterine cervix.

Basement membrane-specific antigens (BMSA) were found by immunofluorescence (IFL) in 20 of 33 tumour samples from patients with squamous carcinoma of the uterine cervix. The BMSA were demonstrated to be unrelated to condensation of both reticulin and subepithelial ground substance (as evidenced by antireticulin IFL antibodies, silver stain and PAS reaction, respectively), the age of the patient, the clinical stage and the histological differentiation of the tumour. The finding that BMSA secreting and non-secreting cervical tumours does not correlate with the degree of differentiation, was somewhat unexpected.


Big cones and little cones.

To investigate whether the amount of tissue removed at conization could influence the frequency of inadequate excision of cervical atypias, 354 cone specimens were measured. The data indicate that the frequency of lesions incompletely removed at conization increased with decreasing size (ie. length and volume) of the specimen. The parameters influencing the detection of epithelial atypias at the surgical margin of the specimens are discussed. A plea is made for international standardization in reporting results of conization (size of cone and number of sections) in order to permit objective comparison between clinics of the results of the conization procedure.


State rate review: a critical assessment.

In response to rising hospital costs, several states established rate setting programs, which now affect about one-fourth of the nation's hospitals.


The transition in frequency of Y chromatin in males during the neonatal period.

The frequency of Y chromatin, visualized as fluorescent bodies in cell nuclei from lymphocytes in blood smears, was significantly less in newborn males than in three-month-old male infants and adults. The frequency of Y chromatin-positive cells on day 0 was 36.16 +/- 9.11% and then increased daily. At one month after birth the frequency was 55.07 +/- 9.29%, which was not significantly different from that in adult males (57.08 +/- 5.97%).


Study of human male meiosis. II-Q-banding in pachytene bivalents.

In continuation of the research on male human meiosis within the study of pachytene bivalents, results from the analysis of 125 cells are presented. The aim of this work is to establish Q-banding patterns and make a detailed study of chromomere counting and distribution. To obtain Q-bands Quinacrine and Pseudoisocyanine staining techniques have been used.


Comparison of silver staining of nucleolus organizer regions in human lymphocytes and fibroblasts.

Ag-staining of the nucleolus organizer region (NOR) was studied in the acrocentric chromosomes identified by Q-banding in repeated lymphocyte and skin fibroblast cultures from three different individuals. A similar pattern of Ag-stainability of NORs was found in the two tissues in each individual. Small differences concerning, in each case, only one of the acrocentric chromosomes were found between repeated lymphocyte cultures, as well as between lymphocyte and fibroblast cultures of the same individual without indication of any prevalence of one tissue type in a certain direction. The possibility that these differences are caused by different stages of NOR activation is discussed.


A rapid technique for producing silver-stained nucleolus organizer regions and trypsin-giemsa bands on human chromosomes.

A simple and rapid technique is described whereby the nucleolus organizer regions (NORs) of human chromosomes can be differentially stained with silver. This staining is followed by trypsin-Giemsa banding on the same metaphase chromosomes. The metaphases simultaneously exhibit silver-stained NORs and G bands, allowing for the unequivocal identification of all chromosomes and greatly facilitating studies involving the NOR-bearing acrocentrics.


Selection of mouse X hamster hybrids using HAT medium and a polyene antibiotic.

In the present study we tested the feasibility of utilizing a structurally modified polyene antibiotic, amphotericin B methyl ester (AME), as a half-selection agent for isolating somatic cell hybrids. By using HAT medium supplemented with AME we have isolated interspecific mouse-hamster hybrids from mixed cultures of mouse (TK-C1 ID or HPRT-A9) and hamster (BHK/C 13) cells fused with Sendai virus, lysolecithin or polyethylene glycol. Hybrid cells proliferated and clones were isolated after 2 to 3 weeks growth in three changes of HAT-AME medium and subsequent growth in HAT medium alone. In contrast, genetically deficient parental C1 1D or A9 cells and AME-sensitive BHK/C 13 cells were killed using a similar growth protocol. The described technique is simple, efficient and permits one to use a cell line without a genetic defect in combination with a genetically deficient cell type in hybrid formation.


A comparison of methods for detecting bacteriophage contamination of tissue culture sera.

Detection of bacteriophage contamination of tissue culture sera by direct plating has been compared with detection methods based on batch enrichment and on the Poisson distribution (PD plating). Batch enrichment is extremely sensitive for detecting the presence of phage contamination. PD plating combines sensitivity with isolation of each contaminating phage in pure culture. Both batch enrichment and PD plating are more sensitive than direct plating. Neither method requires highly trained personnel or specialized equipment.


Synchronization of Drosophila cells in culture.

We synchronized Drosophila cell lines (Schneider's line 2 and Kc) by allowing the cells to enter the stationary phase of growth and then diluting them into fresh culture medium. The cells of both cell lines entered S phase, after an 8- to 14-hr delay, in a state of partial synchrony; 60 to 80% of the cell population accumulated in S phase. Measurements of the cell cycle phases of Schneider's line 2 cells (S = 14 to 16 hr; G2 = 6 to 8 hr; M = 0.4 hr) were similar to those of Kc cells.


Artificial capillary perfusion cell culture: metabolic studies.

Glucose, lactic-acid, and oxygen metabolism of BHK and L929 cells on artificial capillary perfusion units have been studied using several different modes of perfusion. After 7 to 10 days, cells planted in the extracapillary compartment of culture units containing 80 to 150 fibers reached populations that used 0.073 +/- 0.025 mumol per min glucose and 0.76 +/- 0.26 microliter per min oxygen and excreted 0.078 +/- 0.038 mumol per min lactic acid. From these data it is estimated that these units contain approximately 2 x 10(7) cells. The metabolic rate of cultures perfused through the capillaries or through the extracapillary compartment was not affected significantly by change in flow rate except at perfusion flow rates less than or equal to 0.05 ml per min. The cell population, as measured by metabolic activity, did not increase significantly when the serum content of the medium was less than or equal to 1%. No major differences were found in glucose utilization rates of equal numbers of cells on artificial capillaries, on short-term suspension culture, or as monolayers in plastic flasks. Artificial capillary perfusion may provide a simple system for studying metabolism of mammalian cells in culture.


Growth of animal cells on glass fiber filters and their utilization for biosynthetic analysis.

HeLa and L-M cells can be effectively grown directly on glass fiber filters to yield replicate cultures that allow easy analysis of biosynthetic capabilities through measurement of radioactive precursor uptake and incorporation. The glass fiber filters are superior to glass cover slips, growth in scintillation vials, and growth on Millipore filters in the ease of handling during experimental treatment and in the amount of radioactivity incorporated during the labeling period. These parameters are experimentally established and a typical application of the procedure that demonstrates the hydroxyurea inhibition of DNA synthesis is presented.


Immune mechanisms and the action of interferon in chronic hepatitis B virus infection.

Cytotoxic T lymphocytes can eliminate cells infected with hepatitis B virus. A defect of T lymphocyte-mediated cytolysis seems to exist in chronic hepatitis B infection. T cell-mediated lysis is dependent on HLA antigens of the infected host and this may explain the increased or decreased frequency of particular HLA antigens in chronic carriers of hepatitis B virus. This virus may decrease the concentration of HLA antigens expressed on liver cells. Interferon increases the HLA antigen expression on T lymphocytes and hepatocytes, thereby enhancing T lymphocyte-mediate lysis of infected liver cells and elimination of the hepatitis B virus.


Nonspecific immunostimulant activities of synthetic trehalose-6,6'-diesters (lower homologs of cord factor).

Mycobacterial cord factors (6,6'-diesters of trehalose with mycolic acids ranging from C80 to C90) have been shown to protect mice effectively against infection with Klebsiella pneumoniae or with Listeria monocytogenes. Our present findings indicate that the low-molecular-weight cord factor of Corynebacterium diphtheriae (with corynomycolic acids ranging from C28 PTO C36) is equally active. Moreover, its synthetic analog (with synthetic C32 mycolic acid) has the same activity. Two lower synthetic 6,6'-diesters of trehalose with C22 acids, which are described here for the first time, as well as dipalmitate and a dioleate of sucrose, were found inactive. The synthetic C76 trehalose diesters, which are capable of enhancing nonspecific resistance to infection, increase the immune response in mice, even when injected in metabolizable oil. They induce in the injected paws an inflammatory process weaker and more transient than the natural cord factor.


Effect of systemic candidiasis on blastogenesis of lymphocytes from germfree and conventional rats.

Germfree and conventional rats were challenged (intravenously) with Candida albicans and sacrificed at various times after infection, and their spleen cells were harvested to examine the effect of disseminated candidiasis on in vitro lymphocyte hypersensitivity to Candida antigens (CA). Results showed that conventional rat splenocytes, initially responsive in vitro to stimulation by CA, manifested a depression in CA-specific responsiveness after challenge with viable C. albicans (days 3 to 6 postchallenge). In addition, the latter splenocyte response to phytohemagglutinin (PHA) and concanavalin A (ConA) was suppressed by 3 to 6 days after challenge with Candida. In contrast to conventional rats, the response of germfree rat splenocytes to CA was insignificant before challenge with C. albicans, and it was increased at 9 days after infection. The response of uninfected germfree rat splenocytes to PHA and ConA was significantly lower than that of unchallenged conventional rats. Challenge with viable C. albicans did not result in a suppression of gnotobiotic rat splenocyte responses to PHA and ConA, but rather, the disseminated infection resulted in as much as fivefold increases in PHA or ConA-induced blastogenesis. These findings suggest that disseminated candidiasis is capable of suppressing blastogenesis in immunologically mature conventional rats and of improving lymphocyte blastogenesis from immunologically immature germfree rats.


Adherence of group A streptococci to human epithelial cells.

The adherence of group A streptococci to epithelial cells was studied by using streptococcal strains labeled with [(3)H]uridine or fluorescein isothiocyanate. The ability of the labeled organisms to adhere to Detroit 562 epithelial cells, derived from a human pharyngeal carcinoma, as well as to epithelial cells scraped from the oral cavity was determined. Adherence to monolayer cultures or cell suspensions of Detroit cells compared favorably with adherence to suspensions of human oral epithelial cells. Initial experiments to determine the optimal conditions for adherence showed that adherence was temperature dependent and that the optimal incubation time was 15 min for adherence to epithelial cells in suspension and 30 to 60 min for monolayer cultures. Both streptococci and epithelial cells exhibited specificity in the adherence process. Different streptococcal strains varied in their ability to adhere. Adherence was also affected by the growth stage of the bacterial cultures. Trypsin treatment of the streptococci slightly decreased adherence, whereas hyaluronidase treatment increased the adherence of some strains. Streptococci were found to adhere to only about half of the epithelial cells. Those epithelial cells apparently have a limited number of receptor sites since they can be saturated by adding increasing concentrations of bacteria. Further support for limited receptor sites was provided by competition experiments. Adherence was inhibited by trypsin treatment of the epithelial cells, suggesting that proteins in the epithelial cell membrane may play a role in streptococcal adherence.


Platelet migration inhibtion as an indicator of immunologically mediated target cell injury in canine ehrlichiosis.

A platelet migration inhibition test was devised to determine the presence of antiplatelet activity in serum collected from experimentally produced and natural cases of canine ehrlichiosis. The maximum platelet migration inhibition effect was observed during the acute phase of the disease and before the appearance of specific humoral antibody, measured by the indirect fluorescent-antibody test. Platelet migration inhibition may be one of the earliest events leading to pancytopenia. In most cases, sera positive for humoral antibodies also were positive for platelet migration inhibition, although no direct correlation was evident between the serological titer and the degree of platelet migration inhibition. Inoculation of dogs with uninfected canine blood did not induce the production of inhibition factor or antibody activity, which precluded a histocompatibility response to the cellular elements in the inoculum. Scanning electron microscopy indicated that the platelet inhibition factor interfered with platelet migration by inhibiting pseudopod formation. Affected platelets became rounded and showed evidence of clumping and leakage.


Inhibition of macrophage phagocytic activity by group A streptococcal cell walls.

Group A streptococcal cell walls inhibited phagocytosis of polystyrene latex particles by rat peritoneal macrophages in vitro. This inhibition was not accompanied by a measurable loss of cell viability. Group D streptococcal cell walls were relatively ineffective in altering phagocytosis. The effectiveness of group A variant streptococcal cell walls and peptidoglycan derived from group A cell walls was increased if cell wall preparations were added to macrophage cultures 12 h before, rather than simultaneously with, latex particles.


Cryptococcus neoformans: pseudohyphal forms surviving culture with Acanthamoeba polyphaga.

During experiments on the gastrointestinal tract as a possible portal of entry for Cryptococcus neoformans, we occasionally observed the free-living amoeba, Acanthamoeba polyphaga, growing in the presence of C. neoformans cultured from mouse feces. Examination of the amoebic trophozoites revealed that they were engorged with yeast cells. Over a period of 2 to 3 weeks of incubation, the amoebae apparently killed most of the yeast cells. Some of the surviving C. neoformans cells formed atypical colonies which contained pseudohyphae. Seven other strains have since been cultured with this amoeba. Pseudohyphal forms were found among the surviving colonies in all strains tested. Virulence studies were performed on one randomly selected pseudohyphal isolate from each of the eight strains of C. neoformans. Pseudohyphal isolates from seven of the eight strains failed to kill mice 30 days after intracranial inoculation. The potential role of soil amoebae in the control of C. neoformans in nature is discussed.


Candida albicans endocarditis: ultrastructural studies of vegetation formation.

Candida albicans endocarditis was established in rabbits after transaortic catherization. Within 30 to 90 min after infection, C. albicans was observed by scanning electron microscopy on the valve surface. The organisms were predominantly associated with host deposits of erythrocytes, phagocytes, platelets, and fibrinous-appearing material, which collectively appeared on the valve surface in response to trauma. Within 48 h after infection, vegetations composed of these same host components were observed on the heart valves, although Candida cells were not demonstrable on the valve or vegetation surface. When the vegetations were examined by transmission electron microscopy, Candida blastospores were only observed within phagocytic cells (predominantly monocytes) enmeshed within the vegetation matrix. Many of the intracellular organisms were undergoing degradation, as evidenced by a reduction in electron density of the cell wall. Other fungi had highly electron-dense cell walls and germ tubes. Phagocytic cells containing germinating Candida were highly vacuolated and were observed at various stages of cell lysis. After 7 days of infection, the vegetation contained a dense meshwork of fibrin and Candida pseudohyphae with 10(8) to 10(9) colony-forming units/g of vegetation. The mature vegetation was devoid of phagocytic cells and continued to grow until the death of the animal.


Virulence of Nocardia asteroides during its growth cycle.

Cells of Nocardia asteroides undergo structural and chemical changes, especially in the cell wall, during growth in brain heart infusion broth. Experiments were devised to determine whether these changes affected the virulence of Nocardia for mice. It took, on the average, 1,380 times the number of colony-forming units at the stationary phase to achieve the same mortality induced by the log-phase cells. Cells in either the lag phase or early stationary phase of growth were intermediate in the numbers of colony forming units required to kill mice. Dry-weight determinations at different stages of growth demonstrated that the log-phase organisms were approximately 10 times heavier than stationary-phase cells. Thus, on the basis of dry-weight (micrograms) values, the average colony-forming unit of log phase is approximately 130 times more virulent than in stationary-phase cultures. Therefore, the stage of growth affects greatly the virulence of N. asteroides.


Enteropathogenicity of Escherichia coli isolated from hamsters (Mesocricetus auratus) with hamster enteritis.

Escherichia coli isolated from ilea of hamsters with hamster enteritis were tested for enteropathogenicity in intestinal loops prepared in both adult and weanling hamsters. E. coli isolated from hamsters with hamster enteritis caused dilatation of loops in weanling hamsters but not in adult hamsters.


Effects of age and ambient temperature on the responses of infant mice to heat-stable enterotoxin of Escherichia coli: assay modifications.

The response of infant mice to heat-stable enterotoxin from Escherichia coli was affected by the age of the mice (2, 4, 6, and 8 days) and by the ambient temperature (25, 30, and 37 degrees C) after exposure to the enterotoxin. The younger mice and/or mice held at lower temperatures tended to accumulate intestinal fluid (high gut weight/body weight ratios), but older mice and/or mice held at higher temperatures tended to respond with diarrhea and low gut weight/body weight ratios. The standard infant mouse assay forheat-stable E. coli enterotoxin can be simplified, without loss of sensitivity or reliability, by holding the mice at 37 degrees C after exposure and using diarrhea as the index of response. Diarrhea can be detected easily by incorporating dye in the inocula and (at the end of the assay) checking for dye mixed with feces on the rear quarters of the mice or on a sheet of white paper placed under them during incubation.


Modulations of myelopoiesis in vivo by chemically pure preparations of cell wall components from gram-negative bacteria: effects at different stages.

Modulation of myelopoiesis by chemically pure preparations of different cell wall components from gram-negative bacteria was investigated in vivo. The effects of lipid A, outer membrane lipoprotein, and murein were evaluated at several distinct stages: induction of colony-stimulating activity (CSA) in the serum, increase in the number of committed splenic precursor cells (CFU-C) forming granulocyte-macrophage colonies in vitro, and triggering into the cell cycle of noncommitted hemopoietic stem cells (CFU-S) from bone marrow. The results reveal different patterns of activity of the bacterial cell wall components (BCWC) tested. (i) In C57Bl/6 mice and C3H/Bom mice, all three preparations were potent inducers of CSA. In C3H/HeJ mice, CSA was only induced by lipoprotein and murein and not by lipid A. After injection of lipid A or lipoprotein, but not murein, the number of CFU-C in spleens of C57Bl/6 mice was increased up to 100-fold. In C3H/Bom and C3H/HeJ mice, not only murein but also lipoprotein were much less potent in this respect. (iii) In C57Bl/6 mice, both lipid A and lipoprotein, but not murein, were capable of inducing the proliferation of CFU-S, as demonstrated by a hot thymidine cytocide technique. Thus, induction of CSA and changes in the pool size of splenic CFU-C after administration of BCWC may be unrelated events. On the other hand, the increase of CFU-C might reflect the mitogenicity of BCWC for CFU-S.


Association of hemolysin production, hemagglutination of human erythrocytes, and virulence for chicken embryos of extraintestinal Escherichia coli isolates.

One hundred forty-two strains of Escherichia coli isolated from extraintestinal infections were examined for colicin V (ColV) and hemolysin (Hly) production. For comparison, 20 strains isolated from the feces of normal individuals and 12 enteropathogenic strains of E. coli were tested for these properties. Thirty-five to 59% of extraintestinal isolates were Hly+, but only one fecal strain was Hly+. Colicin V biosynthesis was found for 12% of blood culture isolates, 7% of urine culture isolates and 16% of the strains from other extraintestinal infections. None of the fecal isolates was ColV+. Selected strains were tested for virulence in 13-day-old chicken embryos; these same strains were tested for their ability to hemagglutinate chicken or human erythrocytes. Of 22 extraintestinal isolates, 13 (59%) killed greater than or equal to 60% of the embryos within 72 h. Only one of eight normal fecal isolates and two of three enteropathogenic strains tested were virulent. About 80% of the virulent strains were Hly+. The most striking finding, however, was the hemagglutination of human erythrocytes by virulent extraintestinal isolates. It seems possible that the hemagglutination property reflects a specific common adherence factor.


Increased bone marrow production of granulocytes and mononuclear phagocytes induced by mycobacterial adjuvants: improved recovery of leukopoiesis in mice after cyclophosphamide treatment.

The effects of complete Freund adjuvant (CFA) or Mycobacterium bovis BCG on leukopoiesis and on leukopoietic recovery from cyclophosphamide treatment in mice was studied. CFA injected subcutaneously or intraperitoneally resulted in increased blood granulocyte and monocyte counts, increased numbers of bone marrow granulocyte and mononuclear phagocyte progenitors, and increased hematopoietic colony-stimulating factor in the serum. Furthermore, the quantitative cellular response within 24 h to an induced sterile intraperitoneal inflammation (thioglycolate) was augmented by subcutaneous CFA. In mice given CFA subcutaneously, blood granulocyte counts, as well as the peritoneal granulocyte and macrophage response to intraperitoneal thioglycolate, recovered more quickly than did those of the controls after a 250-mg/kg dose of cyclophosphamide. CFA-treated mice consistently maintained blood granulocyte and monocyte counts 1.3-to 4-fold higher than those of the controls for 2 weeks while receiving 75 mg of cyclophosphamide per kg every other day. Mice pretreated with CFA intraperitoneally had higher numbers of bone marrow colony-forming units in culture and higher levels of serum colony-stimulating factor after 250-mg/kg injections of cyclophosphamide. Similarly, BCG resulted in increased bone marrow colony-forming units in culture, increased serum colony-stimulating factor, and a faster return of the peritoneal inflammatory response after cyclophosphamide injection. These results show that mycobacterial adjuvants accelerate recovery of leukopoietic functions after cyclophosphamide treatment and suggest a mechanism whereby such adjuvants afford nonspecific protection against infection in immunosuppressed mice.


Protection against ascending Escherichia coli pyelonephritis in rats and significance of local immunity.

Acute pyelonephritis in rats caused by Escherichia coli O6K13H1 produced serum and urinary antibodies against O6 and K13 antigens. This was also seen after intravesical immunization with Formalin-killed bacteria. Both intraperitoneal and intravesical immunization with these Formalin-killed bacteria protected against ascending urinary tract infection induced by homologous bacteria. Passive transfer of urine containing both O6 and K13 antibodies also protected against infection. By absorption experiments it was shown that K13 antibodies were especially important.


Experimental murine candidiasis: cell-mediated immunity after cutaneous challenge.

Attempts were made to isolate an antigen(s) from Candida albicans suitable for detecting hypersensitivity in a murine model of candidiasis. Using footpad reactivity in cutaneously infected animals as the assay, comparisons were made of two commercial extracts and cell wall and cytoplasmic preparations made in the laboratory. An extract of the cell wall, a glycoprotein (GP) removed with ethylenediamine, and an extract prepared from the membrane fraction of disrupted C. albicans blastospores proved most useful in demonstrating delayed hypersensitivity in the murine model. The activity of the GP fraction was considerably reduced by oxidation with periodate and was abrogated entirely by digestion with proteolytic enzymes. The extract from the membrane fraction was obtained by incubating the insoluble membrane fraction with phosphate-buffered saline, pH 7.4, at 50 degrees C, and the proteins in the extract were subsequently precipitated with ammonium sulfate to yield a test preparation that was approximately 75% protein and 25% carbohydrate. The precipitated extract was designated ppt-HEX. Footpad reactivity to ppt-HEX could be transferred with cells and not with serum if the cells were taken from animals at the appropriate time after sensitization. Since the membrane and GP fractions appear to elicit true delayed hypersensitivity reactions, further investigations into their specificity and biochemistry seem warranted.


Production and partial purification of Salmonella enterotoxin.

By using a strain of Salmonella typhimurium, we detected the presence of an enterotoxin, as determined by the rabbit ileal loop assay, in various complex and defined media. The enterotoxin was concentrated by ultrafiltration of culture supernatant fluids and eluted in and adjacent to the void volume of a Sephadex G-100 column. This suggested that the enterotoxic factor was of a relatively high molecular weight, and additional evidence indicated it was heterogeneous in size. Further chromatography, using a diethylaminoethyl-cellulose anion exchanger, facilitated at least a 50-fold purification of the Salmonella enterotoxin.


Infectious agents in immunodeficient murine models: pathogenicity of Nocardia asteroides in congenitally athymic (nude) and hereditarily asplenic (Dh/+) mice.

Congenitally athymic (Nu/Nu), hereditarily asplenic (Dh/+), and littermate control mice were given intravenous injections of homogeneous cell suspensions of the virulent Nocardia asteroides GUH-2. Kill curve, 50% lethal dose, and kidney clearance data were obtained over a period of 3 months postinfection. N. asteroides initiated both an acute infectious process and a chronic, progressive disease in these animals when given intravenously. The heterozygous (Nu/+) mice appeared to be slightly more susceptible to the acute phase of infection than their nude littermates. In contrast, nude mice were at least 50 times more susceptible to chronic nocardial infection than were the heterozygous (Nu/+) controls. Swiss Webster specific pathogen-free mice were similar to heterozygous (Nu/+) mice in their susceptibility to N. asteroides. The hereditarily asplenic (Dh/+) mice were not as susceptible to lethal infection as were nude mice. However, asplenic mice demonstrated an inability to eliminate nocardia from infected kidneys, whereas their littermate control (+/+) mice were able to mount an effective response and destroy most of the organisms within the kidneys. Similar observations were noted when nude and heterozygous (Nu/+) littermate mice were infected in the footpad. The nude mice developed a systemic infection and died within 4 weeks with little inflammation of the footpad and no macroscopic lesions. In contrast, heterozygous (Nu/+) mice developed extensive local abscesses in the foot that persisted for at least 4 weeks. There was no animal death and no evidence of dissemination. The data presented herein indicate that T cells are essential for adequate host response against infection with a virulent strain of N. asteroides.


Ideal target organism for quantitative bactericidal assays.

We have developed a target organism which permits quantitative bactericidal assays. The organism is an Escherichia coli mutant which cannot grow at the temperature of the assay (37 degrees C), but retains full colony-forming potential for subsequent quantitation at 25 degrees C. We show that quantitative data on the bactericidal capacity of polymorphonuclear leukocytes and alveolar macrophages can be obtained when this mutant is used as a target. The procedure used to generate the strain is described in detail and should be applicable to many bacterial species. Characterization of the properties of the mutant indicates that it has a strong potential for use in other in vivo and in vitro investigations of host responses to microbial invasion.


Pathogenesis, lethality, and immunizing effect of experimental cutaneous cryptococcosis.

Mice were subcutaneously inoculated with small numbers of virulent Cryptococcus neoformans and divided into groups. Numbers of viable yeasts at the site were estimated at weekly intervals for 5 weeks on the basis of cultures of minced tissue excised from sacrificed animals. Organisms multiplied at the site for at least 4 weeks and were still detectable after the 5th week, although in reduced numbers. Agglutinins appeared within a week, but these antibodies were not detectable during the 2nd through the 5th week. Cryptococcal polysaccharide began to appear in the sera at 3 weeks, persisting through the duration of 5 weeks. All animals appeared healthy, but a few sickened after many months and died of systemic cryptococcosis. All of these events were observed in many separate experiments. The immunizing capacity of a cutaneous lesion was tested by challenging some of the above animals with viable C. neoformans after various intervals of time, either subcutaneously at a site distant from that of the vaccination or intravenously. Although we were unable to demonstrate reduced multiplication of yeasts in the brains, lungs, and spleens of intravenously challenged animals, it was possible to show that multiplication was inhibited at the site of subcutaneous challenge. It was noted also that vaccinated animals lived longer after lethal intravenous challenge than did nonvaccinated animals. The latter protection was observed, however, only when challenge followed vaccination by 3 weeks or longer, and it was effective only against a relatively low challenge dose. Mice were protected against a higher dose if they had previously received killed cryptococci, alternating subcutaneous and intraperitoneal inoculations, one of which contained a microbial adjuvant. No protection was observed in animals that were subcutaneously vaccinated with inert materials such as chitin, latex spheres, or even cryptococcal cell walls themselves.


Lactoperoxidase, peroxide, thiocyanate antimicrobial system: correlation of sulfhydryl oxidation with antimicrobial action.

The antimicrobial activity of the lactoperoxidase, peroxide, thiocyanate system against Escherichia coli was directly related to the oxidation of bacterial sulfhydryls. Lactoperoxidase catalyzed the oxidation of thiocyanate, which resulted in the accumulation of hypothiocyanite ion, OSCN-. A portion of the bacterial sulfhydryls were oxidized by OSCN- to yield sulfenic acid and sulfenyl thiocyanate derivatives. The remaining sulfhydryls were not oxidized, although OSCN- was present in large excess. The oxidation of sulfhydryls to sulfenyl derivatives inhibited bacterial respiration. This inhibition could be reversed by adding sulfhydryl compounds to reduce the sulfenyl derivatives and the excess OSCN-. Also, this inhibition could be reversed by washing the cells so as to remove the excess unreacted OSCN-. After washing, the bacteria underwent a time-dependent recovery of their sulfhydryl content. This recovery resulted in recovery of the ability to respire. The inhibited cells were viable if diluted and plated shortly after the incubation with the lactoperoxidase, peroxide, thiocyanate system. On the other hand, long-term incubation in the presence of the excess OSCN- resulted in loss of viability. Also, the inhibition of respiration became irreversible. During this long-term incubation, the excess OSCN- was consumed and the sulfenyl derivatives disappeared.


Heterogeneity of group A streptococcal pyrogenic exotoxin type B.

Streptococcal pyrogenic exotoxin type B purified from culture filtrates of either the NY-5 or T-19 strain of group A streptococcus was found to be heterogeneous in charge. Three protein fractions with isoelectric points of 8.0, 8.4, and 9.0 were isolated by differential solubility in ethanol and acetate-buffered saline followed by isoelectric focusing and shown to be antigenically identical to streptococcal pyrogenic exotoxin type B. The molecular weights of all three fractions were approximately 17,500, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with aggregates forming in the presence of hyaluronic acid. Only the pI 8.4 fraction showed the characteristic activities of streptococcal pyrogenic exotoxin in rabbits: pyrogenicity and ability to enhance susceptibility to lethal endotoxin shock. The pI 8.0 and pI 9.0 fractions were not pyrogenic, but could be used to immunize against pyrogenicity. These two fractions failed either to enhance lethal endotoxin shock or to immunize against enhancement activity. When the isolated fractions were electrofocused again they appeared heterogeneous, suggesting an instability of the B toxin molecular forms.


Relationship between delayed-type hypersensitivity and the progression of Mycobacterium lepraemurium infection.

The relationship between the level of delayed-type hypersensitivity (DTH) and the progression of Mycobacterium lepraemurium infection was examined after inoculation of mice with 10(8) M. lepraemurium in the left hind footpad. The expression of DTH developed over the first 4 weeks of infection, remained high up to week 8, and then dropped to a low level at which it remained for 12 more weeks. The development of DTH was concordant with an initial swelling of the inoculated foot, the appearance of a mononuclear infiltrate at this site, and a prevention of any increase in the number of mycobacteria in this foot and in other tissues studied. A decay of DTH reactivity was associated with a progressive increase in the number of M. lepraemurium initially at the original site of inoculation and subsequently in all other tissues. Although the expression of DTH was lost, adoptive immunization experiments showed that a population of sensitized lymphocytes persisted within host. Further experimentation offered evidence to suggest that the level of systemic antigen may be in part responsible for the loss of DTH reactivity.


Characterization of plasmids that encode for the K88 colonization antigen.

K88 antigen, and important virulence factor in porcine enteropathogenic Escherichia coli (EEC), can be transferred along with the ability to ferment the trisaccharide raffinose (Raf). The plasmids from a number of EEC strains that encode these two properties were isolated and characterized. In most strains the K88 and Raf genes were found on a single nonconjugative plasmid approximately 50 x 10(6) daltons in size. This plasmid core was conserved with only slight variation among the strains tested. In some transconjugants, larger conjugative plasmids were observed that were apparently recombinants between the Raf/K88 plasmid and a transfer fa(tor. Occasionally plasmids carrying only the raffinose fermentation genes arose by deletion of a deoxyribonucleic acid segment of about 20 x 10(6) daltons that included the K88 antigen gene(s).


Isolation and characterization of a Streptococcus mutans bacteriocin inhibitor from Streptococcus pyogenes.

An inhibitor of Streptococcus mutans strain GS5 bacteriocin was released from a bacteriocin-sensitive strain of S. pyogenes by sonic treatment and partly purified by gel filtration on a Sepharose 6B column. The inhibitory factor was sensitive to heat, trypsin, and periodate and possessed a molecular weight of approximately 93,000. This factor may be the receptor for the S. mutans bacteriocin.


Immediate cytotoxicity of Chlamydia trachomatis for mouse peritoneal macrophages.

The toxicity of Chlamydia trachomatis was studied with mouse peritoneal macrophage culture. Inoculation of 30 inclusion-forming units of trachoma B/TW-5/OT organisms and 250 inclusion-forming units of lymphogranuloma venereum L2/434/Bu organisms per cell caused immediated toxicity, with the killing of 40 to 90% of the macrophages within 6 h after inoculation. Inhibition of phagocytosis by adsorption at 0 degrees C or by NaF pretreatment of macrophages prevented the toxicity, indicating that chlamydiae must be phagocytized to induce toxicity. Infectivity and toxicity could be dissociated, since ultraviolet-inactivated chlamydiae were still toxic. However, the toxicity was destroyed by heating the organisms at 56 degrees C for 10 min. Tetracycline, and antichlamydial drug, did not prevent toxicity, indicating that multiplication of the organisms was not required to induce toxicity. Toxicity was not prevented by treatment of macrophages with hydrocortisone. The toxicity of trachoma TW-5 was reduced by the rabbit immune serum of trachoma TW-5 but not by the rabbit immune serum of psittacosis meningopneumonitis.


Enhancement of lymphocyte blastogenic and delayed hypersensitivity skin responses by indomethacin.

Incubation of Mycobacterium bovis-sensitized bovine peripheral blood lymphocytes with concanavalin A or M. bovis purified protein derivative and indomethacin caused a consistent, statistically significant increase in [3H] thymidine uptake as compared to cultures without indomethacin. The kinetics of  the response showed that indomethacin must be added to lymphocyte cultures within hours after mitogen or antigen addition for enhancement of [3H]thymidine uptake to occur. Lymphocyte blastogenic responses to purified protein derivative were enhanced in both normal and M. bovis-sensitized lymphocyte cultures. However, enhancement of sensitized lymphocyte responses was significantly (P less than 0.01) greater than that in normal animals. Additionally, indomethacin treatment of M. bovis-sensitized guinea pigs singnificantly augmented delayed skin reactions to tuberculin. Delayed hypersensitive skin reactions were only enhanced when indomethacin was administered simultaneously with tuberculin.


Enzyme-linked immunosorbent assay for measurement of serological response to respiratory syncytial virus infection.

An enzyme-linked immunosorbent assay was applied to the detection of serum antibodies against respiratory syncytial virus. The end points of the various sera tested in the assay were approximately 100 times higher than in the complement-fixation test and 2 to 4 times higher than in the plaque reduction test. In addition, the immunosorbent assay appeared to be more efficient than the plaque reduction and complement-fixation techniques for detecting a serological response in young infants (1 to 6 months old) with serous respiratory syncytial virus lower respiratory disease. The simplicity, sensitivity, and rapidity of the enzyme-linked immunosorbent assay make it a useful tool for immunological studies with respiratory syncytial virus.


Relationship of macrophages to cell-mediated immunity in experimental Nocardia asteroides infection.

Marked in vivo intracellular killing of Nocardia asteroides occurred in the peritoneal macrophages obtained 72 h after an intraperitoneal challenge with N. asteroides, in guinea pigs either actively immunized with ribonucleic acid protein or passively immunized by immune spleen cell transfer from actively immunized donor guinea pigs. This specific killing of N. asteroides in immune macrophages persisted for at least up to 60 days. Administration of antimacrophage sera before intravenous challenge with N. asteroides in the immune guinea pigs produced an early death of the animals, and the total tissue counts of N. asteroides in the liver, spleen, lungs, and heart remained the same in them as in unimmunized controls.


Specific and nonspecific resistance in mice immunized with irradiated Myobacterium leprae.

Following subcutaneous inoculation of irradiated Mycobacterium leprae (I-ML) into the left hind footpad of mice, there was increased resistance to Listeria monocytogenes, indicative of macrophage activation, at the immunization site. In spite of the high level of localized macrophage activation which was proportioned to the immunizing dose of I-ML, no such activity could be demonstrated systemically in these mice, as evidenced by the absence of increased resistance to an intravenous challenge with L. monocytogenes. Under these conditions, I-ML-immunized mice were nonetheless resistant to intravenous infection with either M. tuberculosis or M. bovis BCG, and this immunity was transferred to normal recipients using spleen or lymph node cells. Neonatal thymectomy completely abolished the development of antimycobacterial immunity after vaccination with I-ML, but immunity was restored by an intraperitoneal infusion of syngeneic thymocytes. Systemic nonspecific resistance could be generated in I-ML-immunized mice by an intravenous injection of disrupted I-ML. This study reveals that, after subcutaneous vaccination with I-ML, there is local accumulation of activated macrophages at the inoculation site and a widespread distribution of lymphocytes which are sensitized to mycobacterial antigens. Nonspecific resistance is mediated by the former cells and specific antimycobacterial immunity by the latter.


Water-soluble adjuvant obtained from Bacterionema matruchotii.

The adjuvant effect of a butanol-extracted water-soluble adjuvant (bu-WSA) obtained from Bacterionemia matruchotii, a gram-positive oral bacteria, was studied on the antibody response at the plaque-forming cell (PFC) level in murine spleens. Intraperitoneal injection of Bu-WSA caused significant increase in direct PFC numbers in spleens 1 to 3 days after the antigenic stimulation with sheep erythrocytes (SRBC). Injection of 100 to 800 microgram of Bu-WSA was effective, and 400 microgram of Bu-WSA seemed to be the optimum for induction of the adjuvant effect. The adjuvant effect was strongest when Bu-WSA  was injected at the same time as the SRBC, but some effect was still observed when Bu-WSA was injected 7 days before or 1 day after the immunization. The adjuvant effect of Bu-WSA was greatest at high dose of antigen. The mice injected with Bu-WSA at the time of priming SRBC and then immunized with trinitrophenylated SRBC showed greater anti-trinitrophenyl PFC response than controls without the injection of Bu-WSA. These findings suggest that a part of the adjuvant effect of Bu-WSA depends on thymic cell function and another part does not.


Mechanisms of lipopolysaccharide-initiated rabbit platelet responses: alternative complement pathway dependence of the lytic response.

Experiments were performed to examine the relationship of endotoxin-initiated complement activation and rabbit platelet lysis. The results of these experiments supported the concept that activation of the alternative pathway is required for endotoxin-initiated complement-dependent rabbit platelet lysis. Our data demonstrated that preparations of endotoxin or isolated lipid A, which activate selectively the classical pathway, are incapable of initiating platelet lysis. Essentially equivalent results were obtained in citrated or heparinized plasma, although the latter anticoagulated plasma appeared to be more efficient in supporting lysis. Additional data support the concept that natural antibody to either the polysaccharide or the lipid A region of the lipopolysaccharide, which might be present in rabbit plasma, probably did not play a prominent role in the complement-mediated lytic response.


Meningeal exudate cells in vaccinia meningitis of mice: role of local T cells.

Intracerebral inoculation of vaccinia virus into adult DDD mice produced intensive meningitis several days after infection. The inflammatory reaction could be quantitated by counting cells obtained from this inflammatory lesion. The local virus titer increased until day 5 and subsequently decreased rapidly with time. Concomitant with this titer decrease, numerous meningeal exudate cells appeared in the lesion. The cytotoxic activity of these cells against vaccinia virus infected cells was studied after removal of glass-adherent cells. The results showed that these meningeal exudate cells possessed cytotoxic activity against the virus-infected cells and moreover inhibited plaque formation by vaccinia virus. The magnitude of this activity was much larger than that of spleen cells obtained from the same animals. After treatment with antithymocyte serum, or with antitheta serum plus complement, the meningeal cells lost their inhibitory activity, suggesting that the cells which exerted the effect were mainly T lymphocytes. The meningeal exudate cells obtained on day 7 postinfection were further characterized. A greater part, approximately 80%, of the cell population was composed of theta-positive cells. Less than 1% carried immunoglobulin, 7% possessed neither theta antigen nor immunoglobulin on the surface, and 12% represented glass-adherent cells.


Dichotomy between opsonization and serum complement activation by encapsulated staphylococci.

Previous studies have demonstrated that encapsulated Staphylococcus aureus strains are not effectively opsonized by the serum complement system. Encapsulated staphylococci thereby "resist phagocytosis." To test whether this phenomenon might be explained by an inability of encapsulated strains to activate complement, the relationship between staphylococcal opsonization and serum complement activation was studied. Although encapsulation was found to interfere with opsonization by pooled human serum (human polymorphonuclear leukocytes phagocytized significantly fewer encapsulated bacteria than unencapsulated bacteria after incubation in this opsonic source), encapsulated (S. aureus M and Smith diffuse) and unencapsulated (S. aureus M variant and Smith compact) strains had similar capacities for complement activation as measured by C3-C9 consumption. When C2-deficient and immunoglobulin-deficient sera were studied, again C3-C9 consumption was not influenced by the presence or absence of a capsule. In addition, C3 was detected on the surface of both S. aureus M and M variant strains after incubation in pooled serum and staining with fluorescein-conjugated anti-C3 antibody. Thus, encapsulated staphylococci are not effectively opsonized even though complement is activated and C3 is present on the bacterial surface. The exact mechanism by which the capsule interferes with opsonization is still not known; however, inhibition of complement activation appears not to be the explanation of this phenomenon.


Protective effect of glucan against systemic Staphylococcus aureus septicemia in normal and leukemic mice.

The reticuloendothelial stimulant glucan, a beta-1,3-polyglucose component of the cell wall of Saccharomyces cerevisiae, was evaluated for its ability to modify Staphylococcus aureus-induced lethality in normal and leukemic mice. In normal mice the intravenous injection of glucan (0.45 mg per mouse) 7 and 4 days prior to intravenous challenge with S. aureus (1.0 x 10(9)) resulted in a significantly increased survival. Histological examination of the kidneys revealed that glucan significantly inhibited renal necrosis associated with systemic staphylococcal diseases. Further studies indicated that glucan administration not only enhanced survival of leukemic mice, but also increased survival of leukemic mice with experimentally induced staphylococcal speticemia. These data denote that glucan enhances nonspecific resistance to S. aureus sepsis, promotes survival during leukemic episodes, and increases survival time of leukemic mice with experimentally induced staphylococcal infection.


Role of the surface coat in in vitro attachment and phagocytosis of Plasmodium berghei by peritoneal macrophages.

Evidence is presented to indicate that Plasmodium berghei merozoites, but not trophozotites, have an antiphagocytic capsule. The capsule appears to form around the developing merozoties of the schizont in the parasitophorous vacuole. Serum from animals immune to P. berghei reacts with this capsule. After reaction with immune serum, the antiphagocytic action of the capsule is lost. By the process of binding serum protein, the capsule becomes electron dense and can be readily visuallzed as the surface coat by electron microscopy. At physiological temperatures, phagocytosis by macrophages rapidly follows adhesion of antibody-coated parasites. Both tight and loose phagosomes are formed.


In vitro interaction of alveolar macrophages and pneumocytes with feline respiratory viruses.

Feline alveolar macrophages and feline pneumocytes were inoculated in vitro with low multiplicities of either feline calicivirus or feline viral rhinotracheitis virus. Pneumocytes were permissive for both viruses. High titers were attained, and characteristic cytopathic effects developed. Alveolar macrophages were permissive for feline viral rhinotracheitis virus, although the cycle of replication was delayed. Infection of macrophages with feline calicivirus resulted in the production of viral antigens and cytopathic effects; however, viral particles were not detected by electron microscopy, and viral infectivity titers rose only slightly and then fell to undetectable levels by 96 h. The differences in viral susceptibility between these two peripheral pulmonary cell populations that were demonstrated in vitro probably contribute to the differences in pathogenesis of viral rhinotracheitis and calicivirus infections in cats.


Measles virus nucleocapsids: large-scale purification and use in radioimmunoassays.

Nucleocapsids in quantities approaching 1 mg were purified from 109 measles virus-infected cells. They contained one polypeptide species with a molecular weight of 59,000. Antiserum was raised in rabbits against purified nucleocapsids and used in a competitive radioimmunoassay. Because of their instability, purified nucleocapsids were not suitable for use in such an assay. Instead partially purified nucleocapsids from HEp-2 cells persistently infected with measles virus and labeled in vitro with [35S]methionine were used as the source of radioactive antigen. The radioimmunoassay thus developed measured less than 5 ng of nucleocapsids in infected cells.


Bronchopulmonary cellular response to aluminum and zirconium salts.

The bronchopulmonary cellular immunological response to repeated intratracheal inoculation of aluminum chlorhydrate, sodium zirconium lactate, and zirconium aluminum glycine was examined in rabbits. Results of a dose-response experiment using 0.1, 1.0, and 10.0-mg intratracheal inoculations of each metallic salt demonstrated significant bronchopulmonary histopathology in the 10.0-mg dose-response groups only. Acute lesions were histologically characterized by an inflammatory response centered around respiratory bronchioles. Although epithelioid cell formation was evident in 10.0 mg of aluminum salt (aluminum chlorhydrate and zirconium aluminum glycine) -injected animals, no well-defined granulomas characterized by an orderly arrangement of epithelioid cells, lymphocytes, and giant cells were evident in any of the experimental groups employed. All three metallic salts induced "activated" bronchopulmonary macrophages as determined by an in vitro phagocytic assay. This activation was likely nonimmunological since no measurable differences were observed in metallic salt-induced delayed skin reactivity or migration inhibition factor production between inoculated and uninoculated rabbits. The above observations suggest that aluminum and zirconium salts administered in comparatively high dosage via the respiratory tract route can induce respiratory bronchiolitis and activation of alveolar macrophages in the absence of demonstrable delayed hypersensitivity.


Serotyping of Chlamydia: antibodies to lymphogranuloma venereum strains compared by microimmunofluorescence and neutralization tests.

The microimmunofluorescence test with hyperimmune rooster sera against lymphogranuloma venereum strains gave the same serotype patterns obtained in the standard mouse serum system. Results in a plaque reduction test appeared similar when only 3 prototype strains were tested, but differed when a battery of 11 strains were tested.


Coupling of Escherichia coli lipopolysaccharide to epoxy-activated Sepharose 6B.

Escherichia coli O111:B4 lipopolysaccharide was coupled to epoxy-activated Sepharose 6B. The bound lipopolysaccharide was immunogenic and immunoadsorptive although at less efficiency than free lipopolysaccharide.


Inactivation of polymorphonuclear-neutrophil derived eosinophil chemotactic factor by human serum.

A low-molecular-weight eosinophil chemotactic factor (ECF) can be released from human neutrophils on stimulation with the calcium ionophore (A 23187) and during phagocytosis. The presence of human serum suppresses the chemotaxis of eosinophils by ECF significantly. The inhibition is not cell-directed but affects the ECF either by binding or enzymatic cleavage. The inactivators belong to a heterogeneous class of serum components in the 19s, 7s and 4s molecular weight range. Highly purified human IgM and IgA, but neither IgG nor human serum albumin inhibit eosinophil chemotaxis.


Demonstration of Fc receptors on the surface of B lymphocytes.

Human blood lymphocytes were tested by an immunofluorescence technique for surface immunoglobulin and by a rosette test with IgG-sensitised red cells for Fc receptors. From combined tests and experiments involving fractionation of lymphocyte populations it is concluded that Fc receptors are demonstrable on most B lymphocytes by the rosette test, but only if the red cells are optimally sensitised. These observations are advanced as an explanation of the discrepant results of surface marker tests on B cells.


The beat goes on: status of the artificial heart, 1977.

After two decades of continuous research on the artificial heart, survival times of experimental animals indicate that clinical application of such a device is definitely feasible. However, a number of problems remain to be solved. Durability of the device, infection, pannus formation at the interface between the device and natural tissue and thrombosis within the device appear to be the major problems. Development of an implantable power source is also an area of important research. Questions of efficiency, cosmetic and psychological acceptability and cost are just beginning to be considered.


Apomorphine revived: fortified, prolonged, and improved therapeutical effect.

An historical review is presented of the evolution of the clinical apomorphine treatment. Some of the results from the last 10 years of psychopharmacological research have led us to the hypothesis that there exists a close relationship between abstinence and craving symptoms in drug and alcohol addicts, and that anxiety, depression, and tremor symptoms in Parkinsonism (and dementia senilis), are due to disturbances of the same, mainly dopaminergic, pathways in the CNS. In such cases, by means of effective preparations for oral use, we have utilized the synergistic effect of small amounts of apomorphine, L-dopa, and decarboxylase inhibitor with considerable therapeutic effect.


Covert sensitization in group treatment of adolescent drug abusers.

Seven self-referred males with long-standing histories of excessive use of amphetamines, LSD, and marijuana participated in an experiment dealing with the behavioral procedures of covert sensitization in group treatment. LSD usage did not change during a 14-week precovert-sensitization treatment program. The group then received covert sensitization in 11 sessions within a 6-week period, with all members reporting no LSD usage or desire to take LSD. A 6 to 18-month follow-up indicated that six subjects had not taken LSD. Two subjects reported self-administration of covert sensitization procedures at home to develop aversions to other drugs and were completely drug-free. The seventh subject could not be located. The practical advantages of using covert sensitization procedures in group treatment of drug users within the school setting are discussed.


Clonazepam: its efficacy in association with phenytoin and phenobarbital in mental patients with generalized major motor seizures.

In a 36-week controlled study, the efficacy of clonazepam administered with phenytoin and phenobarbital was evaluated in twenty-four epileptic mental patients suffering from major motor seizures. The patients were distributed in two strata according to the presence or the absence of chlorpromazine. As compared with placebo treatment in the chlorpromazine-free patients, a significant reduction in the frequency of the tonic-clonic seizures was observed during the 24-week clonazepam treatment. This effect could not be observed in the patients requiring the antipsychotic drug. The EEG performed before and at the end of the experimental period did not show any significant change. The most common adverse reactions to clonazepam were drowsiness and ataxia; they diminished with continued treatment.


Comparison of terbutaline and salebutamol aerosols in patients with bronchial asthma.

The authors have compared the activity of Terbutaline with that of Salbutamol in 14 subjects with partially reversible airways obstruction. In this within patients trial FEV1 resulted significantly increased with both compounds and was still significant 240 minutes after Terbutaline.  After Terbutaline there was in addition a better situation in regard to blood gases. Tolerability with both compounds was very good.


A double blind study with melperone and placebo in the treatment of chronic alcoholics.

In a 2-wk randomized double blind study 60 chronic alcoholics were treated with either melperone (Buronil) or placebo. The patients were assessed daily using a scale including 4 items: tension, depression, craving and sleep. Statistically significant improvement was achieved in the placebo group only for "tension" and "sleep"; whereas, in the melperone group all four items improved significantly. Comparison between the groups revealed statistically significant superiority of melperone over placebo for the item "craving".


Methylergometrine: comparison of plasma concentrations and clinical response of two brands.

There were no significant differences between the two brands of methylergometrine (methylergonovine), Methergin and Myomergin, in the radioimmunologically measured serum concentrations nor in the AUC after an oral 0.250 mg dose determined on the third or sixth postpartum day during a continuous treatment with methylergometrine 0.125 mg t.i.d., nor were there significant differences in the clinical response to this oxytocic drug. Peak serum concentrations were obtained at 3 hours (Methergin 6.3 nmol/1 and Myomergin 6.0 nmol/1), indicating a delayed gastrointestinal absorption in postpartum females in comparison with healthy male volunteers [6]. Spontaneous complaints of side-effects were rare and mild.


Are there schizophrenics for whom drugs may be unnecessary or contraindicated?

This study reports that there are schizophrenics who do relatively well long term without the routine or continuous use of antipsychotic medication. Specially selected young males undergoing an acute schizophrenic episode were followed, after hospitalization, for up to three years. While hospitalized they were assigned randomly to either placebo or chlorpromazine treatment. Many unmedicated-while-in-hospital patients showed greater long-term improvement, less pathology at follow-up, fewer rehospitalizations and better overall function in the community than patients who were given chlorpromazine while in the hospital. Factors related to post-hospital outcome were good premorbid history and short-lived paranoid characteristics. Considerations which may have an effect on the successful management of acute schizophrenic patients not on medication are mentioned. The findings underline the need for further study of how to utilize antipsychotic medication more selectively in the treatment of schizophrenia.


Acetylator phenotype in relation to monoamine oxidase inhibitor antidepressant drug therapy.

The MAOI antidepressant drug phenelzine has a molecular structure closely related to drugs known to be acetylated, specifically isoniazid. Acetylator phenotype is genetically determined and shows wide variation between different ethnic groups. Acetylator phenotype could be a major factor as a determinant of response to phenelzine therapy and the findings from three studies are discussed. The implications for other (non-psychotropic) drug therapy are briefly referred to.


Clinical prediction of antidepressant response.

Depression is not a unitary disease and not all cases of depression can be treated alike. Therefore, predictors of antidepressant response to different drugs need to be identified. Premorbid personality, age and sex and family history are useful in prediction. As an example, only females respond to the addition of triiodothyronine to the antidepressant therapy. Endogenous depressions respond favourably to antidepressants, so also do psychotic depressions. On the other hand, delusional depressions do not respond to antidepressants. While atypical depressions are best treated with MAOI, motor retardation improves with imipramine and obsessive symptoms with chlorimipramine. In addition to clinical variables, biochemical and statistical approaches have also yielded significant results in prediction.


Inhibition of macrophage migration by choroidal malignant melanoma-associated antigens in patients with unveal melanoma.

The specific cell-mediated immunity of the lymphocytes of eight patients with choriodal malignant melanoma (MM) to four extracts of choroidal MM-associated antigens was tested with the aid of the MIF technique. Seven of the patients with choroidal MM responded to at least one of the four extracts used, whereas patients with choroidal nevus or carcinoma as well as healthy controls did not respond to any of the MM choroidal extracts. There was no response to iris extracts obtained from the enucleated eyes with MM in any of the subjects tested.


Coxsackievirus aggregates in muscle cells of a polymyositis patient.

Muscle biopsy specimens of a patient with polymyositis showed crystalline structures resembling picornavirus aggregates within muscle cells. The patient's serum reacted in an indirect immunofluorescence assay with autologous muscle cells. A strongly positive immunofluorescence staining was also noted when a section of muscle tissue was reacted with coxsackievirus A9 antiserum, and a weakly positive reaction was noted with coxsackievirus B2 antiserum. No staining was observed after treatment with antiserum to poliovirus type 1 or echovirus types 11 and 22.


Defective neutrophil chemotaxis and hyperimmunoglobulinemia E in a child with recurrent infections.

The clinical and immunologic features of an adolescent boy with recurrent pyogenic infections are presented. A cellular defect in neutrophil chemotaxis, hyperimmunoglobulinemia E, and a negative delayed cutaneous reaction to antigen from Candida albicans were found.


Effect of lung volume on expiratory time in the newborn infant.

The effect of an increased end-expiratory lung volume on inspiratory and expiratory duration was examined in 13 term infants at 4, 30, and 70 h of age. This was accomplished by the administration of a continuous positive airway pressure (CPAP) of 0, 3, and 6 cmH2O by use of a face mask connected to a pneumotachometer, and by measurement of the timing of the respiratory cycle over 1-min intervals. At increasing functional residual capacity (FRC) there was a progressive increase in expiratory time (TE) and fall in respiratory rate, with a variable effect on inspiratory time (TI). As CPAP increased from 0 to 6 cmH2O, the TI/TE ratio fell from 0.75 to 0.62 (P less than 0.01), 0.90 to 0.66 (P less than 0.001), and 0.87 to 0.64 (P less than 0.001) at 4, 30, and 70 h, respectively. We conclude that alterations in end-expiratory lung volume significantly alter expiratory duration in the newborn infant at term. This may be analogous to the vagally mediated tonic control of expiratory time with changing FRC recently described in anesthetized animals.


Growth of transplants of rat humerus following circumferential division of the periosteum.

The growth of transplants of young rat humeri has been studied following circumferential division of the periosteum at either the proximal or the distal end of the shaft. Periosteal division resulted in bones that were longer than those in which the periosteum was not cut. Following proximal division the experimental bones grew 30% more at both ends than the control bones and 16% more at the proximal end following distal division. There was, however, no difference between the growth of the experimental and control bones at the distal end following distal division of the periosteum. The experimental humeri had less new subperiosteal bone deposited over their diaphyses than the control humeri. These findings support the view that the periosteum is under tension as a result of the activity of the growth plates, and that changes in periosteal tension affect both the growth in length and the deposition of subperiosteal bone.


Globomycin, a new peptide antibiotic with spheroplast-forming activity. II. Isolation and physico-chemical and biological characterization.

The peptide antibiotic globomycin was extracted from the culture filtrate of Streptomyces halstedii No. 13912, purified on silica-gel columns and crystallized from acetonitrile to give colorless needles. It is a neutral substance with m.p. of 115 degrees C and a molecular formula of C32H57N5O9. On amino-acid analysis, it gave serine, threonine, glycine and an unidentified amino acid. It is soluble in methanol, ethyl acetate and chloroform but sparingly soluble in water. The antimicrobial spectrum of globomycin revealed its specific activity against Gram-negative bacteria. Formation of spheroplasts was observed when Escherichia coli was grown in the presence of globomycin, indicating inhibition of the bacterial cell wall synthesis as its mode of action.


Kaspar Hauser's recovery and autopsy: a perspective on neurological and sociological requirements for language development.

The feral children literature has frequently been cited for relevance to understanding historical antecedents of autism. Kaspar Hauser, who appeared in Nuremberg, Germany in 1828, is one of these children, raised under conditions of extreme deprivation. His case history and gradual acquisition of language after age 17 years are summarized. There is strong evidence that he was the prince of Baden, abducted from his cradle in 1812. Findings of postmortem examination, conducted after his assassination, are discussed. Hauser's postadolescent recovery of language contradicts the notion of a "critical period" for language development.


Escherichia coli gene that controls sensitivity to alkylating agents.

A new type of Escherichia coli mutant which shows increased sensitivity to methyl methane sulfonate but not to UV light or to gamma rays was isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. The mutant is unable to reactivate phage lambdavir or double-stranded phiX174 DNA (replicative form) that had been treated with methyl methane sulfonate. The mutant is sensitive to other alkylating agents, such as ethyl methane sulfonate, mitomycin C, and N-methyl-N'-nitro-N-nitrosoguanidine, as well. It grows normally and exhibits almost normal recombination proficiency. The mutant possesses normal levels of DNA polymerase I, exonuclease I, exonuclease V, endonuclease specific for methyl methane sulfonate-treated DNA, and 3-methyladenine-DNA glycosidase activities. The genetic locus responsible has been named alk and is located near his on the chromosome.


Evidence for the specific association of the chromosomal origin with outer membrane fractions isolated from Escherichia coli.

DNA-envelope complexes isolated from osmotically lysed spheroplasts of Escherichia coli contained 0.2 to 1% of the total cellular DNA after labeling with [3H]thymidine. Molecular weight determinations indicated that the amount of bound DNA was equivalent in most cases to a maximum of three binding sites per chromosome. Bound DNA from E. coli B/r was distributed approximately equally between inner and outer membrane components when envelopes were fractionated on sucrose equilibrium gradients. Outer membrane-DNA complexes, in particular, fraction H1, with a density of 1.24 g/cm3, were quite stable against shearing and against Sarkosyl NL97. In the case of E. coli B/r, H1-DNA was also relatively resistant to deoxyribonuclease. Inner membrane-DNA complexes, in contrast, were quite labile and readily dissociated to release free DNA. The outer membrane fractions did not appear to contain replication fork DNA, but small amounts may have been present in the inner membrane complexes. A two- to eightfold enrichment for chromosomal origin DNA in the envelope was obtained when cultures of E. coli K-12, synchronized for DNA replication, were pulse labeled at different times in the replication cycle. This enrichment was found invariably in the outer membrane fractions. However, the data do not exclude the possibility that this DNA is bound to regions of adhesion between inner and outer membranes which sediment with a density indistinguishable from that of the outer membrane.


Functional interaction of the tonA/tonB receptor system in Escherichia coli.

Host range mutants of phage T1 (T1h), which productively infected tonB mutants of Escherichia coli, were isolated. The phage mutants were inactivated by isolated outer membranes of E. coli in contrast to the wild-type phage, which only adsorbed reversibly. For the infection process, the tonB function is apparently only required for the irreversible adsorption of the phage T1, but not for the transfer of the phage DNA through the outer membrane and the cytoplasmic membrane of the cell. Mutants of the tonA gene expressing normal amounts of outer membrane receptor proteins were isolated and found to be partially sensitive to phage T5 and resistant to the phages T1 and T1h, colicin M, and albomycin and unable to take up iron as a ferrichrome complex. One tonA mutant remained partially sensitive to T5, colicin M, and albomycin and supported growth of T1h (not of T1) with the same plating efficiency as the parent strain. Only a small region of the tonA receptor protein seems to function for all the very different substrates. A newly isolated host range mutant of T5 (T5h) adsorbed faster to tonA(+) cells than did wild-type T5 and infected tonA missense mutants resistant to wild-type T5. The interplay of the tonA with the tonB function was observed with phage T5 infection, although T5 required only the tonA receptor. Ferrichrome inhibited plaque formation of T5 only when plated on tonB mutants. Adsorption of T5 to cells in liquid medium was influenced by ferrichrome as follows: complete inhibition by 0.1 muM ferrichrome with tonB mutants, not more than 35% inhibition by 1 to 100 muM ferrichrome with the tonB(+) parent strain in the presence of glucose as energy source, and 90% inhibition by 1 muM ferrichrome with partially starved parent cells. We conclude that there exist different functional states of the receptor protein that depend on the energy state of the cell and the tonB function. The latter seems to be required only for translocation processes with outer membrane proteins involved.


Escherichia coli mutants defective in membrane phospholipid synthesis: binding and metabolism of 1-oleoylglycerol 3-phosphate by a plsB deep rough mutant.

Mutants of Escherichia coli containing a defective sn-glycerol 3-phosphate acyltransferase are conditionally defective in the synthesis of acylglycerol phosphate (acylglycerol-P). Incubation of a deep rough derivative of one of these plsB strains with 1-[3H]oleoylglycerol-32P resulted in the binding of up to 70 nmol of oleoylglycerol-P per 100 nmol of cellular phospholipid. The binding was dependent on time, oleoylglycerol-P concentration, and the quantity of cells employed. The rate and extent of oleoylglycerol-P binding was affected by the deep rough mutation. The altered phospholipid composition due to oleoylglycerol-P binding was without consequence on cell growth and viability, but caused the appearance of intracellular multilamellar structures. Use of the double-labeled oleoylglycerol P demonstrated that the entire molecule was bound to the cell. Intact [3H]-oleoylglycerol-32P was converted to phosphatidylethanolamine and phosphotidyl-glycerol at a rate about 40% of that of de novo phospholipid synthesis. These data demonstrate the transmembrane movement of oleoylglycerol-P to the inner surface of the cytoplasmic membrane and suggest that it may become possible to supplement plsB strains of E. coli with acylglycerol-P's.


Bacteriophage P22 is not a likely probe for zones of adhesion between the inner and outer membranes of Salmonella typhimurium.

Thin-section electron micrographs of plasmolyzed Salmonella typhimurium infected with bacteriophage P22 demonstrated that phage adsorbed to cells over sites of inner- and outer-membrane contact. Efforts were made to isolate such adsorption sites by infection of cells with 35S- and 32P-labeled phage and by separation of the membranes on sucrose gradients. At 37 degrees C, about 75% of the 35S radioactivity could be recovered in a region of intermediate density between the inner and outer membranes. This region (phi band) did not contain 32P. The gradient profile was independent of the multiplicity of infection (between 0.2 and 50) and of the presence or absence of chloramphenicol, dinitrophenol, or cyanide. However, ethylenediaminetetraacetate, when present during the infection step, prevented the formation of phi band. The density of phi band was at least 1.30 g/cm3, as demonstrated by prolonged centrifugation on a D2O-sucrose gradient. phi Band was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopy to contain empty phage heads and contaminating cellular debris. In purified preparations, phage heads were the only structures, visible by negative staining, and very little cellular phospholipid or protein was associated with the phage proteins (less than 2% and 30% by weight, respectively, as determined by using [3H]glycerol or [3H]leucine). The residual cellular protein included all of the major outer-membrane proteins rather than any one specific protein. These results are interpreted as indicating that phi band probably does not contain adhesion site material stably associated with phage heads.


Noncoordinate control of RNA, lipopolysaccharide, and phospholipid syntheses during amino acid starvation in stringent and relaxed strains of Escherichia coli.

The syntheses of RNA, lipopolysaccharides, and phospholipids were measured simultaneously in stringent and relaxed cells of Escherichia coli during normal growth or starvation for amino acids. The synthesis of all these molecules was inhibited by amino acid starvation, but the reduction in synthesis was not coordinated.


Derepression of colicin E1 synthesis in the constitutive tif mutant strain (spr tif sfi) and in a tif sfi mutant strain of Escherichia coli K-12.

We show here that expression of the colicin gene of the ColE1 plasmid is greatly derepressed in Escherichia coli K-12 strain DM1187 spr tif sfi, which is a constitutive tif mutant, altered in the lexA gene, and which shows constitutive expression of various pathways of the recA-dependent, lexA-blocked (SOS) repair system. In this strain colicin E1 synthesis is at least 100-fold greater than that observed in uninduced control strains (spr+ tif sfi and spr+ tif+ sfi). This result confirms the regulatory role of the lexA product in colicin E1 synthesis. Colicin yields by the uninduced strain DM1187 are as high as the maximum yields from mitomycin-induced control strains and often are several-fold higher. When the nonconstitutive tif sfi strain GC467 is raised to 43 degrees C to induce the SOS system, a low level of colicin synthesis is observed which is less than one-tenth of the yield obtained by induction with mitomycin C. Addition of adenine at the time of shift-up can increase the colicin yield of tif sfi to about one-third of the yield obtained with mitomycin C. We have also found that colicin overproduction can be detected by altered colony appearance in an overlay assay with colicin-sensitive bacteria. In addition, the lethality of the process of colicin synthesis is observed here without the use of bacteriostatic inducing agents.


Position of the extra amino acid sequence in the precursor arabinose-binding protein of Escherichia coli.

The extra amino acid sequence in the precursor arabinose-binding protein was shown to be either close to or at the N-terminus.


Complementation analysis and deletion mapping of Escherichia coli mutants defective in chemotaxis.

Motile, but generally nonchemotactic (che) mutants of Escherichia coli were examined for complementation and recombination with specialized lambdafla transducing phages. The complex complementation behavior of these mutants found previously in F-prime tests could largely be accounted for by intragenic complementation and by polarity effects. Mutants of the "cheA" class defined two genes, cheA and cheW, which appeared to be contranscribed. Mutants of the "cheB" class defined four genes, cheX, cheB, cheY, and cheZ, which also constituted a transcriptional unit. Mutants defective in cheA, cheW, cheX, or cheY function swam smoothly, with little or no tumbling, whereas cheB or cheZ mutants exhibited very high tumbling rates. These functions are probably involved in initiating of controlling changes in flagellar rotation in response to chemotactic stimuli.


Molecular events during the release of delta-aminolevulinate dehydratase from catabolite repression.

Transfer of exponential-phase cells of Saccharomyces cerevisiae, previously grown in 2% glucose, to a derepression medium resulted in a prompt increase in the level of delta-aminolevulinate dehydratase, the rate-limiting enzyme of heme biosynthesis under these conditions. This derepression exhibited a lag of 35 min at 23 degrees C and required the participation of both RNA and protein syntheses. Dissection of the molecular events during this lag period disclosed that RNA synthesis, rnal gene function (messenger RNA transport from nucleus to cytosol), and initiation of protein synthesis were completed within less than 10, 18, and 24 min, respectively. The potential regulation of derepression by mitochondrial gene products and mitochondrial function was probed by means of a series of isogenic, respiration-deficient (rho-, pet-, and mit-) mutants; no such regulation was found.


Site of inhibition of peptidoglycan biosynthesis during the stringent response in Escherichia coli.

The site of inhibition of peptidoglycan synthesis during the stringent response in Escherichia coli was determined in strains which were auxotrophic for both lysine and diaminopimelic acid (DAP). Cells were labeled with [(3)H]DAP for 30 to 60 min in the presence and absence of required amino acids, and the cellular distribution of [(3)H]DAP was determined. In both stringent (rel(+)) and relaxed (relA) strains, amino acid deprivation did not inhibit the incorporation of [(3)H]DAP into the nucleotide precursor and lipid intermediate fractions. The amount of [(3)H]DAP incorporated into the peptidoglycan fraction by the amino acid-deprived relA strain was over 70% of the amount incorporated in the presence of required amino acids. In contrast, the amounts of labeled peptidoglycan in amino acid-deprived rel(+) strains were only 20 to 44% of the amounts synthesized in the presence of amino acids. These results indicate that a late step in peptidoglycan synthesis is inhibited during the stringent response. The components of the lipid intermediate fraction synthesized by rel(+) strains in the presence and absence of required amino acids were quantitated. Amino acid deprivation did not inhibit the synthesis of either the monosaccharide-pentapeptide or the disaccharide-pentapeptide derivatives of the lipid intermediate. Thus, the reaction which is most likely inhibited during the stringent response is the terminal one involving the incorporation of the disaccharide-pentapeptide into peptidoglycan.


Effects of adenosine triphosphate and magnesium chloride on affinity elution of aminoacyl-transfer ribonucleic acid synthetases from phosphocellulose with transfer ribonucleic acids.

Aminoacyl-tRNA synthetases of bakers' yeast (Saccharomyces cerevisiae) were adsorbed to a phosphocellulose (P-cellulose) column, and those specific for tyrosine [EC 6.1.1.1], threonine [EC 6.1.1.3], valine [EC 6.1.1.9], and isoleucine [EC 6.1.1.5] were eluted with several specific tRNAs. Elutions of these synthetases were affected by ATP and/or MgCl2. The effects of ATP and MgCl2 differ with synthetases. Elutions of tyrosyl- and valyl-tRNA synthetases with their cognate tRNAs were more specific in the presence of MgCl2. Isoleucyl-tRNA synthetase was eluted with its cognate tRNA in the presence of both ATP and MgCl2. On the other hand, threonyl-tRNA synthetase was eluted in the absence of ATP and MgCl2 with unfractionated tRNA but not with some non-cognate tRNAs. This suggests that elution of threonyl-tRNA synthetase is highly specific. The present data on the effects of ATP or MgCl2 or both on this affinity elution will be useful for simple and rapid purification of the synthetases.


Disulfide bonds of purothionine, a lethal toxin for yeasts.

Purothionin isolated from commercial wheat flour contained several components and two of them (A-I and A-II) were isolated in pure form by CM-52 column chromatography. Each component contained 45 amino acid residues with a 4 disulfide bonds. Purothionin A-II was digested with trypsin and thermolysin to isolate cystine peptides. These were separated and purified by chromatography on an SP-Sephadex column, and paper electrophoresis and chromatography. A peptide containing a -Cys-Cys- sequence was hydrolyzed with 10 N sulfuric acid. Amino acid compositions and partial sequence studies of the cystine peptides and their performic acid-oxidized peptides revealed the positions of all 4 disulfide bonds in purothionin A-II. They were formed between residues 3 and 39, 4 and 31, 12 and 29, and 16 and 25. The results of a partial study of purothionin A-I are also presented.


Relaxation effect of chloramphenicol on the stringent control in Escherichia coli.

In 10B601 (rel+) strain possessing a temperature-sensitive valyl-tRNA synthetase, chloramphenicol prevented the formation of guanosine-3'-diphosphate-5'-diphosphate (ppGpp) as well as the stringent control of stable RNA synthesis, under the conditions where the incorporation of valine into protein was still detectable i.e. at the lower restrictive temperatures. On the other hand, the effect of chloramphenicol was not observed at higher restrictive temperatures above 42 degrees C where the incorporation of valine was completely absent. Pretreatment of 10B601 cells with chloramphenicol before transfer to a high restrictive temperature (43.5 degrees C) did retard the onset of accumulation of ppGpp after the shift-up. Duration of the lag period was dependent on the concentration of chloramphenicol added. In parallel with the inability of the cells to accumulate ppGpp, stable RNA synthesis was permitted to continue at that high temperature. These results suggest that chloramphenicol traps aminoacyl-tRNA at the A-sites of ribosomes by damming-up the small flow of aminoacyl-tRNA under the restrictive supply of amino acids. Unchanged tRNA which has been located at the A-site is replaced by the charged one, thus resulting in the suppression of ppGpp formation and in the restoration of stable RNA synthesis.


Synthesis of bacteriophage phiC DNA in dna mutants of Esherichia coli.

Host dna functions involved in the replication of microvirid phage phiC DNA were investigated in vivo. Although growth of this phage was markedly inhibited even at 35-37 degrees C even in dna+ host, conversion of the infecting single-stranded DNA into the double-stranded parental replicative form (stage I synthesis) occurred normally at 43 degrees C in dna+, dnaA, dnaB, dnaC(D), and dnaE cells. In dnaG mutant, the stage I synthesis was severely inhibited at 43 degrees C but not at 30 degrees C. The stage I replication of phiC DNA was clearly thermosensitive in dnaZ cells incubated in nutrient broth. In Tris-casamino acids-glucose medium, however, the dnaZ mutant sufficiently supported synthesis of the parental replicative form. At 43 degrees C, synthesis of the progeny replicative form DNA (stage II replication) was significantly inhibited even in dna+ cells and was nearly completely blocked in dnaB or dnaC(D) mutant. At 37 degrees C, the stage II replication proceeded normally in dna+ bacteria.


2'-Versus 3'-OH specificity in tRNA aminoacylation. Further support for the "secondary cognition" proposal.

Purified Escherichia coli tRNAAla and tRNALys were each converted to modified species terminating in 2'- and 3'-deoxyadenosine. The modified species were tested as substrates for activation by their cognate aminoacyl-tRNA synthetases and for misacylation with phenylalanine by yeast phenylalanyl-tRNA synthetase. E. coli alanyl- and lysyl-tRNA synthetases normally aminoacylate their cognate tRNA's exclusively on the 3'-OH group, while yeast phenylalanyl-tRNA synthetase utilizes only the 2' position on its own tRNA. Therefore, the finding that the phenylalanyl-tRNA synthetase activated only those modified tRNAAla and tRNALys species terminating in 3'-deoxyadenosine indicated that the position of aminoacylation in this case was specified entirely by the enzyme, an observation relevant to the more general problem of the reason(s) for using a particular site for aminoacylation and maintaining positional specificity during evolution. Initial velocity studies were carried out using E. coli tRNAAla and both alanyl- and phenylalanyl-tRNA synthetases. As noted in other cases, activation of the modified and unmodified tRNA's had essentially the same associated Km values, but in each case the Vmax determined for the modified tRNA was smaller.


Affinity labeling of succinyl-CoA synthetase from porcine heart and Escherichia coli with oxidized coenzyme A disulfide.

Incubation of oxidized coenzyme A disulfide (produced by oxidation of reduced CoA with 1 eq of sodium periodiate or of CoA disulfide with 1 eq of peracetic acid) with succinyl-CoA disulfide with 1 eq of peracetic acid) with succinyl-CoA synthetase from either porcine heart or Escherichia coli led to the formation of inactive enzyme containing 1 mol of CoA per alphabeta dimer. The bound CoA was attached through a disulfide bond to a sulfhydryl group of the beta subunit. Release of CoA and restoration of activity was achieved by incubation of the modified enzyme with thiols, such as dithiothreitol. Interaction of oxidized CoA disulfide with enzyme was inhibited competitively by desulfo-CoA, which is a competitive inhibitor of the enzyme with respect to CoA. These data are evidence that oxidized CoA disulfide is an affinity label for the CoA binding site of succinyl-CoA synthetase and are the first positive results implicating the beta subunit in the catalytic mechanism of the enzyme.


Transcription of T7 DNA containing modified nucleotides by bacteriophage T7 specific RNA polymerase.

The interaction of bacteriophage T7 specific RNA polymerase with its cognate promoter sites has been probed by selectively replacing bases in one T7 promoter site with base analogs. Base analogs such as 2,6-diaminopurine or hypoxanthine, which alter residues appearing in the minor groove of the DNA helix, prevent utilization of the promoter by T7 RNA polymerase. These analogs do not affect transcription which starts outside of the modified region. In contrast, base analogs that have alterations that appear in the major groove of the DNA helix, such as uracil, 5-bromouracil, 5-methylcytosine, 5-hydroxymethylcytosine, and [5-HgSR]pyrimidines, do not prevent utilization of the promoter. The deoxyribonucleoside analog 5'-imino-5'-deoxythymidine, an alteration appearing in the deoxyribose-phosphodiester backbone of the DNA helix, does not prevent promoter recognition. Haemophilus aegyptius restriction endonuclease III, which cleaves DNA at the sequence 5'GGCC3', does not act at sites in which the guanine residues in one of the two DNA strands have been substituted with hypoxanthine. This implicates the guanine amino group in the minor groove of the DNA helix as a possible recognition point for this restriction endonuclease.