vendredi 24 avril 2009
par   G. Grégori

An GH, Bielich J, Auerbach R, Johnson EA (1991) Isolation and characterization of carotenoid hyperproducing mutants of yeast by flow cytometry and cell sorting. Biotechnology (N Y) 9 :70-73


The carotenoid pigment astaxanthin (3,3’-dihydroxy-beta,beta-carotene-4,4’-dione) is an important component in feeds of aquacultural animals. It is produced as a secondary metabolite by the yeast Phaffia rhodozyma, and the isolation of rare mutants that produce increased quantities is limited by the lack of genetic selections. As a model system for enriching mutants increased in production of secondary metabolites, we have used quantitative flow cytometry/cell sorting (FCCS) to isolate astaxanthin hyperproducing mutants of the yeast. Experimental conditions were developed that gave a quantitative correlation of fluorescence and carotenoid content. In mutated populations, a 10,000-fold enrichment of carotenoid-overproducing yeasts was obtained. Distinctive differences were detected by FCCS in fluorescence and forward scatter values of mutant and wild-type populations of yeasts. Comparison of wild-type and mutant clones by fluorescence confocal laser microscopy showed that the mutants had more intense fluorescence throughout the cell than the wild-type. Quantitative FCCS is a sensitive method to isolate and characterize carotenoid overproducing mutants and should be useful as a general method for the isolation of mutants increased in other fluorescent metabolites.

Berglund DL, Starkey JR (1991) Introduction of antibody into viable cells using electroporation. Cytometry 12 :64-67


Conditions for labelling an intracellular antigen, p21ras, using electroporation to introduce a fluorescent antibody, are described. Following labelling, cells were evaluated for p21ras associated fluorescence by flow cytometry. Electroporation, sorting, and cell handling parameters were varied to determine optimal conditions for cell viability. Cells were best held in serum containing growth medium both before and after electroporation, while antibody introduction during the electroporation phase was most efficient when carried out in a balanced saline solution. For maximum efficiency of antibody internalization, the antibody needed to be present during electroporation, and medium needed to be replaced several times in the first few hours after electroporation to ensure good cell survival.

Bhakdi S, Martin E (1991) Superoxide generation by human neutrophils induced by low doses of Escherichia coli hemolysin. Infect Immun 59 :2955-2962


Escherichia coli hemolysin (Hly) was isolated from bacterial culture supernatants by polyethylene glycol precipitation and centrifugation in glycerol density gradients. The toxin preparations contained less than 1 mol of lipopolysaccharide per 10 mol of protein, and they had no fatty acids. The capacity of purified hemolysin to stimulate superoxide anion production in polymorphonuclear leukocytes was monitored kinetically in a lumimeter by using the lucigenin assay and was correlated with the kinetics of transmembrane pore formation. When applied to leukocytes suspended in protein-free buffer, very low concentrations (0.02 to 0.1 HU/ml) of the toxin strongly stimulated the production of superoxide anions ; shortly thereafter, irreversible membrane permeabilization occurred. When the toxin was applied at concentrations exceeding 0.2 to 0.3 HU/ml, membrane permeabilization was so rapid that the cells were unable to mount a respiratory burst. When applied in the narrow range of 0.05 to 0.1 HU/ml, E. coli hemolysin rivaled phorbol myristate acetate in its capacity to stimulate production of superoxide anions. Additionally, hemolysin applied at doses that elicited no pore formation (0.01 to 0.02 HU/ml) primed leukocytes for an augmented response to subsequent challenge by the phorbol ester. These data demonstrate that very low doses of E. coli hemolysin can evoke cellular reactions that appear independent of and precede transmembrane pore formation and cell death.

Bilej M, Rossmann P, VandenDriessche T, Scheerlinck JP, De Baetselier P, Tuckova L, Vetvicka V, Rejnek J (1991) Detection of antigen in the coelomocytes of the earthworm, Eisenia foetida (Annelida). Immunol Lett 29 :241-245


Earthworms, Eisenia foetida, are able to respond to antigenic stimulation by the formation of the antigen-binding molecules by coelomocytes—the effector cells of annelids’ defence reactions. The ability to react with gold-labelled antigen was detected in agranular coelomocytes by electron microscopy. Furthermore, flow cytometry analysis used for quantitative evaluation of antigen binding showed significant increase of both antigen-binding cells and the amount of antigen bound per cell after stimulation. The antigen binding was inhibited by preincubation of cells with several similar proteins, although the most potent inhibitor was the immunizing antigen.

Daley MJ, Oldham ER, Williams TJ, Coyle PA (1991) Quantitative and qualitative properties of host polymorphonuclear cells during experimentally induced Staphylococcus aureus mastitis in cows. Am J Vet Res 52 :474-479


Polymorphonuclear cells have a critical role in the pathogenesis of bovine mastitis. We have documented that experimentally induced Staphylococcus aureus mastitis is associated with cyclic increase and decrease in the quantity of viable bacteria shed in the milk. Concomitant with this cycling of bacteria is an inverse cycling of the hosts cells within the milk. Such somatic cells were determined to be greater than or equal to 95% polymorphonuclear cells. The quality of these cells was evaluated by measuring their relative efficiency of bacterial killing and phagocytosis at various times during an infection. Host polymorphonuclear cells had as much as 10,000-fold variation in the bactericidal failure rate for staphylococci during cell cycling. The most efficient bactericidal effect was observed at or near the peak of the somatic cell count (SCC). The ability of these cycling cells to ingest fluorescent beads was also quantitated by use of flow cytometry. The percentage of phagocytic polymorphonuclear cells that ingested fluorescent latex beads ranged from 15 to 80% of the total cell population during cell cycling, and tended to be optimal at or near peak SCC. In addition, the average number of beads ingested varied between 1 and 2 particles/polymorphonuclear cell, with as many as 17% of the phagocytic cells ingesting 4 or more beads at maximal efficiency. Polymorphonuclear cells from quarters infected with S aureus varied quantitatively (total SCC) and qualitatively (bactericidal activity and phagocytic ability) during the course of an infection. Not only is the quantity of host’s phagocytic cells in the mammary gland central to the defense mechanism against infection, but the biological activation state appears to be equally important. The role of these cells in the pathogenesis of a cycling infection is presented in a model to explain the cyclic nature of mastitis.

Dasgupta S, Bernander R, Nordstrom K (1991) In vivo effect of the tus mutation on cell division in an Escherichia coli strain where chromosome replication is under the control of plasmid R1. Res Microbiol 142 :177-180


The phenotypic effect of the tus ::kan mutation in an Escherichia coli strain, where the chromosome is replicated unidirectionally by an integrated R1 miniplasmid, was examined by flow cytometry and phase fluorescence microscopy. The tus+ cells exhibited perturbed cell division, as indicated by the presence of many elongated cells and filaments. Inactivation of the tus gene led to a reduction in the frequency of such elongated cells, presumably by eliminating Tus-mediated polar arrests of replication forks at ter sites, thereby shortening the time required for completion of chromosome replication.

Davis WC, Ellis JA (1991) Individual antigens of goats. Vet Immunol Immunopathol 27 :121-131


Farkas-Himsley H, Freedman J, Read SE, Asad S, Kardish M (1991) Bacterial proteins cytotoxic to HIV-1-infected cells. Aids 5 :905-907


Gomez Jorge JT, Estrada C, Gonzalez ZA, Morales-Otero LA, Lavergne J, Santiago-Delpin EA (1991) Flow cytometric analysis of urine sediment after kidney transplantation. Transplant Proc 23 :1764-1765


Gottlieb S, Altboum Z, Savage DC, Segal E (1991) Adhesion of Candida albicans to epithelial cells effect of polyoxin D. Mycopathologia 115 :197-205


Data from our previous studies suggested that the fungal cell wall component, chitin, is involved in the adhesion of Candida albicans to mucosal surfaces. In the present study, we investigated the effect of polyoxin D, an inhibitor of chitin synthase, on the interaction of the fungus with epithelial cells. The effect of polyoxin D on Candida was evaluated in in vitro assays for its capacity to adhere to buccal epithelial cells (BEC), and by fluorescent-microscopy photometry and flow cytometry using cells stained with cellufluor (CF), a fluorochrome with affinity for chitin. C. albicans grown with and without polyoxin D was stained with CF and examined in a fluorescent microscope equipped with a photometer. Measurements of fluorescence revealed a wide range of intensity among C. albicans cells and a decreased intensity in polyoxin D treated cultures. Flow cytometry analyses of yeasts revealed 2 peaks of fluorescence intensity, and pointed to differences between polyoxin D treated and non-treated microorganisms. C. albicans stained with CF were separated into 2 subpopulations by flow cytometry according to fluorescence intensity. In vitro adhesion of each subpopulation to BEC was similar. Polyoxin D treated fungi showed significantly reduced adherence to BEC, as evaluated by a radioactivity assay with radiolabelled yeasts and by microscopic readings. The reduction in adhesion was Polyoxin D concentration dependent. These observations support our previous findings suggesting involvement of chitin in the attachment process of C. albicans (CBS562) to epithelial cells.

Hansen FG, Atlung T, Braun RE, Wright A, Hughes P, Kohiyama M (1991) Initiator (DnaA) protein concentration as a function of growth rate in Escherichia coli and Salmonella typhimurium. J Bacteriol 173 :5194-5199


The DnaA protein concentration was determined in five different Escherichia coli strains and in Salmonella typhimurium LT2 growing at different growth rates. The DnaA protein concentration was found to be invariant over a wide range of growth rates in the four E. coli K-12 strains and in S. typhimurium. In E. coli B/r the DnaA protein concentration was generally higher than in the K-12 strains, and it increased with decreasing growth rates. For all the strains, there appears to be a correlation between the DnaA protein concentration and the initiation mass. This supports the concept of the concentration of DnaA protein setting the initiation mass and, thus, that the DnaA protein is a key molecule in the regulation of initiation of chromosome replication in members of the family Enterobacteriaceae.

Harris RW, Sims PJ, Tweten RK (1991) Evidence that Clostridium perfringens theta-toxin induces colloid-osmotic lysis of erythrocytes. Infect Immun 59 :2499-2501


Clostridium perfringens theta-toxin was shown to lyse target erythrocytes by a colloid-osmotic mechanism. Analysis showed the onset of lysis of erythrocytes by theta-toxin could be temporarily stabilized with 0.3 M sucrose. Flow cytometry analysis of the size distribution of theta-toxin-treated erythrocytes showed swelling of the erythrocytes prior to lysis.

Lapinsky SE, Glencross D, Car NG, Kallenbach JM, Zwi S (1991) Quantification and assessment of viability of Pneumocystis carinii organisms by flow cytometry. J Clin Microbiol 29 :911-915


Analysis of drug efficacy in animal models of Pneumocystis carinii pneumonia requires an accurate method of quantification of organisms, as well as a means of assessing viability. Lung homogenates were prepared from a colony of athymic nude F344 rats experiencing a spontaneous outbreak of P. carinii pneumonia. With the fluorescent nucleic acid stain propidium iodide, flow cytometric analysis was able to quantify P. carinii cysts and trophozoites reproducibly. As this stain is excluded by living cells, this method was also used to assess the viability of organisms. Application of this technique to analysis of bronchoalveolar lavage specimens was demonstrated.

Lutton DA, Patrick S, Crockard AD, Stewart LD, Larkin MJ, Dermott E, McNeill TA (1991) Flow cytometric analysis of within-strain variation in polysaccharide expression by Bacteroides fragilis by use of murine monoclonal antibodies. J Med Microbiol 35 :229-237


The reactivity of four different monoclonal antibodies (MAbs) with populations of Bacteroides fragilis NCTC 9343, enriched by density gradient centrifugation for a large capsule, small capsule and electron-dense layer (EDL) only visible by electronmicroscopy, was examined. The MAbs reacted strongly with polysaccharides present in both the large capsule- and EDL-enriched populations but not in the small capsule-enriched populations. The pattern of labelling was determined by immunoblotting, immunofluorescence and immuno-electronmicroscopy, and flow cytometry. The MAbs labelled cell membrane-associated epitopes in the large capsule- and EDL-enriched populations and cell-free material in the EDL population. By immunoblotting, ladders of repeating polysaccharide subunits were evident in the EDL population but not in the large capsule population. The proportion of cells labelled within each population was determined by flow cytometry. The reactivity of another MAb with the small capsule population was confirmed by flow cytometry. A qualitative indication of epitope expression was obtained by examination of the flow cytometric profiles. Differential expression of the same saccharide epitope was observed both between and within structurally distinct B. fragilis populations. The MAbs were species-specific and cross-reacted with several recent clinical isolates. These polysaccharides may be relevant to the virulence of B. fragilis.

Manafi M, Kneifel W (1991) Fluorogenic and chromogenic substrates—a promising tool in microbiology. Acta Microbiol Hung 38 :293-304


During the last few years the use of fluorogenic and chromogenic substrates for rapid and sensitive detection of bacteria has proved to be a powerful alternative to traditional methods. These sophisticated substrates might find widespread application in, for instance, the assay of clinically important enzymes, flow cytometry, and direct epifluorescent filter technique. Specific enzyme detection offers another approach to differential identification and characterization of viable bacteria from a sample. The use of some chromogenic and fluorogenic substrates specific for bacterial enzymes and their applications to microbial identification is reported. Particular emphasis is given to the examination of Escherichia coli and the description of the different techniques as used in routine analysis.

Mellencamp MW, O’Brien PC, Stevenson JR (1991) Pseudorabies virus-induced suppression of major histocompatibility complex class I antigen expression. J Virol 65 :3365-3368


The ability of pseudorabies virus (PrV) to down-modulate expression of major histocompatibility complex class I antigens in murine and porcine cells was investigated. When quantified by flow cytometry, surface expression of class I Kk and Dk antigens on PrV-infected cells decreased by 60% or more. Down-modulation was associated with a decrease in total cellular class I antigens, indicating regulation at the transcriptional or posttranscriptional level. PrV did not suppress expression of transferrin receptor, suggesting a selective regulatory mechanism.

Morgan JA, Cranwell PA, Pickup RW (1991) Survival of Aeromonas salmonicida in lake water. Appl Environ Microbiol 57 :1777-1782


The survival of Aeromonas salmonicida subsp. salmonicida in lake water was investigated by using a variety of techniques. They included acridine orange epifluorescence, respiration, cell culture, cell revival, flow cytometry, plasmid maintenance, and membrane fatty acid analysis. During a 21-day study, A. salmonicida became nonculturable in sterile lake water samples. Flow cytometry and direct microscopy indicated that cells were present. Although the nonculturable cells could not be revived, the recovery method did indicate that the presence of low numbers of culturable cells within samples could produce misleading results. Plasmid DNA, genomic DNA, and RNA were maintained in the nonculturable cells ; in addition, changes in the fatty acid profiles were also detected. Although viability could not be proven, it was shown that the morphological integrity of nonculturable cells was maintained.

Nelson D, Bathgate AJ, Poxton IR (1991) Monoclonal antibodies as probes for detecting lipopolysaccharide expression on Escherichia coli from different growth conditions. J Gen Microbiol 137 :2741-2751


Monoclonal antibody (mAb) probes were used to investigate the expression of lipopolysaccharide (LPS) on four Escherichia coli strains, grown under a variety of conditions in batch culture which mimicked some of the in vivo environmental conditions of an infected host. Techniques of silver staining, immunoblotting, whole cell ELISA and flow cytometry were all used to monitor the expression of LPS on the bacteria and the binding of the anti-LPS mAbs. Growth in heat-inactivated sheep serum and magnesium-depleted conditions demonstrated increased expression of LPS core and subsequent increased binding of anti-core mAbs. Magnesium-depleted conditions also resulted in decreased production of O-polysaccharide material. Iron-depleted bacteria showed only minor changes in LPS expression, although increased binding of anti-core mAbs was observed. Nitrogen-deficient/high-carbon conditions, chosen to promote capsule production, resulted in increased expression of O-polysaccharide and decreased binding of anti-core mAbs.

O’Gorman MR, Hopfer RL (1991) Amphotericin B susceptibility testing of Candida species by flow cytometry. Cytometry 12 :743-747


We have developed an 8 hr flow cytometry (FCM) method for assessing susceptibility of yeasts to amphotericin B (AmpB). The method detects both high-level and relative-resistance to the drug. Variables found to affect fluorescence of control and AmpB treated cells included pH, presence of glucose, incubation conditions, concentration and length of exposure to both AmpB and ethidium bromide (ETBR), and the degree of resistance to AmpB. The FCM method was optimized based on increased red fluorescence intensity (RF), decreased forward angle light scatter (FALS), and a negative gating technique. A dose response was seen between 0.1 and 10 micrograms AmpB/ml for the susceptible control strain. Greater than 50% of cells from all susceptible strains tested transfer into the negative gate when exposed to 2.5 micrograms Amp B/ml while fewer than 5% of cells of the highly resistant C. tropicalis (ATCC 28707) are affected at concentrations up to 20 micrograms/ml. This method may provide a more accurate assessment of Amp B susceptibility than conventional tube dilution methods.

Ohbo K, Takeshita T, Asao H, Kurahayashi Y, Tada K, Mori H, Hatakeyama M, Taniguchi T, Sugamura K (1991) Monoclonal antibodies defining distinct epitopes of the human IL-2 receptor beta chain and their differential effects on IL-2 responses. J Immunol Methods 142 :61-72


We have established and characterized five new monoclonal antibodies (mAbs) which specifically immunoprecipitate the human interleukin-2 receptor beta chain (IL-2R beta). One of them, TU30, recognizes the intracytoplasmic ’serine-rich region’ of IL-2R beta that is critical for IL-2 signal transduction. The others, TU12, TU21, TU23 and TU25, completely inhibit IL-2 binding, as does the previously characterized TU27. However, reciprocal binding competition assays show that the epitopes recognized by the individual mAbs are different from each other. The mAbs inhibit the growth of IL-2-dependent cells. The magnitude of their inhibitory effects is dependent on not only the affinities of the mAbs for IL-2R beta but also upon the number of IL-2R alpha subunits expressed on IL-2-dependent cells. These mAbs should be useful in studying the structure and function of the IL-2R.

Rodriguez de Cuna C, Kierszenbaum F, Wirth JJ (1991) Binding of the specific ligand to Fc receptors on Trypanosoma cruzi increases the infective capacity of the parasite. Immunology 72 :114-120


The infective capacity of Trypanosoma cruzi was significantly increased after treatment with monoclonal IgG1 antibodies, whether or not specific for the parasite ; minimal or no change in infectivity was seen after treatment with IgG2a, IgG2b or IgG3 monoclonal antibodies. The stimulatory effect was evidenced by elevated numbers of trypanosomes invading mammalian host cells in vitro compared to parasites treated with medium alone. Greater infectivity was also induced by pure human Fc, suggesting a role for Fc receptors on the organism. This inference received support in the fact that protein A inhibited the stimulatory effect of Fc. In addition, Fc-treated parasites incubated with fluorescein-labelled F(ab’)2 from goat anti-human IgG exhibited fluorescence detectable by both ultraviolet microscopy and flow cytometry. 125I-Fc binding to T. cruzi was found to be saturable at 0 degrees and was inhibited by cold Fc but not by bovine serum albumin (BSA) or orosomucoid. Interestingly, 125I-Fc binding was greater at 37 degrees and it was not saturable with the concentrations that did saturate at 0 degrees. Possibly, Fc might up-regulate expression of its own receptor and greater endocytosis could take place at 37 degrees. Significant increases in infectivity were detectable after a 40 min pretreatment with Fc—hinting that Fc could trigger a chain of biochemical events underlying the phenomenon—and were reversible, becoming undetectable 2 hr after Fc removal. The average number of Fc receptors per parasite, determined at 0 degrees (at which binding saturation was possible), was estimated as 5 x 10(5), the dissociation constant was of the order of 10(-6)-10(7)M. The present results define an important biological role for an Fc-binding T. cruzi surface component and expose the capacity of this organism to exploit even elements of the immune system in its quest to attain intracellular localization, required for multiplication.

Roman MJ, Crissman HA, Samsonoff WA, Hechemy KE, Baca OG (1991) Analysis of Coxiella burnetii isolates in cell culture and the expression of parasite-specific antigens on the host membrane surface. Acta Virol 35 :503-510


Coxiella burnetii isolates may be classified into several groups based on plasmid character. These groups may also be correlated with disease syndrome—chronic or short-term acute. L929 mouse fibroblast cells were exposed, independently, to two members of the three major C. burnetii groups, and their growth/morphological characteristics analysed by light and electron microscopy, including High Voltage Electron Microscopy. The fates of the isolates were followed. Two acute isolates [Nine Mile (RSA 493) and Henzerling (RSA 331), QpH1-type plasmids] and two chronic isolates (S Q217 and L Q216, plasmidless) readily infected L929 cells in static culture. Priscilla (Q177) and F (Q228) isolates (QpRS-type plasmid, and implicated in causing chronic Q fever) took longer to infect cells, and, unlike the members of the other two groups, gradually disappeared when shifted to suspension culture. Cells infected with Q177 and Q228 exhibited a higher degree of vacuolation than cells infected with the other isolates. Parasite-specific antigens on the surfaces of the host cells were analysed by immunofluorescence/flow cytometry. The acute and plasmidless isolates caused the display of significantly more C. burnetii-specific antigen on the host cell membrane than the two QpRS plasmid-containing isolates. This host cell model system clearly reveals biological differences among the C. burnetii groups.

Rutz JM, Abdullah T, Singh SP, Kalve VI, Klebba PE (1991) Evolution of the ferric enterobactin receptor in gram-negative bacteria. J Bacteriol 173 :5964-5974


Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of iron-deficient and replete cell envelopes, 59Fe-siderophore uptake studies, and Western immunoblots and cytofluorimetric analyses with monoclonal antibodies (MAbs), we surveyed a panel of gram-negative bacteria to identify outer membrane proteins that are structurally related to the Escherichia coli K-12 ferric enterobactin receptor, FepA. Antibodies within the panel identified FepA epitopes that are conserved among the majority of the bacteria tested, as well as epitopes present in only a few of the strains. In general, epitopes of FepA that are buried in the outer membrane bilayer were more conserved among gram-negative bacteria than epitopes that are exposed on the bacterial cell surface. The surface topology and tertiary structure of FepA are quite similar in E. coli and Shigella flexneri but differ in Salmonella typhimurium. Of the 18 different genera tested, 94% of the bacteria transported ferric enterobactin, including members of the previously unrecognized genera Citrobacter, Edwardsiella, Enterobacter, Haemophilus, Hafnia, Morganella, Neisseria, Proteus, Providencia, Serratia, and Yersinia. The ferric enterobactin receptor contains at least one buried epitope, recognized by MAb 2 (C. K. Murphy, V. I. Kalve, and P. E. Klebba, J. Bacteriol. 172:2736-2746, 1990), that is conserved within the structure of an iron-regulated cell envelope protein in all the bacteria that we have surveyed. With MAb 2, we identified and determined the Mr of cell envelope antigens that are immunologically related to E. coli FepA in all the gram-negative bacteria tested. Collectively, the library of anti-FepA MAbs showed unique patterns of reactivity with the different bacteria, allowing identification and discrimination of species within the following gram-negative genera : Aeromonas, Citrobacter, Edwardsiella, Enterobacter, Escherichia, Haemophilus, Hafnia, Klebsiella, Morganella, Neisseria, Proteus, Providencia, Pseudomonas, Salmonella, Serratia, Shigella, Vibrio, and Yersinia.

Sandberg K, Eloranta ML, Johannisson A, Alm GV (1991) Flow cytometric analysis of natural interferon-alpha producing cells. Scand J Immunol 34 :565-576


Herpes simplex virus-infected cells induce high interferon-alpha (IFN-alpha) production in infrequent cells among peripheral blood mononuclear cells (PBMC), designated natural IFN-alpha producing (NIP) cells. The properties of such NIP cells were compared with defined populations of leucocytes by means of flow cytometric analysis and sorting. The NIP cells are characterized as a discrete population of cells with high forward and low to intermediate orthogonal light scattering, similar to that of early progenitors of myeloid and lymphoid cells. However, they appear to lack the stem cell-associated molecule CD34. Furthermore, NIP cells cannot be localized to the myeloid line of cell differentiation, because they do not express the CD33, CD13, CD11b, CD15 or CD14 antigens. Neither do they express CD10 and CD19 antigens which are present in all stages of B-cell differentiation plasma cells excepted, nor CD7 antigens expressed on early T cells. In combination with previous results, our data support the view that the NIP cell is a unique and distinct cell type in peripheral blood, possibly with a physiological role in the defence against certain viral infections.

Smith MM (1991) Mutations that affect chromosomal proteins in yeast. Methods Cell Biol 35 :485-523


Sodora DL, Eisenberg RJ, Cohen GH (1991) Characterization of a recombinant herpes simplex virus which expresses a glycoprotein D lacking asparagine-linked oligosaccharides. J Virol 65 :4432-4441


Glycoprotein D (gD) is an envelope component of herpes simplex virus essential for virus penetration. gD contains three sites for addition of asparagine-linked carbohydrates (N-CHO), all of which are utilized. Previously, we characterized mutant forms of herpes simplex virus type 1 gD (gD-1) lacking one or all three N-CHO addition sites. All of the mutants complemented the infectivity of a gD-minus virus, F-gD beta, to the same extent as wild-type gD. Here, we show that recombinant viruses containing mutations in the gD-1 gene which eliminate the three N-CHO signals are viable. Two such viruses, called F-gD(QAA)-1 and F-gD(QAA)-2, were independently isolated, and the three mutations in the gD gene in one of these viruses were verified by DNA sequencing. We also verified that the gD produced in cells infected by these viruses is devoid of N-CHO. Plaques formed by both mutants developed more slowly than those of the wild-type control virus, F-gD(WT), and were approximately one-half the size of the wild-type. One mutant, F-gD(QAA)-2, was selected for further study. The QAA mutant and wild-type gD proteins extracted from infected cells differed in structure, as determined by the binding of monoclonal antibodies to discontinuous epitopes. However, flow cytometry analysis showed that the amount and structure of gD found on infected cell surfaces was unaffected by the presence or absence of N-CHO. Other properties of F-gD(QAA)-2 were quite similar to those of F-gD(WT). These included (i) the kinetics of virus production as well as the intracellular and extracellular virus titers ; (ii) the rate of virus entry into uninfected cells ; (iii) the levels of gB, gC, gE, gH, and gI expressed by infected cells ; and (iv) the turnover time of gD. Thus, the absence of N-CHO from gD-1 has some effect on its structure but very little effect on its function in virus infection in cell culture.

Steed LL, Setareh M, Friedman RL (1991) Intracellular survival of virulent Bordetella pertussis in human polymorphonuclear leukocytes. J Leukoc Biol 50 :321-330


Little is known regarding the interaction of Bordetella pertussis with polymorphonuclear leukocytes (PMNL) or the role PMNL play as an initial line of defense against B. pertussis infection. An in vitro system was developed to establish conditions for the study of phagocytosis and killing of virulent B. pertussis by human PMNL. Phagocytosis of B. pertussis strains BP504, BP165, and BP338 occurred by opsonization with anti-B. pertussis antibody, while autologous normal human sera did not induce significant phagocytosis. In PMNL bacterial killing assays virulent B. pertussis strains survived PMNL bactericidal activities while Escherichia coli controls were readily killed. Electron microscopy studies using acid phosphatase as a lysosomal marker strongly suggested that B. pertussis inhibits phagosome-lysosome fusion in PMNL. These results indicate that virulent B. pertussis strains are capable of surviving intracellularly within PMNL and that such survival may be due to inhibition of phagosome-lysosome fusion.

Telford WG, King LE, Fraker PJ (1991) Evaluation of glucocorticoid-induced DNA fragmentation in mouse thymocytes by flow cytometry. Cell Prolif 24 :447-459


The ability of glucocorticoids to induce apoptosis or programmed cell death in mouse thymocytes is well-established. Measurement of apoptosis-associated internucleosomal DNA fragmentation through determination of the percentage of fragmented DNA by electrophoresis or centrifugation of whole cell lysates is by far the most common means of quantifying apoptosis. Since these methods measure DNA fragmentation in whole cell lysates rather than intact cells, they have severe limitations, particularly with heterogeneous cell populations. When mouse thymocytes were incubated with glucocorticoids, fixed, stained with propidium iodide and analysed flow cytometrically for cell cycle distribution, a distinct subpopulation of cells was observed to form below the G0/G1 region, denoted as the A0 region. The presence of cells in this region was consistent with the presence of internucleosomal DNA fragments as determined by gel electrophoresis. Inhibitors of transcription, translation and endonuclease activity, and a glucocorticoid receptor antagonist prevented accumulation of cells in this region. Irradiation of mouse thymocytes also produced a population in the A0 region. Cells in this region are believed to have undergone glucocorticoid-induced DNA fragmentation. This method represents a useful alternative to whole cell lysate assays, since apoptosis can be evaluated on an individual cell basis.

von Freiesleben U, Rasmussen KV (1991) DNA replication in Escherichia coli gyrB(Ts) mutants analysed by flow cytometry. Res Microbiol 142 :223-227


We have investigated the initiation of DNA replication in Escherichia coli gyrB (Ts) mutants and find that initiations in single cells, even those that occur at non-permissive temperature, are synchronous. Furthermore, our results indicate that the gradual arrest of DNA replication at non-permissive temperature reflects a general decrease in transcription activity and not an initiation-specific function of the DNA gyrase. At an intermediate temperature, the only effect observed was a lack of segregation (decatenation) of replicated chromosomes in a sizeable fraction of the cell population.

Whitt MA, Buonocore L, Prehaud C, Rose JK (1991) Membrane fusion activity, oligomerization, and assembly of the rabies virus glycoprotein. Virology 185 :681-688


The spike glycoprotein (G protein) of rabies virus (CVS strain) expressed in HeLa cells from cloned cDNA mediated membrane fusion after exposure to pHs of 6.1 or below. Chemical crosslinking showed that the rabies G protein, like the vesicular stomatitis virus (VSV) G protein, could be crosslinked to dimers and trimers, indicating that rabies G protein is a trimer. However, unlike the VSV G protein, rabies G protein trimers were not stable to sedimentation in sucrose gradients, even at a mildly acidic pH which stabilizes the VSV G protein trimers. In addition, we report that the expressed rabies virus G protein was functional because it could assemble into VSV particles (tsO45) lacking VSV G protein and rescue infectivity. These VSV (rabies) pseudotypes were neutralized only by an antibody to the rabies G protein. We also examined the properties of a hybrid protein containing the extracellular domain of the rabies virus glycoprotein and the transmembrane and cytoplasmic domains of the VSV G protein. This protein was transported to the cell surface and could be crosslinked to form dimers and trimers, but had little or no detectable membrane fusion activity. The lack of fusion activity was paradoxical because the hybrid protein could rescue VSV infectivity, although the titers were lower than those obtained with the wild-type rabies G protein.

Whitt MA, Rose JK (1991) Fatty acid acylation is not required for membrane fusion activity or glycoprotein assembly into VSV virions. Virology 185 :875-878


We have investigated the role of fatty acid acylation on two properties of the glycoprotein (G protein) from the Indiana serotype of vesicular stomatitis virus (VSV). Using a mutated G protein described previously (CS-2) that is not palmitylated, we found that fatty acid acylation was not required for the low pH-induced membrane fusion activity of VSV G protein. Transient expression of CS in HeLa cells resulted in syncytia formation that was indistinguishable from that induced by wild-type G protein. In addition, we found that expression of CS complemented a temperature-sensitive mutant of VSV (tsO45) as well as the wild-type protein. These results indicate that the presence of palmitate on the cytoplasmic domain of VSV G protein is not required for any step in the life cycle of the virus.

Wyatt CR, Goff W, Davis WC (1991) A flow cytometric method for assessing viability of intraerythrocytic hemoparasites. J Immunol Methods 140 :23-30


We have developed a rapid, reliable method of evaluating growth and viability of intraerythrocytic protozoan hemoparasites. The assay involves the selective uptake and metabolic conversion of hydroethidine to ethidium by live parasites present in intact erythrocytes. The red fluorescence imparted by ethidium intercalated into the DNA of the parasite permits the use of flow cytometry to distinguish infected erythrocytes with viable parasites from uninfected erythrocytes and erythrocytes containing dead parasites. Comparison of the fluorochromasia technique of enumerating the number and viability of hemoparasites in cultured erythrocytes with enumeration in Giemsa-stained films and uptake of [3H]hypoxanthine demonstrated the fluorochromasia technique yields comparable results. Studies with the hemoparasite, Babesia bovis, have shown the fluorochromasia technique can also be used to monitor the effect of parasiticidal drugs on parasites in vitro. The cumulative studies with the fluorochromasia assay suggest the assay will also prove useful in investigations focused on analysis of the immune response to hemoparasites and growth in vitro.