vendredi 24 avril 2009
par   G. Grégori

Accotto GP, Mullineaux PM, Brown SC, Marie D (1993) Digitaria streak geminivirus replicative forms are abundant in S-phase nuclei of infected cells. Virology 195 :257-259


We have analyzed the replication of Digitaria streak geminivirus (DSV) using flow cytometry. Nuclei from infected plants were sorted on the basis of their DNA content in the different phases of the cell cycle and viral DNA forms in them were analyzed. DSV replicative forms were much more abundant (up to 20 times in one experiment) in S-phase nuclei than in G0-G1 and G2 nuclei, while single-stranded viral DNA did not show such dramatic increase during any phase. DSV replicative forms constitute almost 90% of total viral DNA forms in S-phase nuclei. DSV replication seems to be synchronized with host DNA replication and might be primarily under control of host factors becoming available only at the G1/S boundary.

Atlung T, Hansen FG (1993) Three distinct chromosome replication states are induced by increasing concentrations of DnaA protein in Escherichia coli. J Bacteriol 175 :6537-6545


The DnaA protein concentration in Escherichia coli was increased above the wild-type level by inducing a lacP-controlled dnaA gene located on a plasmid. In these cells with different DnaA protein levels, we measured several parameters : dnaA gene expression ; cell size, amount of DNA per cell, and number of origins per cell by flow cytometry ; and origin-to-terminus ratio and the frequencies of five other markers on the chromosome by Southern hybridization. The response of the cells to higher levels of DnaA protein could be divided into three states. From the normal level to a level 1.5-fold higher, DnaA protein had little effect on dnaA gene expression and the rate of DNA replication but led to nearly proportional increases in DNA and origin concentrations. Between 1.5- and 3-fold, the normal DnaA protein concentration, dnaA gene expression was gradually decreased. In this interval, the origin concentration increased significantly ; however, the replication rate was severely affected, becoming slower—especially near the origin—the higher the DnaA protein concentration, and as a result, the DNA concentration was constant. Further increases in the DnaA protein concentration did not lead to an increased origin concentration. Thus, the initiation mass was set by the DnaA protein from the normal level to an at least twofold-increased level, but the increased initiation did not lead to a large increase in the amount of DNA per unit of mass because of the inhibition of replication fork velocity.

Avery VM, Gordon DL (1993) Characterization of factor H binding to human polymorphonuclear leukocytes. J Immunol 151 :5545-5553


Previous studies indicate that factor H (fH) binds to a number of cell types and may have functions other than C regulation. We have examined for fH binding to PMN using flow cytometry and radiolabeled binding assays. Binding of fH was demonstrated to be specific and saturable with approximately 6 x 10(4) binding sites/polymorphonuclear leukocytes (PMN) and a Ka value of 3.3 x 10(8) L/M. Binding of fH to PMN was ionic strength dependent, required divalent cations, and was enhanced by PMN stimulation with FMLP and calcium ionophore, A23187. The 38-kDa N-terminal tryptic fragment of fH bound to PMN and blocking experiments with mAb suggested a receptor binding site was located within the fifth SCR of fH. fH binding was not due to associations with surface-bound C3 or to CR3. Binding of fH to U937 and Raji cells, but not to T cells was also demonstrated. These studies provide presumptive evidence for a fHR on PMN. Binding of fH by fHR could enhance recognition of opsonized targets, trigger secondary intracellular events or contribute to intrinsic protection of cells against C.

Bowden RA, Verger JM, Grayon M, Limet JN, Dubray G (1993) Simultaneous expression of smooth and rough phase properties related to lipopolysaccharide in a strain of Brucella melitensis. J Med Microbiol 39 :363-370


Brucella strains exhibit either a rough (R) or a smooth (S) colonial phase identifiable by bacteriological methods. This depends on the biosynthesis and translocation to the surface in S but not in R strains, of the O-polysaccharide chain of the lipopolysaccharide (LPS) molecule. B. melitensis biovar 1 strain EP exhibited simultaneously both S and R characteristics in relation to colonial morphology, agglutination by monospecific anti-M and anti-R sera, activity of bacteriophages lytic for rough Brucella spp. (phage R/C) and for smooth B. melitensis (phage Iz). B. melitensis strain EP expressed fewer O-chains with a similar distribution of molecular weights than B. melitensis reference strain 16M by SDS-PAGE and immunoblotting, but higher amounts of R-LPS. Quantitative determination of S-LPS by a turbidimetric latex inhibition immunoassay with monoclonal antibodies confirmed the limited expression of S-LPS in strain EP. As with other gram-negative bacteria, the phenomenon could be attributed to a deficiency in one step of the biosynthetic assembly of the O-chains.

Caruso A, Tinti M, Peroni L, Cabibbo E, De Rango C, Manca N, Turano A (1993) Flow cytometric indirect immunofluorescence assay with high sensitivity and specificity for the detection of antibodies to HSV-1 and HSV-2. Eur J Epidemiol 9 :547-552


Cells infected with HSV-1 or HSV-2 develop viral antigens which can be detected by immunofluorescence. We developed a flow cytometric indirect immunofluorescence assay to detect and quantitate antibodies to HSV-1 and HSV-2 in human sera. Results obtained by flow cytometry for detecting antibodies against HSV-1, when compared with results obtained by ELISA, showed an index of overall agreement of 100%. The correlation between the antibody titers obtained with each method was found to be highly significant. An index of overall agreement equal to 94.1% was observed between results obtained by flow cytometry and by immunofluorescence as concerns the discrimination of HSV-2 positive from negative samples. However, the correlation between antibody titers was found to be not statistically significant. The flow cytometric assay proved to be type-specific.

Chaffin WL, Collins B, Marx JN, Cole GT, Morrow KJ, Jr. (1993) Characterization of mutant strains of Candida albicans deficient in expression of a surface determinant. Infect Immun 61 :3449-3458


Monoclonal antibody (MAb) 17E4 reacts with a surface carbohydrate determinant and agglutinates cells of Candida albicans. Using this MAb, we have isolated N-methyl-N’-nitro-N-nitrosoguanidine-induced nonagglutinating mutants. Eleven of these were characterized for the presence and expression of the surface antigen recognized by MAb 17E4 by immunoblot analysis of whole cells and by fluorescence flow cytometry. Soluble cell wall extracts from five mutant strains were negative by immunoblot analysis. The reactivities of the strains with several other MAbs and commercial antisera (Candida Check ; Iatron Laboratories, Tokyo, Japan) which also recognize carbohydrate determinants were examined by immunoblot analysis of whole cells. Mutant strains showed no or reduced expression of the MAb 17E4 antigen and could be placed into at least two distinct phenotypic classes. Recognition by the other MAbs tested showed a similar pattern, while recognition by the commercial antisera was unchanged in the mutant strains. 1H and 13C nuclear magnetic resonance spectral analysis of mannan prepared from the wild type and nonexpressing mutant-strain 4A showed that the spectra from the mutant strain were simpler than those of the wild type. Most of the beta-1,2 linkages and all of the C-1 phosphate linkages were absent in the 4A mannan spectra, which suggested that the mutant mannan lacked the phosphate-bound beta-1,2-linked mannooligosaccharides. The effect of the surface defect on the ability of mutant strain 4A to adhere and to invade host tissue was examined in two murine models. In ex vivo binding assays, strain 4A showed reduced binding to the marginal zone and increased binding to the white pulp of splenic tissue, decreased binding to kidney tissue, and no change in binding to liver tissue compared with the wild type. In vivo, no difference was observed in translocation of the wild type or strain 4A to liver following immuno-compromising treatment of infant mice which had been challenged with either strain by the oral-intragastric route.

Chemin I, Vermot-Desroches C, Baginski I, Lamelin JP, Hantz O, Jacquet C, Rigal D, Trepo C (1993) Monitoring of early events of experimental woodchuck hepatitis infection : studies of peripheral blood mononuclear cells by cytofluorometry and PCR. FEMS Immunol Med Microbiol 7 :241-249


The peripheral blood mononuclear cells (PBMC) of woodchucks experimentally infected by woodchuck hepatitis virus (WHV) were examined simultaneously for the presence of membrane associated WHV antigens by cytofluorometry, and for WHV DNA and RNA sequences by the polymerase chain reaction (PCR). Four woodchucks were inoculated : two with a well-defined infectious inoculum and two with an inoculum obtained from an animal at the late incubation phase, which was positive for WHV DNA by PCR but still devoid of WHV markers. Infection was demonstrated in all four inoculated woodchucks by the appearance at different times of WHV DNA and WHV antigens in both leucocytes and serum. WHV DNA was first detected by PCR either in the serum (two cases) or in leucocytes (two cases). The mean percentage of cells positive for membrane associated WHsAg or WHcAg detected by cytofluorometry were 37% +/- 25 and 17% +/- 15 respectively. After 8 weeks, all inoculated animals were WHsAg positive in serum. These data suggest that PBMC are involved in the early events of hepadnavirus infection. They also show that sera which are positive by PCR for WHV DNA may transmit viral infection even while still seronegative for WHV markers and for WHV DNA by dot blot.

Curiel RE, Miller MH, Ishikawa R, Thomas DC, Bigley NJ (1993) Does the gender difference in interferon production seen in picornavirus-infected spleen cell cultures from ICR Swiss mice have any in vivo significance ? J Interferon Res 13 :387-395


Splenocyte cultures from female ICR Swiss mice produced greater interferon (IFN) levels, particularly IFN-gamma, than did cultures from males by 12 h post-infection (pi) with the D variant of encephalomyocarditis virus (EMCV-D). This early IFN-gamma is produced by natural killer (NK)-like cells and is dependent on plastic adherent cells and IFN-alpha/beta. In this study, we evaluated the significance of this observation on the innate resistance of ICR Swiss females to EMCV-D-mediated disease. Treatment of females with rabbit anti-mouse IFN-alpha/beta serum rendered them susceptible to the diabetogenicity of EMCV-D. Although sera from both sexes of ICR Swiss mice exhibited peak IFN levels day 3 pi, IFN-gamma was present in the sera of males at only 1 day pi and in the sera of females at days 1-3 pi. Females cleared virus from the circulation by day 2 pi, 1 day earlier than did males. Flow cytometric evaluations of lymphoid cell phenotypes in spleens and pancreata of infected mice revealed that percentages of L3T4+ cells were significantly decreased only in spleens from males at day 1 pi and were diminished along with Ly2+ cells in pancreata of males at 7 days pi, suggesting that T-cell responses were impaired in virus-infected males.

Diago ML, Estepa A, Lopez-Fierro P, Villena A, Coll JM (1993) The in vitro infection of the hematopoietic stroma of trout kidney by hemorrhagic septicemia rhabdovirus. Viral Immunol 6 :185-191


Viral hemorrhagic septicaemia virus (VHSV) infected the hematopoietic stromal cells (7,8) derived from pronephritic tissue of the rainbow trout, Oncorhynchuss mykiss, W., at their ninth passage in vitro. Viral infection resulted in the development of lytic cytopathic effects on confluent in vitro tridimensional network stromal cell cultures. Replication of VHSV in the stromal cell cultures was demonstrated by the increase in infectivity by epithelioma papulosum cyprini (EPC) cell culture assays and by the increase of the nucleoprotein antigen of VHSV by ELISA. By using anti-VHSV monoclonal antibodies (MAbs), flow cytometry studies demonstrated that only the infected stromal cells contained cytoplasmic viral antigens. The lytic infection of trout hematopoietic stromal cells in vitro could be relevant to the hemorrhagic pathology seen in the kidney of fish infected with VHSV.

Fouchet P, Jayat C, Hechard Y, Ratinaud MH, Frelat G (1993) Recent advances of flow cytometry in fundamental and applied microbiology. Biol Cell 78 :95-109


This review focuses on the recent applications of flow cytometry (FCM) in microbiological research (1987-mid 1992). It tries to give a scope of the important breakthroughs which occurred in this field during this period. The technical difficulties of microorganism analysis by flow cytometry is briefly appraised. The significance and the limits of the different microbial cell parameters attainable by flow analyses are systematically evaluated : light scatter for cell size and structure, fluorescence measurements for quantification of cellular components, microbial antigen detection and cell physiological activity estimation. Emphasis is given on the new technological advances which appeared in the last two years. The second part of the review is devoted to the analysis of the usefulness of flow cytometric approach in the different fields of microbiology : fundamental studies in microbial physiology, differentiation, microbial ecology and aquatic sciences, medical microbiology, parasitology, microbial pharmacology and biotechnology.

Freistadt MS, Fleit HB, Wimmer E (1993) Poliovirus receptor on human blood cells : a possible extraneural site of poliovirus replication. Virology 195 :798-803


In order to determine whether any primary human blood cells have the ability to replicate poliovirus (PV), peripheral blood cell components were isolated and analyzed for their cell surface expression of the poliovirus receptor (PVR). Following two-color immunfluoresence staining with lineage-specific markers, the cells were analyzed by flow-cytometric methods. PVR cell surface expression was detected on most mononuclear cells expressing CD14, a marker for mononuclear phagocytes. There was no PVR cell surface expression on platelets and extremely low levels on polymorphonuclear leukocytes. Mononuclear leukocytes from Ficoll density centrifugation were found to support PV replication. The finding of PVR on mononuclear phagocytes and the ability of primary human blood cells to support PV replication in the absence of cultivation has implications for both the normal and pathogenic role of PVR.

Gant VA, Warnes G, Phillips I, Savidge GF (1993) The application of flow cytometry to the study of bacterial responses to antibiotics. J Med Microbiol 39 :147-154


Experiments were performed to determine whether a modern flow cytometer could be used to study bacterial populations in suspension, with particular reference to their morphological characteristics and their responses to antibiotics. The FACScan, a commercial benchtop flow cytometer fitted with an air-cooled laser, designed primarily for the study of eukaryotic peripheral blood mononuclear cells, yielded reproducible data relating to bacterial shape and internal architecture. It was sensitive enough to detect changes in bacterial morphology on entry into the growth cycle and after exposure to antibiotics. Antibiotic-induced morphological changes affecting subpopulations of bacteria were sufficiently specific to allow differentiation between antibiotics with different cell-wall enzyme targets. Simultaneously, the effect of such antibiotics on the integrity of the outer cell membrane of Escherichia coli was assessed by measurement of the association of the nucleic acid-binding dye propidium iodide with the bacteria. These experiments demonstrated complex patterns of probable cell-wall leakage, related to the modes of action of the antibiotics. The FACScan is a useful and sensitive tool for the study of the morphology and physiology of bacterial populations in suspension, and is especially applicable to the study of antibiotic action.

Garcia-Armesto MR, Prieto M, Garcia-Lopez ML, Otero A, Moreno B (1993) Modern microbiological methods for foods : colony count and direct count methods. A review. Microbiologia 9 :1-13


Over the last years methods for enumeration of microorganisms in foods are changing rapidly. Techniques based on totally new concepts as well as instruments and miniaturized systems that allow the automation and simplification of existing microbiological procedures have been developed. These rapid methodologies should satisfy the increasing requirements for effective quality assurance of foods. In the present paper we review some of the more interesting methods based on colony count or direct bacterial count.

Gordon DL, Sadlon T, Hefford C, Adrian D (1993) Expression of CD59, a regulator of the membrane attack complex of complement, on human astrocytes. Brain Res Mol Brain Res 18 :335-338


The present study demonstrates that human astrocytes synthesize and express CD59, a regulator of the membrane attack complex of complement. This was shown by flow cytometry following staining of astrocytes with MAb to CD59, and Western blotting of astrocyte lysates, which revealed the characteristic 18-23,000 M(r) band of CD59. Synthesis of CD59 by astrocytes was confirmed by detection of CD59 specific mRNA by polymerase chain reaction. A low level of C3 deposition occurred on astrocytes following exposure to autologous serum. CD59 may prevent subsequent damage from C5b-9 and protect astrocytes during inflammatory and infectious disorders of the nervous system.

Helgeland K, Nordby O (1993) Cell cycle-specific growth inhibitory effect on human gingival fibroblasts of a toxin isolated from the culture medium of Actinobacillus actinomycetemcomitans. J Periodontal Res 28 :161-165


A toxin isolated from the growth medium of Actinobacillus actinomycetemcomitans by ammonium sulfate precipitation was shown to inhibit irreversibly the multiplication of human gingival fibroblasts. DNA histograms from flow cytometric measurements showed that the cells accumulated preferentially in the G2 phase of the cell cycle. Such cells exhibited sheetlike protrusions, and an increased frequency of micronuclei was evident in cells treated with low concentrations of the toxin. Toxin-treated cells were viable for several weeks, as shown by staining with trypan blue and fluorescein diacetate, and the general cell metabolism as measured by oxygen consumption was unimpaired.

Houle JJ, Hoffmann EM (1993) Antibodies specific for human albumin function as blocking antibodies when attached to erythrocyte-bound albumin. Biochem Biophys Res Commun 194 :1161-1166


Albumin is shown to be firmly bound to human red blood cells using the techniques of flow cytometry, immunoblotting, and complement fixation. The interactions between antibodies attached to the cell bound albumin and the complement system are examined. Antibodies specific for human serum albumin bind to albumin on erythrocytes and activate both homologous and heterologous complement in the absence of hemolysis. Moreover, treatment of erythrocytes with anti-albumin antibodies renders the cells resistant to classical pathway mediated lysis initiated by a passive lysis system. Thus, erythrocyte-bound anti-albumin antibodies appear to function in a manner similar to "blocking" antibodies described in some bactericidal systems.

Ibrahim L, Dominguez M, Yacoub M (1993) Primary human adult lung epithelial cells in vitro : response to interferon-gamma and cytomegalovirus. Immunology 79 :119-124


Primary human adult lung epithelial cells (ALEC) were established in culture using the most distal parts of the lung to avoid the airways. Immunocytochemical peroxidase staining and semiquantitative flow cytometry were used to characterize the cells in conjunction with a panel of monoclonal antibodies (mAb). The cells showed a constitutive expression of major histocompatibility complex (MHC) class I antigens, patchy expression of intercellular adhesion molecule-1 (ICAM-1) and a weak patchy expression of MHC class II antigens (detected using immunocytochemical staining). Incubation of the primary ALEC with interferon-gamma (IFN-gamma) (250 U/ml) stimulated an up-regulation of the expression of these three antigens to varying degrees ; expression of MHC class I antigens and ICAM-1 molecules showed an up-regulation at 10 hr after the start of the treatment, reaching a peak at 48 hr, maintaining it for the next 24 hr and then, steadily and progressively, losing it towards the end of the experiment at 96 hr. Expression of HLA-DR showed an up-regulation at 17 hr after the start of the treatment, reaching a peak at 72 hr and maintaining it for the next 24 hr. Cytomegalovirus (CMV) infection of ALEC in culture caused an up-regulation of expression of class I antigens and ICAM-1, but not DR. However, when the infected cells were incubated with IFN-gamma, an up-regulation in the expression of DR took place. Therefore, within the micro-environment of the transplanted lung the presence of cytokines (IFN-gamma) produced by infiltrating activated mononuclear cells, may render the lung epithelial cells capable of acting as antigen-presenting cells, expressing high levels of class I antigens, ICAM-1 and class II antigens, activating CD8 and CD4 cells thus playing a major part in the process of rejection of the lung allograft ; themselves becoming a primary target in the process.

Ivanov VN, Pindrus AA (1993) Alternation of exo- and endotrophy during the mitotic cycle of the yeast cells. Mikrobiol Zh 55 :28-37


The utilization of the intracellular and extracellular sources of carbon and energy during the mitotic cycle of yeasts Saccharomyces cerevisiae, Kluyveromyces marxianus, Candida boidinii, Candida tropicalis has been studied. Increase in the consumption rate of carbon and energy sources and in the exogenous respiration rate at G1- and G2-phases of the mitotic cycle is shown. The rate of the endogenous respiration of the cells at these phases decreased. The hypothesis has been proposed that during the mitotic cycle of the yeast cell the regular alternation of exotrophy (the utilization of the extracellular carbon and energy sources by a cell) and endotrophy (the process of the utilization of the intracellular carbon and energy sources by a cell) occurs. It is possible to reveal the exotrophic cells by the cytological method which is based on the calculation of dead cells after incubation of the yeast suspension in amyl alcohol solution. This method has revealed that exotrophic and endotrophic processes do not predominate one over another but alternate at the mitotic cycle. Exotrophy and endotrophy are phase-specific processes. The G1- and G2-phases are exotrophic processes, phases S and M are endotrophic ones.

Jespersen L, Lassen S, Jakobsen M (1993) Flow cytometric detection of wild yeast in lager breweries. Int J Food Microbiol 17 :321-328


A flow cytometric method for detection of wild yeast infections in breweries is reported. It is based on selective enrichment in Malt extract Yeast extract Glucose Peptone broth (MYGP) at 37 degrees C and in MYGP with 200 ppm CuSO4 at 25 degrees C, staining with a fluorochrome precursor and flow cytometry. In experiments with several types of wild yeast isolated from breweries and two different strains of lager yeast it has been possible to detect one wild yeast per 10(6) culture yeast after 48-72 h of incubation and, in some cases, after 24 h.

Laplace-Builhe C, Hahne K, Hunger W, Tirilly Y, Drocourt JL (1993) Application of flow cytometry to rapid microbial analysis in food and drinks industries. Biol Cell 78 :123-128


In food and drinks industries, the time required for conventional tests can lead to substantial delays in product release to the market. Flow cytometry (FCM) has been used in conjunction with viability markers for rapid counting of yeast, mould and bacterial cells in food products. A single-parameter flow cytometer has proved applicable to the rapid detection of low numbers of microbial contaminants in finished products. The excellent correlation between FCM results and product quality shelf-life expiry date has allowed the establishment of realistic quality control criteria for rapid positive release of product. Used for the monitoring of microbial biomass during manufacturing processes, flow cytometry allowed a direct assessment of bacterial growth. The reproducibility of the results and the proven correlation with standard plate count method obtained in industrial conditions make FCM a good predictive method for product and process quality control.

Lehman JM, Friedrich TD, Laffin J (1993) Quantitation of simian virus 40 T-antigen correlated with the cell cycle of permissive and non-permissive cells. Cytometry 14 :401-410


These studies examined cell cycle progression and quantitative changes in T-antigen following infection by SV40. Single cells were assayed by multiparameter flow cytometric analysis (FCM) for DNA content and T-antigen expression. Conditions were used which permitted permissive, semi-permissive, and non-permissive cells to be monitored through two rounds of DNA synthesis induced by SV40. The permissive cells included the monkey kidney cell lines ; CV-1, Vero and BSC-1 and the COS-1 and COS-7 which are CV-1 cells transformed with an origin defective SV40. The non-permissive cell strains included mouse embryo fibroblasts, Chinese hamster fibroblasts, and IMR-90, a human diploid fibroblast. Cell types differed in the maximal amount of T-antigen expressed per cell. Additionally, all cell types expressed a limited quantity of T-antigen for each cell cycle phase and the quantity increased in each successive phase. The level in each phase was increased only two-fold when 100 times more virus was used. Thus, for an infected population the quantity of T-antigen was dependent on cell cycle distribution. High levels of T-antigen were not required for permissive infection ; however, permissive cells were distinguished from non-permissive cells by the G2 levels. Permissive G2 cells had more than double the T-antigen content expressed in G1, while nonpermissive G2 cells had less than a two-fold increase over G1 levels. The appearance of cells with tetraploid DNA content and the failure to undergo mitosis correlated to the higher T-antigen levels in the G2 of the permissive cells. Two other strains of SV40, 776, and VA45 exhibit similar values for T-antigen expression and movement into tetraploid DNA content. This study establishes the levels of T-antigen correlated to the cell cycle and cell type.

Lim EL, Amaral LA, Caron DA, DeLong EF (1993) Application of rRNA-based probes for observing marine nanoplanktonic protists. Appl Environ Microbiol 59 :1647-1655


The use of small-subunit rRNA-based oligonucleotides as probes for detecting marine nanoplanktonic protists was examined with a ciliate (an Uronema sp.), a flagellate (a Cafeteria sp.), and mixed assemblages of protists from enrichment cultures and natural seawater samples. Flow cytometry and epifluorescence microscopy analyses demonstrated that hybridizations employing fluorescein-labeled, eukaryote-specific probes intensely stained logarithmically growing protists, whereas these same protist strains in late stationary growth were barely detectable. The fluorescence intensity due to probe binding was significantly enhanced by the use of probes end labeled with biotin, which were detected by fluorescein-labeled avidin. The degree of signal amplification ranged from two- to fivefold for cultured protists in both logarithmic and stationary growth phases. Mixed assemblages of heterotrophic protists from enrichment cultures were also intensely labeled by rRNA-targeted oligonucleotide probes by the biotin-avidin detection system. Protists in late stationary growth phase and natural assemblages of protists that were otherwise undetectable when hybridized with fluorescein-labeled probes were easily visualized by this approach. In the latter samples, hybridization with multiple, biotin-labeled probes was necessary for detection of naturally occurring marine protists by epifluorescence microscopy. The signal amplification obtained with the biotin-avidin system should increase the utility of rRNA-targeted probes for identifying protists and facilitate characterization of the population structure and distribution of protists in aquatic environments.

MacNeill SA, Nurse P (1993) Mutational analysis of the fission yeast p34cdc2 protein kinase gene. Mol Gen Genet 236 :415-426


The p34cdc2 protein serine-threonine kinase plays an essential role in the life cycle of fission yeast, being required for both the G1-S and G2-M transitions during mitotic growth, and also for the second meiotic nuclear division. Functional homologues of p34cdc2 (each ca. 60% identical to the fission yeast prototype) have been isolated from organisms as diverse as humans, insects and plants, and there is now considerable evidence supporting the view that fundamental aspects of the cell cycle controls uncovered in fission yeast will prove to be conserved in all eukaryotes. By comparing the amino acid sequences of fission yeast p34cdc2 with its higher eukaryotic counterparts it is possible to identify conserved residues that are likely to be centrally important for p34cdc2 function. Here the effects are described of mutating a number of these conserved residues. Twenty-three new mutant alleles have been constructed and tested. We show that replacing cysteine 67 with tryptophan renders the resulting mutant protein p80cdc25-independent (while neither leucine, isoleucine nor valine has this effect) and that several of the amino acids within the highly conserved PSTAIRE region are not absolutely required for p34cdc2 function. Five acidic amino acids have also been mutated within p34cdc2, which are invariant across the eukaryotic protein kinase family. Acid-to-base mutations at three of these residues resulted in a dominant-negative, cell cycle arrest phenotype while similar mutations at the other two simply abolished p34cdc2 protein function. The results are discussed with reference to the predicted tertiary structure of the p34cdc2 enzyme.

Morgan JA, Rhodes G, Pickup RW (1993) Survival of nonculturable Aeromonas salmonicida in lake water. Appl Environ Microbiol 59 :874-880


The survival of Aeromonas salmonicida subsp. salmonicida was investigated in sterile and untreated lake water. In sterile lake water (filtered and autoclaved), it was found that cells of A. salmonicida entered a nonculturable but viable condition. Viability was determined by flow cytometry with the dye rhodamine 123, which is taken up and maintained within cells with a membrane potential. For survival studies in untreated lake water, A. salmonicida was marked with the xylE gene by using the plasmid pLV1013. Marked cells were detected by growth on tryptone soy agar and tryptone soy agar supplemented with kanamycin. Cells were also detected by polymerase chain reaction DNA amplification of the xylE gene and a chromosomal DNA fragment specific for A. salmonicida (pLV1013). The results indicated that A. salmonicida entered a nonculturable condition in untreated lake water over a 21-day study. The viability of nonculturable cells could not be determined in mixed samples ; however, the presence of nonculturable cells containing both chromosomal and plasmid DNA was confirmed.

Nelson D, Delahooke TE, Poxton IR (1993) Influence of subinhibitory levels of antibiotics on expression of Escherichia coli lipopolysaccharide and binding of anti-lipopolysaccharide monoclonal antibodies. J Med Microbiol 39 :100-106


The expression of Escherichia coli lipopolysaccharide (LPS) and the binding capacity of anti-LPS monoclonal antibodies (MAbs) to E. coli grown in the presence or absence of subinhibitory concentrations of various antibiotics was studied. Four E. coli strains (three clinical blood-culture isolates and an isogenic, non-capsulate mutant of the O18:K1 parent) were grown in the presence of the beta-lactam antibiotic, ampicillin, the aminoglycoside gentamicin, the fluoroquinolone ciprofloxacin and chloramphenicol. The 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. Treatment with ampicillin, chloramphenicol and ciprofloxacin resulted in enhanced binding of anti-core reactive MAbs to most E. coli strains. Overall, treatment with gentamicin produced the least effect on MAb binding. The presence of chloramphenicol decreased the expression of high molecular mass O-antigen or increased the expression of low molecular mass substituted E. coli LPS or both. These results further illustrate that LPS core, especially the inner-core region, becomes more accessible to antibodies when bacteria are grown in the presence of certain antibiotics. Possible synergy between antibodies and antibiotics for treatment of septicaemia and septic shock remains an intriguing possibility.

Nordstrom K, Austin SJ (1993) Cell-cycle-specific initiation of replication. Mol Microbiol 10 :457-463


The following characteristics are relevant when replication of chromosomes and plasmids is discussed in relation to the cell cycle : the timing or replication, the selection of molecules for replication, and the coordination of multiple initiation events within a single cell cycle. Several fundamentally different methods have been used to study these processes : Meselson-Stahl density-shift experiments, experiments with the so-called ’baby machine’, sorting of cells according to size, and flow cytometry. The evidence for precise timing and co-ordination of chromosome replication in Escherichia coli is overwhelming. Similarly, the high-copy-number plasmid ColE1 and the low-copy-number plasmids R1/R100 without any doubt replicate randomly throughout the cell cycle. Data about the low-copy-number plasmids F and P1 are conflicting. This calls for new types of experiments and for a better understanding of how these plasmids control their replication and partitioning.

Porter J, Edwards C, Morgan JA, Pickup RW (1993) Rapid, automated separation of specific bacteria from lake water and sewage by flow cytometry and cell sorting. Appl Environ Microbiol 59 :3327-3333


The use of fluorescence-activated flow cytometric cell sorting to obtain highly enriched populations of viable target bacteria was investigated. Preliminary studies employed mixtures of Staphylococcus aureus and Escherichia coli. Cells of S. aureus, when mixed in different proportions with E. coli, could be selectively recovered at a purity in excess of 90%. This was possible even when S. aureus composed only approximately 0.4% of the total cells. Cell sorting was also tested for the ability to recover E. coli from natural lake water populations and sewage. The environmental samples were challenged with fluorescently labelled antibodies specific for E. coli prior to cell sorting. Final sample purities of greater than 70% were routinely achieved, as determined by CFU. Populations of E. coli released into environmental samples were recovered at greater than 90% purity. The use of flow cytometry and cell sorting to detect and recover viable target bacteria present at levels of less than 1% within an indigenous microflora was also demonstrated.

Ritchie MH, Oakes JE, Lausch RN (1993) Passive transfer of anti-herpes simplex virus type 2 monoclonal and polyclonal antibodies protect against herpes simplex virus type 1-induced but not herpes simplex virus type 2-induced stromal keratitis. Invest Ophthalmol Vis Sci 34 :2460-2468


PURPOSE. To investigate whether passive transfer of antibodies to viral glycoproteins would protect against herpes simplex virus type 2-induced stromal keratitis. METHODS. Balb/c mice were infected on the scarified cornea with herpes simplex virus types 1 or 2 (HSV-1 and HSV-2, respectively), and monoclonal or polyclonal antibodies were administered intraperitoneally 24 hr later. Eyes were monitored for corneal opacity. Flow cytometry was used to examine the expression of glycoproteins on the surface of HSV-infected cells. RESULTS. Passive transfer of monoclonal antibodies to viral glycoproteins gB, gD, or gE or anti-HSV-2 hyperimmune serum were all highly effective (P < 0.005) at preventing blinding disease induced by HSV-1. In contrast, none of the antibody preparations could prevent stromal keratitis when the animals were challenged with various HSV-2 strains. However, antibody treatment could prevent the development of fatal encephalitis in the majority of HSV-2 infected hosts. Flow cytometry analysis revealed that gD and gB expression on the membranes of HSV-2 infected corneal epithelial cells isolated from excised corneas was substantially less (P < 0.005) than that detected on HSV-1 infected cells at both 24 and 48 hours postinfection. This antigenic difference was not due to the failure of HSV-2 to replicate in corneal epithelial cells in vivo. CONCLUSIONS. Decreased levels of membrane glycoprotein antigen expression may be one factor contributing to the refractiveness of HSV-2-induced ocular disease to humoral immunotherapy.

Rote NS, Ng AK, Dostal-Johnson DA, Nicholson SL, Siekman R (1993) Immunologic detection of phosphatidylserine externalization during thrombin-induced platelet activation. Clin Immunol Immunopathol 66 :193-200


The antiphospholipid antibody syndrome is characterized by circulating antiphospholipid antibodies against cardiolipin (CL) and phosphatidylserine (PS) and clinically associated with a high risk of spontaneous thrombosis. Three monoclonal antibodies that differentiate between CL or PS were tested against resting and thrombin-activated platelets by flow cytometry. Each antibody reacted differently with CL and PS ; 3SB9b reacted with PS, D11A4 reacted with CL, and BA3B5C4 reacted with both CL and PS. Activated platelets bound BA3B5C4 and 3SB9b, but not D11A4. The BA3B5C4-reactive epitope appeared earlier during activation than the epitope reactive with 3SB9b. These data suggest that antibodies against PS are reactive with activated platelets and that two immunoreactive forms of PS are sequentially expressed on platelets during activation.

Saadi AT, Blackwell CC, Raza MW, James VS, Stewart J, Elton RA, Weir DM (1993) Factors enhancing adherence of toxigenic Staphylococcus aureus to epithelial cells and their possible role in sudden infant death syndrome. Epidemiol Infect 110 :507-517


Toxigenic strains of Staphylococcus aureus have been suggested to play a role in sudden infant death syndrome (SIDS). In this study we examined two factors that might enhance binding of toxigenic staphylococci to epithelial cells of infants in the age range in which cot deaths are prevalent : expression of the Lewis(a) antigen and infection with respiratory syncytial virus (RSV). By flow cytometry we demonstrated that binding of three toxigenic strains of S. aureus to cells from nonsecretors was significantly greater than to cells of secretors. Pre-treatment of epithelial cells with monoclonal anti-Lewis(a) or anti-type-1 precursor significantly reduced bacterial binding (P < 0.01) ; however, attachment of the bacteria correlated only with the amount of Lewis(a) antigen detected on the cells (P < 0.01). HEp-2 cells infected with RSV bound significantly more bacteria than uninfected cells. These findings are discussed in context of factors previously associated with SIDS (mother’s smoking, bottle feeding and the prone sleeping position) and a hypothesis proposed to explain some cases of SIDS.

Saalmuller A, Mettenleiter TC (1993) Rapid identification and quantitation of cells infected by recombinant herpesvirus (pseudorabies virus) using a fluorescence-based beta-galactosidase assay and flow cytometry. J Virol Methods 44 :99-108


We recently described construction and use of a beta-galactosidase expression cassette in isolating recombinant pseudorabies virus (PrV) mutants (Mettenleiter and Rauh, 1990). We report here the identification and exact quantitation of cells infected by these mutants using an assay based on the reaction of intracellular beta-galactosidase expressed during infection by the recombinant viruses with the fluorogenic substrate fluorescein di-beta-D-galactopyranoside (FDG) followed by detection of positive cells in flow cytometry (FACS-Gal assay ; Nolan et al., 1988). The detection method is fast, sensitive, and reliable, and yields quantitative results on single cell basis.

Samoszuk M, Ramzi E, Ravel J (1993) Disseminated persistent lymphoid hyperplasia containing Epstein-Barr virus and clonal rearrangements of DNA. Diagn Mol Pathol 2 :57-60


We describe the pathologic, molecular, and clinical features of a 52-year-old man who had a 7-year history of widely disseminated, persistent lymphoid hyperplasia. Exuberant follicular and interfollicular lymphoid hyperplasia with some histologic features of Castleman’s disease were present at various times in the submental and cervical lymph nodes, lacrimal and parotid glands, right and left orbits, mediastinum, and hard palate of this patient. Flow cytometric and immunoperoxidase studies of two of the specimens indicated a slight predominance of cells expressing lambda-immunoglobulin light chain. In one specimen, there were clonal rearrangement of DNA coding for immunoglobulin heavy chain and for the T-cell beta-receptor. When DNA from this specimen was also examined by the polymerase chain reaction technique, Epstein-Barr viral DNA was detected. This case suggests that Epstein-Barr virus may be associated with an unusual form of aggressive and persistent lymphoid hyperplasia that contains clonal rearrangements of DNA.

Schreuer D, Hammerberg B (1993) Modulation of cellular and humoral immunity, and disease manifestation during onset of patency in Brugia pahangi-infected dogs. Immunology 79 :658-666


Recently, it has become possible to obtain as predicted disease manifestation in selectively bred dogs infected with the naturally occurring lymphatic nematode, Brugia pahangi. In this study, an attempt was made to correlate limb oedema with dynamic changes in immune cell responses occurring in the lymph node at the site of infection during onset of patency. Three litters of dogs were selectively bred ; one for the expression of clinical disease, one for the lack of expression of clinical disease and one was of non-specific phenotype. Lymph node cells from 10 of 11 dogs showed a parasite-specific proliferative response at 4-6 weeks post-infection (p.i.), before the onset of patency. In six of 11 dogs, a loss of proliferative response to BpA in the infected node cells was detected around the time of onset of patency. In contrast, there was no reduction in the proliferative response to the mitogen phytohaemagglutinin (PHA). The proliferative response to BpA by unresponsive node cells could be restored by addition of substimulatory amounts of murine or human recombinant interleukin-2 (IL-2) to the culture medium. However, there was no correlation between the proliferative response of lymph node cells from infected limbs and the expression of clinical disease. Similarly, when in vitro parasite-specific antibody production by infected lymph node cells was examined, antibody production manifested by all dogs at 5 weeks p.i. was markedly changed at 8 weeks p.i., but these changes did not correlate with clinical disease. This lack of correlation indicates that the immune response to lymphatic filarial infection, as measured in this study, does not necessarily result directly in disease manifestation, and that other genetically determined factors may influence both the parasite-specific immune response and the clinical outcome of infection.

Schut F, de Vries EJ, Gottschal JC, Robertson BR, Harder W, Prins RA, Button DK (1993) Isolation of Typical Marine Bacteria by Dilution Culture : Growth, Maintenance, and Characteristics of Isolates under Laboratory Conditions. Appl Environ Microbiol 59 :2150-2160


Marine bacteria in Resurrection Bay near Seward, Alaska, and in the central North Sea off the Dutch coast were cultured in filtered autoclaved seawater following dilution to extinction. The populations present before dilution varied from 0.11 x 10 to 1.07 x 10 cells per liter. The mean cell volume varied between 0.042 and 0.074 mum, and the mean apparent DNA content of the cells ranged from 2.5 to 4.7 fg of DNA per cell. All three parameters were determined by high-resolution flow cytometry. All 37 strains that were obtained from very high dilutions of Resurrection Bay and North Sea samples represented facultatively oligotrophic bacteria. However, 15 of these isolates were eventually obtained from dilution cultures that could initially be cultured only on very low-nutrient media and that could initially not form visible colonies on any of the agar media tested, indicating that these cultures contained obligately oligotrophic bacteria. It was concluded that the cells in these 15 dilution cultures had adapted to growth under laboratory conditions after several months of nutrient deprivation prior to isolation. From the North Sea experiment, it was concluded that the contribution of facultative oligotrophs and eutrophs to the total population was less than 1% and that while more than half of the population behaved as obligately oligotrophic bacteria upon first cultivation in the dilution culture media, around 50% could not be cultured at all. During one of the Resurrection Bay experiments, 53% of the dilution cultures obtained from samples diluted more than 2.5 x 10 times consisted of such obligate oligotrophs. These cultures invariably harbored a small rod-shaped bacterium with a mean cell volume of 0.05 to 0.06 mum and an apparent DNA content of 1 to 1.5 fg per cell. This cell type had the dimensions of ultramicrobacteria. Isolates of these ultramicrobacterial cultures that were eventually obtained on relatively high-nutrient agar plates were, with respect to cell volume and apparent DNA content, identical to the cells in the initially obligately oligotrophic bacterial dilution culture. Determination of kinetic parameters from one of these small rod-shaped strains revealed a high specific affinity for the uptake of mixed amino acids (a degrees (A), 1,860 liters/g of cells per h), but not for glucose or alanine as the sole source of carbon and energy (a degrees (A), +/- 200 liters/g of cells per h). The ultramicrobial strains obtained are potentially a very important part of picoplankton biomass in the areas investigated.

Sonnenfeld G, Schaffar L, Schmitt DA, Koebel DA, Smith BA (1993) Interferon production by and leukocyte phenotyping of rhesus monkey lymph node and peripheral blood cells. J Interferon Res 13 :259-265


The ability of peripheral blood leukocytes to produce interferon-gamma (IFN-gamma) and be labeled with monoclonal antibodies against cell-surface markers was determined in this study. Both peripheral blood leukocytes and lymph node cells were able to produce IFN-gamma after challenge with mitogens. The rhesus monkey IFN-gamma was detectable by means of a biological assay but not by means of a radioimmunoassay for human IFN-gamma. Peripheral blood leukocytes and lymph node cells from rhesus monkeys (Macaca mulatta) were treated with fluoresceinated antibodies directed primarily against cell-surface antigens of humans. The degree of binding was determined by means of flow cytometry. Several of the anti-human antibodies did bind to the rhesus monkey peripheral blood leukocytes, as expected. In a novel study, the antibodies bound in a similar fashion to rhesus monkey lymph node cells. Binding of the antibodies was equivalent whether the cells came from inguinal or axillary lymph nodes. Rhesus monkey peripheral blood leukocytes incubated with recombinant human IFN-gamma showed enhanced expression of class II major histocompatibility complex antigens, as detected with anti-HLA-DR antibodies.

Stabnikova EV, Ivanov VN, Gregirchak NN, Dul’gerov AN (1993) [The use of the neustonic forms of bacilli for purifying and decontaminating reservoirs]. Mikrobiol Zh 55 :88-94


It is shown possible to select the bacterial strains which are neuston ones, i.e., concentrating on the water-atmosphere interface. The preparation based on the neuston form of Bacillus megaterium is more efficient for purification of water polluted with oil hydrocarbons than the preparation based on the planktonic form of the same culture. Preparation based on the neuston form of the aerobic spore-forming bacteria is effective for biological decontamination of sewage treated using conventional methods. Application of neuston bacterial forms permits intensifying the microbiological processes in the thin (15-40 microM) surface layer of water bodies.

Talken BL, Tummuru U, Lee DR (1993) Slow egress of a mouse MHC class I molecule to the cell surface despite its strong association with beta 2-microglobulin. Mol Immunol 30 :721-731


Two H-2D region class I genes from the wild-derived mouse strain B10.GAA37 provisionally encoding the Dw16 and Lw16 molecules, respectively, were transfected into mouse L cells, and the expressed gene products were analyzed serologically by flow cytometry. As expected from nucleotide sequence comparisons, these analyses revealed that several Ld-reactive monoclonal antibodies (mAbs) recognize Lw16 and not Dw16. As detected by flow cytometry of intact L.Lw16 cells and B10.GAA37 splenocytes, and by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) of immunoprecipitates from splenocyte lysates, the alpha 2 domain-reactive mAb 30-5-7 detected less Lw16 than did the alpha 3 domain-reactive mAb 28-14-8, suggesting the existence of two populations of Lw16 molecules : 30-5-7+ 28-14-8+ and 30-5-7- 28-14-8+. Sequential immunoprecipitation studies provided further evidence for these two Lw16 subsets ; furthermore, the 30-5-7- 28-14-8+ subset was found predominantly on the cell surface and in association with beta 2-microglobulin (beta 2-m). Pulse-chase studies of B10.GAA37 splenocytes revealed that Lw16, like Ld, is trafficked slowly to the cell surface, whereas Dw16 is trafficked quickly, like most other mouse K and D region class I molecules. Despite these similarities, Lw16 and Ld differ in their association with beta 2-m, in that the immunoprecipitates of Lw16 contained much higher levels of radiolabeled beta 2-m per heavy chain. Together, these studies indicate that the slower trafficking of Lw16 to the surface does not result from a weaker association with beta 2-m, suggesting that other factors, such as peptide ligand-induced assembly, and/or retention by ER-resident proteins play an important role in the trafficking of major histocompatibility (MHC) class I molecules to the cell surface.

Torigoe S, Campbell DE, Torigoe F, Michelson S, Starr SE (1993) Cytofluorographic analysis of effects of interferons on expression of human cytomegalovirus proteins. J Virol Methods 45 :219-228


The appearance of cytomegalovirus (CMV) proteins in infected fibroblasts was determined by flow cytometry. The sequential production of immediate early (IE), early (E), and late (L) proteins reacting with respective monoclonal antibodies (mAbs) E13, 58/5, and 24/4 was determined in fibroblasts infected with the AD-169 strain of CMV. The percentage of cells expressing CMV proteins and the intensity of fluorescence within cells were determined from day 1 to day 7 post-infection. The effect of interferons (IFNs) alpha, beta, gamma on expression of CMV proteins was analyzed using flow cytometry. IFNs inhibited E and L protein production at days 3 and 6 post-infection in a dose-dependent manner. This inhibitory effect on protein expression was associated with a reduction in release of infectious CMV into culture media. The method described here for detection of CMV proteins using flow cytometry may be useful for basic studies of gene expression and for diagnostic purposes.

Troussellier M, Courties C, Vaquer A (1993) Recent applications of flow cytometry in aquatic microbial ecology. Biol Cell 78 :111-121


Microorganisms (unicellular algae, bacteria) constitute fundamental compartments of aquatic ecosystems because of their high concentrations and activities. The evaluation and understanding of their behavior and role raise different problems for which traditional methodologies are often inadequate, whether they refer to global or classical microscopic analyses. Flow cytometry (FCM) has been recently used to study microorganisms in aquatic environments. Although this technology is still applied on a limited scale in our field, a large number of works has been done showing that FCM seems to be a promising tool for aquatic microbial ecology. This paper summarizes, from the literature produced during the last decade and with original data obtained in our laboratory, the main questions related to the cell identification, the evaluation of cell viability, biomasses and productions and the measurements of bacterial and phytoplanktonic activities. The representatives of sampling and observation scales is also discussed within the framework of the FCM measurements.

Van Vliet KE, De Graaf-Miltenburg LA, Verhoef J, Van Strijp JA (1993) A flow cytometric rosetting assay for the analysis of Fc receptors and C3 receptors on HSV-infected cells. J Immunol Methods 157 :57-64


A sensitive and reproducible flow cytometric assay was developed for the analysis of Fc gamma and C3b(i) receptors on HSV-infected cells. The method is based on a rosette technique using fluorochrome-labeled erythrocytes sensitized with IgG or C3b(i). A comparison of flow cytometric and microscopic quantitation demonstrated that the binding of EIgG, EC3b(i) to HSV-infected cells were correlated. Flow cytometric analysis provides the opportunity to study simultaneously the distribution of E per HSV-infected cell and the total binding of E to the whole population of HSV-infected cells. Receptor activity and HSV glycoprotein cell surface expression were shown to be correlated in a linear fashion. The assay could be applied to other Fc gamma R- and C3b(i)R-bearing cells.

Wade BM, Mealey BL, Giardino A, Hallmon WW (1993) Microbial assessments and periodontal diagnosis. Compendium 14 :682-690