1989

vendredi 24 avril 2009
par   G. Grégori

Bernander R, Merryweather A, Nordstrom K (1989) Overinitiation of replication of the Escherichia coli chromosome from an integrated runaway-replication derivative of plasmid R1. J Bacteriol 171 :674-683

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A 16-base-pair fragment, deletion of which completely inactivated oriC, was replaced by a temperature-dependent runaway-replication derivative (the copy number of which increases with temperature) of the IncFII plasmid R1. The constructed strains were temperature sensitive, and flow cytometry revealed a severalfold increase in the DNA/mass ratio following shifts to nonpermissive temperatures. The cell size distribution was broader in the constructed strains relative to that in the wild type because of asynchrony between the chromosome replication and cell division cycles. This difference was more pronounced for counterclockwise initiation of chromosomal replication, in which small DNA-less cells and long filaments were abundant. Following a temperature shift the cell size distributions became even more broad, showing that changes in the frequency of chromosomal replication affect cell division and emphasizing the interplay between these two processes.


Bray RA, Landay AL (1989) Identification and functional characterization of mononuclear cells by flow cytometry. Arch Pathol Lab Med 113 :579-590

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Flow cytometric technology has evolved tremendously over the past 5 years. Among the major advancements have been the development of monoclonal antibodies, fluorescent labels, and computer software that makes it possible to perform routine multiparameter analyses. The clinical application of such analyses requires that standards and quality control procedures be established. Areas that need to be specifically addressed include sample preparation, staining, instrument calibration, and sample and data analysis. This study describes the basic elements involved in establishing such procedures and explores the use of multiparameter flow cytometric analysis in the characterization of peripheral blood mononuclear cells. Two-color, flow cytometric analysis of T, B, and natural killer cells can provide important insights into the biologic features of these cells as well as significant diagnostic information.


Burkhardt JK, Argon Y (1989) Intracellular transport of the glycoprotein of VSV is inhibited by CCCP at a late stage of post-translational processing. J Cell Sci 92 ( Pt 4) :633-642

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The appearance of newly synthesized glycoprotein (G) of vesicular stomatitis virus at the surface of infected BHK cells is inhibited reversibly by treatment with carbonylcyanide m-chlorophenylhydrazone (CCCP). Under the conditions used, CCCP treatment depleted the cellular ATP levels by 40-60%, consistent with inhibition of transport at energy-requiring stages. The G protein that accumulates in cells treated with CCCP is heterogeneous. Most of it is larger than the newly synthesized G protein, is acylated with palmitic acid, and is resistant to endoglycosidase H (Endo H). Most of the arrested G protein is also sensitive to digestion with neuraminidase, indicating that it has undergone at least partial sialylation. A minority of G protein accumulates under these conditions in a less-mature form, suggesting its inability to reach the mid-Golgi compartment. The oligosaccharides of this G protein are Endo-H-sensitive and seem to be partly trimmed. Whereas sialylated G protein was arrested intracellularly, fucose-labelled G protein was able to complete its transport to the cell surface, indicating that a late CCCP-sensitive step separates sialylation from fucosylation. These post-translational modifications indicate that G protein can be transported as far as the trans-Golgi in the presence of CCCP and is not merely arrested in the endoplasmic reticulum.


Buschmann H, Winter M (1989) Assessment of phagocytic activity of granulocytes using laser flow cytometry. J Immunol Methods 124 :231-234

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A laser flow cytometer assay for assessing two functions of polymorphonuclear leucocytes is described. The technique permits the quantitation of phagocytosis and intracellular killing of yeast cells by leucocytes and is illustrated with results obtained using porcine polymorphonuclear leucocytes.


Button DK, Robertson BR (1989) Kinetics of bacterial processes in natural aquatic systems based on biomass as determined by high-resolution flow cytometry. Cytometry 10 :558-563

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The two primary kinetic constants for describing the concentration dependency of nutrient uptake by microorganisms are shown to be maximal rate of substrate uptake and, rather than the Michaelis constant for transport, specific affinity. Of the two, the specific affinity is more important for describing natural aquatic microbial processes because it can be used independently at small substrate concentrations. Flow cytometry was used to evaluate specific affinities in natural populations of aquatic bacteria because it gives a convenient measure of biomass, which is an essential measurement in the specific-affinity approach to microbial kinetics. Total biomass, biomass in various filter fractions, and the specific affinity of the bacteria in each fraction were determined in samples from a near-arctic lake. The partial growth rate of the pelagic bacteria from the 25 micrograms/liter of dissolved amino acids present (growth rate from the amino acid fraction alone) was determined to be 0.78 per day. By measuring activity in screened and whole-system populations, the biomass of the bacteria associated with particles was computed to be 427 micrograms/liter.


Chisari FV, Klopchin K, Moriyama T, Pasquinelli C, Dunsford HA, Sell S, Pinkert CA, Brinster RL, Palmiter RD (1989) Molecular pathogenesis of hepatocellular carcinoma in hepatitis B virus transgenic mice. Cell 59 :1145-1156

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Transgenic mice that overproduce the hepatitis B virus large envelope polypeptide and accumulate toxic quantities of hepatitis B surface antigen (HBsAg) within the hepatocyte develop severe, prolonged hepatocellular injury that initiates a programmed response within the liver, characterized by inflammation, regenerative hyperplasia, transcriptional deregulation, and aneuploidy. This response inexorably progresses to neoplasia. The incidence of hepatocellular carcinoma in this model corresponds to the frequency, severity, and age of onset of liver cell injury, which itself corresponds to the intrahepatic concentration of HBsAg and is influenced by genetic background and sex. Thus, the inappropriate expression of a single structural viral gene is sufficient to cause malignant transformation in this model. These results suggest that severe, prolonged cellular injury induces a preneoplastic proliferative response that fosters secondary genetic events that program the cell for unrestrained growth.


Cohen CY, Sahar E (1989) Rapid flow cytometric bacterial detection and determination of susceptibility to amikacin in body fluids and exudates. J Clin Microbiol 27 :1250-1256

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A flow cytometry-based method for rapid and quantitative detection of bacteria in various clinical specimens and for rapid determination of antibiotic effect is described. Achieving such a measurement with high sensitivity required discrimination between bacteria and other particles which were often present in clinical samples in high concentrations. This discrimination was facilitated by detecting the bacterial characteristic light scatter and fluorescence signals following staining, e.g., with the fluorescent nucleic acid-binding dye ethidium bromide, as well as by measuring bacterial proliferation during short time intervals. Antibiotic susceptibility was measured by observing the inhibition of such proliferation. The method was applied to 43 clinical specimens from various sources, such as wound exudates, bile, serous cavity fluids, and bronchial lavage. Bacterial detection, achieved in less than 2 h, agreed with results of conventional methods with a sensitivity of 74% and a specificity of 88%. Susceptibility to amikacin was detected in 1 h in 92% of 13 positive specimens.


Fronko GE, Long WK, Wu B, Papadopoulos T, Henderson EE (1989) Relationship between methylation status and expression of an Epstein-Barr virus (EBV) capsid antigen gene. Biochem Biophys Res Commun 159 :263-270

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The methylation status of the 160 kD viral capsid antigen (VCA) gene promoter was determined by hybridization analysis. The semi-permissive marmoset cell line FF41-1 lacked cytosine methylation in approximately three quarters of the VCA promoter CpG dinucleotide residues. In the stringently infected HH514CL16 cell line the same CpG residues were methylated in three quarters of the genomes. 5’deoxy-5’-S-isobutyladenosine (SIBA), a DNA methylase inhibitor, was utilized to disrupt the EBV latent state. As determined by flow cytometry, SIBA treatment significantly increased expression of VCA. The VCA promoter was hypomethylated in VCA-positive FF41-1 cells sorted by flow cytometry. While hypomethylation alone was not sufficient for VCA transcriptional activity, the absence of methylation of VCA promoter CpG dinucleotide residues was associated with expression of VCA.


Gordon DL, Rice JL, McDonald PJ (1989) Regulation of human neutrophil type 3 complement receptor (iC3b receptor) expression during phagocytosis of Staphylococcus aureus and Escherichia coli. Immunology 67 :460-465

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Human neutrophils (PMN) express a receptor for iC3b, a cleavage product of C3b. CR3 is an important receptor for phagocytosis of opsonized bacteria and its expression is enhanced by cell activation. We examined PMN CR3 expression during phagocytosis using flow cytometry and a CR3-specific monoclonal antibody. After 30 min phagocytosis of opsonized S. aureus and E. coli, CR3 expression increased to 151% and 221% of controls, respectively. Unopsonized S. aureus had no effect on CR3 ; however, unopsonized E. coli enhanced CR3 expression despite not being phagocytosed. Time-kinetic studies indicated a rapid initial fall in CR3 after addition of bacteria to PMN, followed by enhanced expression within 5-10 min. The initial fall in CR3 probably represented CR3 internalization rather than receptor destruction, as superoxide dismutase, catalase and protease inhibitors had no effect on this. Correlation of CR3 expression with the PMN oxidative response, measured with the intracellular fluorescent probe DCF-DA, demonstrated a dichotomy. Opsonized S. aureus and E. coli caused an oxidative response from PMN but unopsonized E. coli, which caused significant CR3 up-regulation, did not. CR3 up-regulation with unopsonized and opsonized E. coli was markedly inhibited by Polymyxin B, suggesting a role for endotoxin. These experiments indicate that CR3 expression can be regulated during phagocytosis, and the mechanisms responsible are distinct from those involved in the oxidative burst. CR3 up-regulation following exposure to bacteria in vivo may enhance neutrophil function at sites of infection.


Karn J, Watson JV, Lowe AD, Green SM, Vedeckis W (1989) Regulation of cell cycle duration by c-myc levels. Oncogene 4 :773-787

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Early passage murine fibroblasts infected with retroviral vectors carrying human c-myc ’minigenes’ express high levels of c-myc and have a dramatically shortened G1-phase of the cell cycle. Cells infected with viruses where c-myc is expressed from the viral LTR (MSN-4 virus) express more c-myc protein than cells infected with viruses where c-myc is expressed from the SV40 early promoter (NSM-7 virus). Populations of cells were infected with high titre viruses, selected for drug-resistance, pulse labelled with bromodeoxyuridine (BrdUrd) and chased in BrdUrd free media. This allows accurate, simultaneous, measurement of the rate of exit of unlabelled cells from G1 and progression of BrdUrd-labelled cells through S-phase. The length of the G1-phase in cell populations infected with the MSN-4 virus is 4.65 h, a reduction of nearly 30% compared to the G1-phase length of 6.50 h seen in cells infected with the VSN-2 control virus. Cells infected with NSM-7 virus show an intermediate phenotype and have a G1-phase of 5.25 h. The lengths of the S-phase (4.50 to 4.75 h) and G2 + M phases (2.75 h) were not significantly altered by exogenous c-myc expression. When chases are performed in growth-factor free media, the G1-phase of infected and non-infected cells is extended by approximately 2 h. Cells infected with the c-myc viruses continue to cycle more rapidly than uninfected cells. Growth factor-deprived cells, restimulated with serum, show similar alterations of the cell cycle kinetics. MSN-4 and NSM-7 infected cells, expressing high levels of c-myc, enter S-phase 2 to 4 h earlier, but less synchronously, than control cells, and sustain subsequent rounds of DNA synthesis, while control cells do not. However, cells carrying activated c-myc genes have nearly-normal morphologies and are not tumourgenic in syngenic mice. These results demonstrate that c-myc levels are rate limiting for events in G1, and the length of G1 varies proportionally with the level of exogenous c-myc expression.


Kawaguchi S (1989) Reactivity of mouse antibodies against bromelain-treated mouse erythrocytes with thrombin-treated mouse platelets. Immunology 66 :335-338

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The reactivity of mouse antibodies against bromelain-treated mouse erythrocytes (BrMRBC) with mouse platelets before and after thrombin treatment was assessed by flow cytometry. Anti-BrMRBC antibodies could bind to thrombin-treated platelets, although normal platelets were also weakly reactive with the antibodies. The binding of anti-BrMRBC antibodies to platelets was confirmed by complement-dependent lysis. It is suggested that thrombin-activated platelets may be a real target for anti-BrMRBC antibodies.


Kearsley JH (1989) Recent initiatives in head and neck cancer research : an overview. Aust N Z J Surg 59 :367-372

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Legendre L, Yentsch CM (1989) Overview of flow cytometry and image analysis in biological oceanography and limnology. Cytometry 10 :501-510

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Mleczko J, Litke LL, Larsen HS, Chaffin WL (1989) Effect of glutaraldehyde fixation on cell surface binding capacity of Candida albicans. Infect Immun 57 :3247-3249

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The ability of viable and glutaraldehyde-fixed, stationary-phase yeast cells of Candida albicans to bind concanavalin A and monospecific antiserum for antigenic factor 1 was examined. Both fluorescence flow cytometric analysis and transmission electron microscopy indicated that glutaraldehyde-fixed cells bound less of the two reagents than did unfixed viable cells.


Pace J, Chai TJ (1989) Comparison of Vibrio parahaemolyticus grown in estuarine water and rich medium. Appl Environ Microbiol 55 :1877-1887

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Cell envelope composition and selected physiological traits of Vibrio parahaemolyticus were studied in regard to the Kanagawa phenomenon and growth conditions. Cell envelopes were prepared from cells cultured in Proteose Peptone-beef extract (Difco Laboratories, Detroit, Mich.) medium or filtered estuarine water. Protein, phospholipid, and lipopolysaccharide contents varied with culture conditions. The phospholipids present in the cell envelopes were identified as phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. Phosphatidylethanolamine decreased and phosphatidylglycerol increased in cells grown in estuarine water. Profiles of proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated numerous protein species, with four to six predominant proteins ranging from 26,000 to 120,000 in molecular weight. The profile of V. parahaemolyticus cell envelope proteins was unique and might be useful in the identification of the organism. Alkaline phosphatase activity was slightly higher in Kanagawa-negative strains and was higher in cells grown in estuarine water than in cells grown in rich laboratory medium. The DNA levels in estuarine water-grown cells increased, while RNA levels and cell volume decreased. Bacteriophage sensitivity typing demonstrated a close intraspecies relationship. Results indicated that Kanagawa-positive and -negative strains were closely related, but they could be grouped separately and may have undergone starvation-related physiological changes when cultured in estuarine water.


Shiigi S, Wilson B, Leo G, MacDonald N, Toyooka D, Hallick L, Karty R, Belozer ML, McNulty W, Wolff J, et al. (1989) Serologic and virologic analysis of type D simian retrovirus infection in a colony of Celebes black macaques (Macaca nigra). J Med Primatol 18 :185-193

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Celebes macaques were tested for type D simian retrovirus (SRV) infection. SRV infection was first detected in one serum sample collected during 1980. By 1983, 32 of 46 monkeys (70%) were infected. Serotyping of the SRV isolates determined that 0/26 of the isolates were SRV-1 ; 24/26 were SRV-2 ; 1/26 was SRV-5 ; and 1/26 could not be typed. Restriction endonuclease mapping confirmed the SRV-2C and SRV-5 isolates. In addition, two SRV-2C variants were detected.


Van Strijp JA, Van Kessel KP, van der Tol ME, Verhoef J (1989) Complement-mediated phagocytosis of herpes simplex virus by granulocytes. Binding or ingestion. J Clin Invest 84 :107-112

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The role of complement receptors in phagocytosis of herpes simplex virus (HSV) by PMN was examined. Complement components were deposited on the surface of the virus particle in the presence or absence of specific anti-HSV antibodies. Flow cytometry was used to analyze the phagocytosis of fluorescence-labeled viruses and demonstrated that although a virion is able to associate with PMN in the presence of complement alone, the granulocyte is not triggered to mount a metabolic burst. Efficient stimulation of PMN occurs when complexes are formed consisting of virus, specific antibodies, and complement. To address the question whether the viruses were inside or outside the cell, a combined enhancement/quenching method was developed using ammonium chloride as a lysosomotropic agent and trypan blue as a quenching dye. The data indicate that Fc receptor-mediated phagocytosis by PMN results in the ingestion of all cell-associated herpes virions. Interactions of virions through PMN-complement receptors CR1 and CR3 results solely in binding to the PMN but not in internalization. Interactions via both complement and Fc receptors cause synergistic stimulation of the PMN and result in very efficient association of viruses, greater than 80% of which were inside the cell.


Wertz GW, Krieger M, Ball LA (1989) Structure and cell surface maturation of the attachment glycoprotein of human respiratory syncytial virus in a cell line deficient in O glycosylation. J Virol 63 :4767-4776

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The synthesis of the extensively O-glycosylated attachment protein, G, of human respiratory syncytial virus and its expression on the cell surface were examined in a mutant Chinese hamster ovary (CHO) cell line, ldlD, which has a defect in protein O glycosylation. These cells, used in conjunction with an inhibitor of N-linked oligosaccharide synthesis, can be used to establish conditions in which no carbohydrate addition occurs or in which either N-linked or O-linked carbohydrate addition occurs exclusively. A recombinant vaccinia virus expression vector for the G protein was constructed which, as well as containing the human respiratory syncytial virus G gene, contained a portion of the cowpox virus genome that circumvents the normal host range restriction of vaccinia virus in CHO cells. The recombinant vector expressed high levels of G protein in both mutant ldlD and wild-type CHO cells. Several immature forms of the G protein were identified that contained exclusively N-linked or O-linked oligosaccharide side chains. Metabolic pulse-chase studies indicated that the pathway of maturation for the G protein proceeds from synthesis of the 32-kilodalton (kDa) polypeptide accompanied by cotranslational attachment of high-mannose N-linked sugars to form an intermediate with an apparent mass of 45 kDa. This step is followed by the Golgi-associated conversion of the N-linked sugars to the complex type and the completion of the O-linked oligosaccharides to achieve the mature 90-kDa form of G. Maturation from the 45-kDa N-linked form to the mature 90-kDa form occurred only in the presence of O-linked sugar addition, confirming that O-linked oligosaccharides constitute a significant proportion of the mass of the mature G protein. In the absence of O glycosylation, forms of G bearing galactose-deficient truncated N-linked and fully mature N-linked oligosaccharides were observed. The effects of N- and O-linked sugar addition on the transport of G to the cell surface were measured. Indirect immunofluorescence and flow cytometry showed that G protein could be expressed on the cell surface in the absence of either O glycosylation or N glycosylation. However, cell surface expression of G lacking both N- and O-linked oligosaccharides was severely depressed.