vendredi 24 avril 2009
par   G. Grégori

Aran JM, Plagemann PG (1992) Nucleoside transport-deficient mutants of PK-15 pig kidney cell line. Biochim Biophys Acta 1110 :51-58


Previous studies indicated that PK-15 pig kidney cells express solely a nitrobenzylthioinosine-sensitive, equilibrative nucleoside transporter. In the present study, PK-15 cells were mutagenized by treatment with ICR-170 and nucleoside transport-deficient mutants selected in a single step in growth medium containing tubercidin and cytosine arabinoside at a frequency of about 2 x 10(6). The mutants were simultaneously at least 100-times more resistant to tubercidin, cytosine arabinoside and 5-fluorodeoxyuridine than the wild-type parent cells. The mutants failed to transport thymidine and uridine and had lost all high affinity nitrobenzylthioinosine binding sites. Residual low level uptake of thymidine by the mutants was shown to be due to nonmediated permeation (passive diffusion), which explains the sensitivity of the mutants to growth inhibition by high concentrations of the nucleoside drugs. Passive diffusion of thymidine at a concentration of 16 microM was not rapid enough to support the growth of nucleoside transport-deficient mutant cells that had been made thymidine-dependent by treatment with methotrexate, whereas wild-type cells grew normally under these conditions. The nucleoside transport-deficient mutants exhibited about the same growth rate and plating efficiency (60-80%) as wild-type cells, but formed larger colonies than wild-type cells because of a more extensive spread of the cells on the surface of culture dishes. PK-15 cells adhere very strongly to the surface of culture dishes and have been transformed with high efficiency with plasmid DNA either via lipofection or electroporation.

Ayusawa D (1992) [Cell culture and its application current methods for a synchronous culture of mammalian cells]. Gan To Kagaku Ryoho 19 :1935-1940


Synchronization of cells to various phases of the cell cycle in mammalian cells is crucial to analyze cell cycle progression and many other cellular functions. To date, various methods have been developed, such as mitotic selection, use of cell-cycle mutants, use of drugs which inhibit DNA replication (nucleoside analogue, excess thymidine, hydroxyurea, and aphidicolin), elutriation, deprivation of nutrients (amino acid, and serum), and new promising drugs of protein kinase inhibitors. Although above methods have both advantage and disadvantage, a practical and satisfactory method can be chosen from these method if taking into account goals of experiments and cell types to use. In near future, more powerful and reliable ones will be discovered since our understanding of the cell cycle has been increasing quite abruptly.

Beersma MF, Bijlmakers MJ, Geelen JL, Feltkamp TE (1992) HLA-B27 as a receptor for cytomegalovirus. Curr Eye Res 11 Suppl :141-146


Acute anterior uveitis (AAU) is strongly associated with the genetic marker and cell membrane protein HLA-B27. Although also other genetic factors must play a pathogenetic role, the HLA-class I molecule B27 is up to now the only hold. The normal task of HLA class I molecules is to present endogenous, mostly viral, peptides to receptors on cytotoxic T cells. It is possible that HLA molecules at the cell surface serve as viral receptors. Human cytomegalovirus (HCMV) particles have been found to bind beta 2m. This might promote infectivity by a binding to HLA alpha-chains on cell membranes. We studied this mechanism using mouse fibroblasts transfected for human HLA class I molecules. Susceptibility of these cells for HCMV was compared by measuring of HCMV immediate early antigen (IEA) expression. Earlier we observed that cells transfected with HLA-B27 alpha-chains and beta 2m were significantly more infected than cells expressing HLA-A2 + beta 2m or HLA-B7 or HLA-B27 without beta 2m. However, studying another four, separately transfected, cell lines, all expressing HLA-B27 and beta 2m, three of the five B27 cell lines showed low IEA levels. The degree of infectivity was independent of the degree of B27 expression. These results do not support the previous suggestion that HLA-B27 might act as an HCMV receptor.

Borth N, Reiter M, Bluml G, Schmatz C, Gaida T, Katinger H (1992) Determination of division rates of rCHO cells in high density and immobilized fermentation systems by flow cytometry. Cytotechnology 8 :207-214


As most high density and immobilized fermentation systems do not allow the direct quantitative determination of cell density, two flow cytometric methods (the determination of incorporation of bromodeoxyuridine into newly synthesized DNA and the increase in mitotic cells by colchicine blockage) were evaluated as to their suitability to measure true division rates of cells in bioreactors. The BrdU method gave division rates identical to the growth rates measured by cell count, while the colchicine block method gave values that were lower and varied with the cell line. This is due to the cytotoxicity of colchicine and makes a calibration of the method for each cell line necessary. Both methods have been successfully used to measure division rates of rCHO cells immobilized in an alginate matrix as well as in macroporous carriers in a fluidised bed system and in dialysis culture.

Button DK, Robertson BR, McIntosh D, Juttner F (1992) Interactions between marine bacteria and dissolved-phase and beached hydrocarbons after the Exxon Valdez oil spill. Appl Environ Microbiol 58 :243-251


Turnover times for toluene in Resurrection Bay after the Exxon Valdez grounding were determined to be decades, longer than expected considering that dissolved hydrocarbons were anticipated to drift with the current and stimulate development of additional hydrocarbon-utilizing capacity among the microflora in that downcurrent location. These turnover times were based on the recovery of 14CO2 from added [14C]toluene that was oxidized. The concentrations of toluene there, 0.1 to 0.2 microgram/liter, were similar to prespill values. Oxidation rates appeared to be enhanced upstream near islands in the wake of the wind-blown slick, and even more within the slick itself. Specific affinities of the water column bacteria for toluene were computed with the help of biomass data, as measured by high-resolution flow cytometry. They were a very low 0.3 to 3 liters/g of cells.h-1, indicating limited capacity to utilize this hydrocarbon. Since current-driven mixing rates exceeded those of oxidation, dissolved spill components such as toluene should enter the world-ocean pool of hydrocarbons rather than biooxidize in place. Some of the floating oil slick washed ashore and permeated a coarse gravel beach. A bacterial biomass of 2 to 14 mg/kg appeared in apparent response to the new carbon and energy source. This biomass was computed from that of the organisms and associated naphthalene oxidation activity washed from the gravel compared with the original suspension. These sediment organisms were very small at approximately 0.06 microns 3 in volume, low in DNA at approximately 5.5 g per cell, and unlike the aquatic bacteria obtained by enrichment culture but quite similar to the oligobacteria in the water column.(ABSTRACT TRUNCATED AT 250 WORDS)

Diaper JP, Tither K, Edwards C (1992) Rapid assessment of bacterial viability by flow cytometry. Appl Microbiol Biotechnol 38 :268-272


The ability of a flow cytometer to rapidly assess microbial viability was investigated using three vital stains : rhodamine 123 (Rh123) ; 3,3’-dihexyloxacarbocyanine iodide [DiOC6(3)] and fluorescein diacetate (FDA). Rh123 was found to clearly differentiate viable from non-viable bacteria. The methodology for staining bacteria with this dye was optimised. Rh123 was shown to stain and discriminate several different species of viable bacteria although this was not universal. Viable cells of Bacillus subtilis were found to stain better with FDA than with Rh123. The results demonstrate the ability of flow cytometry to rapidly detect and estimate the viability of bacterial populations.

Dolter KE, Goins WF, Levine M, Glorioso JC (1992) Genetic analysis of type-specific antigenic determinants of herpes simplex virus glycoprotein C. J Virol 66 :4864-4873


Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC-1) elicits a largely serotype-specific immune response directed against previously described determinants designated antigenic sites I and II. To more precisely define these two immunodominant antigenic regions of gC-1 and to determine whether the homologous HSV-2 glycoprotein (gC-2) has similarly situated antigenic determinants, viral recombinants containing gC chimeric genes which join site I and site II of the two serotypes were constructed. The antigenic structure of the hybrid proteins encoded by these chimeric genes was studied by using gC-1- and gC-2-specific monoclonal antibodies (MAbs) in radioimmunoprecipitation, neutralization, and flow cytometry assays. The results of these analyses showed that the reactivity patterns of the MAbs were consistent among the three assays, and on this basis, they could be categorized as recognizing type-specific epitopes within the C-terminal or N-terminal half of gC-1 or gC-2. All MAbs were able to bind to only one or the other of the two hybrid proteins, demonstrating that gC-2, like gC-1, contains at least two antigenic sites located in the two halves of the molecule and that the structures of the antigenic sites in both molecules are independent and rely on limited type-specific regions of the molecule to maintain epitope structure. To fine map amino acid residues which are recognized by site I type-specific MAbs, point mutations were introduced into site I of the gC-1 or gC-2 gene, which resulted in recombinant mutant glycoproteins containing one or several residues from the heterotypic serotype in an otherwise homotypic site I background. The recognition patterns of the MAbs for these mutant molecules demonstrated that (i) single amino acids are responsible for the type-specific nature of individual epitopes and (ii) epitopes are localized to regions of the molecule which contain both shared and unshared amino acids. Taken together, the data described herein established the existence of at least two distinct and structurally independent antigenic sites in gC-1 and gC-2 and identified subtle amino acid sequence differences which contribute to type specificity in antigenic site I of gC.

Dunlap NE, Benjamin WH, Jr., Berry AK, Eldridge JH, Briles DE (1992) A ’safe-site’ for Salmonella typhimurium is within splenic polymorphonuclear cells. Microb Pathog 13 :181-190


Following oral or systemic infection with Salmonella typhimurium, the focus of infection is in the liver and spleen. The majority of Salmonella surviving in the liver and spleen by 4 h post infection are already in an environment where they are largely protected from subsequent killing. Previous studies have shown that the majority of surviving Salmonella are intracellular. In the present study we sought to determine the cell type containing most of the cell-associated Salmonella liberated from the spleen. We enriched for Salmonella-containing cells by Ficoll-Hypaque separation followed by fluorescence-activated cell sorting. Approximately 85% of the total intracellular Salmonella were found in Mac-1+/J-11d+ cell fractions of the Ficoll-Hypaque band and pellet. By microscopic examination of stained cells from the sorted cell populations, it was evident that virtually all of the Salmonella were in polymorphonuclear cells (PMN). The numbers of Salmonella observed microscopically were similar in numbers to Salmonella colony forming units detected by plating. Salmonella containing PMN in the Ficoll band generally contained a single bacterium, while those from the probably less healthy cells in the Ficoll pellet generally contained several Salmonella.

Gordon DL, Sadlon TA, Wesselingh SL, Russell SM, Johnstone RW, Purcell DF (1992) Human astrocytes express membrane cofactor protein (CD46), a regulator of complement activation. J Neuroimmunol 36 :199-208


Expression of membrane cofactor protein (CD46) on cultured human astrocytes was demonstrated by indirect immunofluorescence microscopy and flow cytometry following staining with a monoclonal antibody specific for CD46. Western transfer and immunoblotting detected a doublet of Mr 66,000 and 56,000. Analysis of astrocyte mRNA revealed the presence of multiple alternatively spliced transcripts encoding different extracellular regions or cytoplasmic tails of CD46. Astrocytes were also shown to express decay accelerating factor, but not the type 1 complement receptor. Upregulation of astrocyte CD46 occurred following cytomegalovirus infection. These results indicate that astrocytes express proteins involved in regulation of complement activation and protection against autologous complement.

Harris DT, Kapur R, Frye C, Acevedo A, Camenisch T, Jaso-Friedmann L, Evans DL (1992) A species-conserved NK cell antigen receptor is a novel vimentin-like molecule. Dev Comp Immunol 16 :395-403


The role of a novel, evolutionarily conserved function-associated molecule (FAM) in NK cell function has been examined in several species. This molecule has previously been shown to mediate NK and NK-like recognition functions in fish NCC and human NK cells. We now show that this molecule is distributed in those tissues that contain NK cells in mice and rats. Further, we show that this molecule functions as an antigen receptor on NK cells of these species. That is, monoclonal antibodies directed against this FAM inhibit NK cell cytotoxic function and trigger signal transduction pathways in each of the species. Finally, we present evidence that this putative antigen receptor is a vimentin-like molecule which functions to mediate all NK or NK-like recognition functions in a variety of species.

Hietanen S, Auvinen E, Grenman S, Lakkala T, Sajantila A, Klemi P, Maenpaa J (1992) Isolation of two keratinocyte cell lines derived from HPV-positive dysplastic vaginal lesions. Int J Cancer 52 :391-398


Explant cultures were started from human papillomavirus (HPV)-infected genital lesions in order to isolate and propagate abnormally differentiating cells from squamous intraepithelial neoplasia. A medium with high calcium concentration was used to induce terminal differentiation of cells from surrounding normal epithelium. Two cell lines with extended life-spans were established. The UT-DEC-1 cell line was derived from an HPV-33-positive mild vaginal dysplasia (VAIN I). In cultured UT-DEC-1 cells, HPV 33 DNA was detected with Southern-blot hybridization and the polymerase chain reaction (PCR) technique. The restriction pattern of HPV 33 changed during early passages and flow cytometric analysis detected a decrease in chromosomal DNA content. HPV 33 RNA from the E6-E7 region could be amplified by PCR at late passage. UT-DEC-2 cell line was derived from an HPV-16-positive moderate vaginal dysplasia (VAIN II). HPV 16 DNA was also detected in cultured cells by the PCR technique. The senescence of normal keratinocytes and growth selection in favor of aneuploid cells was observed by flow cytometric analysis at subsequent passages. Karyotype analysis showed clonal chromosomal abnormalities in both cell lines. To date, UT-DEC-1 cells have undergone 40 and UT-DEC-2 cells 25 passages. This study shows that the isolation of HPV-infected dysplastic cells can be achieved by culturing the cells in a medium with high calcium concentration. The cell lines presented provide the opportunity of evaluating the early stages of squamous-cell carcinogenesis.

Jacobberger JW, Horan PK, Hare JD (1992) Cell cycle analysis of asexual stages of erythrocytic malaria parasites. Cell Prolif 25 :431-445


Intra-erythrocytic Plasmodium species can be stained with the DNA binding dye, Hoechst 33342, and the distribution of DNA content determined for parasite populations by flow cytometric measurement of fluorescence. Analysis of this distribution will determine the parasitaemia (percentage of erythrocytes infected), and the percentages of trophozoite infected red blood cells, polyparasitized (trophozoite) red blood cells, and schizont/segmenter infected red blood cells. This analysis is based on the hypothesis that the asexual parasites cycle with single G1 period, and effectively, a single S phase with no significant G2/M period except at schizogony when the genome DNA content is equivalent to 8 N or higher, dependent on the species. Data are presented to support this model.

Kollas BB, Hartle HT, Wigdahl B (1992) Effect of gamma-interferon on the expression of major histocompatibility complex class I and II gene products in neural cells isolated from the developing human fetal peripheral nervous system. Fetal Diagn Ther 7 :62-74


The effect of gamma-interferon (gamma-IFN) on the expression of major histocompatibility complex (MHC) gene products was examined in the developing human fetal peripheral nervous system. RNA blot hybridization analysis of total RNA isolated from human fetal dorsal root ganglia (DRG) neural cell populations cultured in vitro for 5 days resulted in the detection of both MHC class I- and class II-specific RNAs. As determined by protein immunoblotting and fluorescence-activated flow cytometry, MHC class I and II proteins were also readily detectable in cultured human fetal DRG neural cell populations 5 days after isolation. In addition, treatment of 3-day human fetal DRG neural cells with gamma-IFN (100 U/ml ; 48 h) resulted in a marked increase in the level of MHC class I- and class II-specific RNA and protein without inhibiting the proliferation of the neural cell population. These results suggest that changes in the levels of selected cytokines such as gamma-IFN may alter the ability of specific neural cell populations present in the developing human nervous system to participate in immune reactions by alteration of MHC class I and II antigen expression which may lead to perturbation in glial cell function and ultimately to nervous system dysfunction in general.

Lambot M, Letesson JJ, Lostrie N, Depelchin A (1992) Streptococcal products and leukocyte activities. Vet Immunol Immunopathol 31 :129-140


Various streptococcal species are directly responsible for udder infections which should normally be countered by polymorphonuclear neutrophils (PMNs). In order to detect a putative inhibition of streptococcal products on the activities of bovine PMNs, we used a combination of four tests which permits an adequate evaluation of PMNs functions, e.g. PMN adherence on endothelial cells, chemotactic assay, phagocytosis of bacteria labelled with fluorescein isothiocyanate (FITC) and measurement of anion superoxide production. The conclusion is that neither of the two pathogenic streptococcal species isolated from mastitis appeared to produce in vitro factors affecting PMN activities.

Link H, Battmer K, Kleine HD (1992) Detection of cytomegalovirus-infected cells by flow cytometry and fluorescence in suspension hybridisation (FLASH) using DNA probes labeled with biotin by polymerase chain reaction. J Med Virol 37 :143-148


A biotin-labeled DNA probe specific for the immediate early gene of the cytomegalovirus (CMV) strain AD 169 was generated with the polymerase chain reaction. Nucleic acid hybridisation was carried out with CMV-infected T-lymphoblastoid cells (MOLT-4) after 4 hr to 6 days of culture. Biotin molecules were made visible with streptavidin coupled with fluorescein. The fluorescence signal of the hybridised probe was measured by flow cytometry in the cell suspension. The number of CMV-positive cells was 7% at 4 hr, 8% after 28 hr, 18% after 2 days, 26% after 3 days, 91% after 4 days, 97% after 5 days, and 98% after 6 days. The first detection of CMV antigen (pp65) was possible with immunoenzymatic labeling by day 4, whereas CMV-DNA was detected by PCR after 4 hr. CMV-specific RNA could be detected in a similar way. The analysis of mononuclear peripheral blood leukocytes in a patient with active CMV infection showed 14.7% CMV DNA-positive cells at day 1 and 7% at day 8, as compared to 0.9% and 0.0% cells which were positive for CMV antigen (pp65) by immunoenzymatic labeling at day 1 and day 8, respectively. We conclude that flow cytometry and fluorescence in suspension hybridisation (FLASH) offers a new tool for analysing exactly and quantifying large numbers of cells for specific DNA or RNA and may be useful for other laboratory and clinical applications.

Lobner-Olesen A, Boye E (1992) Different effects of mioC transcription on initiation of chromosomal and minichromosomal replication in Escherichia coli. Nucleic Acids Res 20 :3029-3036


The mioC gene, which neighbors the chromosomal origin of replication (oriC) in Escherichia coli, has in a number of studies been implicated in the control of oriC initiation on minichromosomes. The present work reports on the construction of cells carrying different mioC mutations on the chromosome itself. Flow cytometry was employed to study the DNA replication control and growth pattern of the resulting mioC mutants. All parameters measured (growth rate, cell size, DNA/cell, number of origins per cell, timing of initiation) were the same for the wild type and all the mioC mutant cells under steady state growth and after different shifts in growth medium and after induction of the stringent response. It may be concluded that the dramatic effects of mioC mutations reported for minichromosomes are not observed for chromosomal replication and that the mioC gene and gene product is of little importance for the control of initiation. The data demonstrate that a minichromosome is not necessarily a valid model for chromosomal replication.

Lobner-Olesen A, Boye E, Marinus MG (1992) Expression of the Escherichia coli dam gene. Mol Microbiol 6 :1841-1851


The Escherichia coli dam gene and upstream sequences were cloned from the Kohara phage 4D4. Five promoters were found to contribute to dam gene transcription. P1 and P2 (the major promoter) were situated approximately 3.5 kb upstream of the structural gene, P3 was within the aroB gene, P4 was within the urf74.3 gene, and P5 was in the urf74.3-dam intergenic region. The nucleotide sequence of 2280 bp of DNA containing P1 and P2 was determined and shown to have the potential to encode a protein of approximately 16 kDa between P1, P2 and the aroB gene. This 16 kDa open reading frame has been identified as aroK, the gene for shikimic acid kinase I. Thus the dam gene is part of an operon containing aroK, aroB, urf74.3, and dam. The transcriptional start points of the promoters were determined. A comparison of their nucleotide sequences suggested that P1-P4 were all recognized by the sigma 70 subunit of the RNA polymerase.

Madelin TM, Johnson HE (1992) Fungal and actinomycete spore aerosols measured at different humidities with an aerodynamic particle sizer. J Appl Bacteriol 72 :400-409


An aerodynamic particle sizer (APS) that uses laser Doppler velocimetry was used to determine aerodynamic diameters of spores of fungal and thermophilic actinomycete species common in mouldy hay, aerosolized at different humidities and temperatures. Results were compared with those obtained from inertial impaction in a cascade impactor. The APS gave slightly smaller measurements than the cascade impactor. Both methods gave aerodynamic diameters generally slightly smaller than the average spore dimensions observed on cascade impactor slides with a microscope. The latter measurements were less than axial dimensions given in the literature. Brief passage of spores through air at 95% relative humidity (RH) and 38 degrees C, compared with 40% RH and 20 degrees C, caused an immediate increase in their aerodynamic diameter and the breaking of chains of spores. Cultures maintained at 75% RH and aerosolized at 98% RH similarly produced larger spore particles than those passed through dry air. These findings have implications for mould-induced asthma and allergic alveolitis since they relate to physical behaviour of airborne spores and particle deposition sites in the lung.

Manning CH, Heise ER (1992) Establishment and characterization of Macaca fascicularis lymphoblastoid cell lines. J Med Primatol 21 :15-23


A panel of cynomolgus macaque lymphoblastoid cell lines (LCL) was established by transforming peripheral blood mononuclear cells (PBMC) with Herpesvirus papio (HVP), and selected lines were examined by flow cytometry. Results indicate that HVP-transformed macaque LCL are phenotypically heterogeneous and resemble human Epstein-Barr virus (EBV)-transformed LCL in the abundant expression of major histocompatibility complex (MHC) class I and class II molecules. At least some lines are of B cell origin.

Martin E, Bhakdi S (1992) Flow cytometric assay for quantifying opsonophagocytosis and killing of Staphylococcus aureus by peripheral blood leukocytes. J Clin Microbiol 30 :2246-2255


We describe a novel flow cytometric method for quantifying opsonophagocytosis and killing of Staphylococcus aureus in cell-rich plasma obtained after dextran sedimentation of erythrocytes. To analyze opsonophagocytosis, phagocytes were labeled with a phycoerythrin-conjugated monoclonal antibody and were incubated with viable staphylococci containing carboxyfluorescein as a vital fluorescent dye. Phagocytosing cells assumed a dual, orange-green fluorescence. The relative numbers of bacteria associating with phagocytes could be determined by quantifying the decrease of free green fluorescent particles. A parallel incubation of fluorescent bacteria with unlabeled cell-rich plasma was performed to assess phagocytic killing. Blood cells were lysed with 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate. This detergent spared viable bacteria, and residual green fluorescent particles were counted. The decrease in the number of these particles relative to the controls yielded the degree of killing. At bacteria-to-phagocyte ratios of 1:1 and 10:1, approximately 36 and 75% of the phagocytes participated in opsonophagocytosis, respectively. Over 90% of the staphylococci were phagocyte associated after 30 to 60 min. Killing rates were on the order of 66% +/- 12% and 80% +/- 7% after 1 and 2 h of incubation, respectively. These numbers, which were confirmed by colony countings, were significantly lower than those reported in the majority of past reports.

Molenaar D, Bolhuis H, Abee T, Poolman B, Konings WN (1992) The efflux of a fluorescent probe is catalyzed by an ATP-driven extrusion system in Lactococcus lactis. J Bacteriol 174 :3118-3124


Many bacteria, both gram positive and gram negative, extrude in an energy-dependent manner the fluorescent pH indicator 2’,7’-bis-(2-carboxyethyl)-5[and -6]-carboxyfluorescein (BCECF) (D. Molenaar, T. Abee, and W. N. Konings, Biochim. Biophys. Acta 1115:75-83, 1991). This efflux was studied in detail in Lactococcus lactis, and several indications that a transport system is involved were found. This transport system is most likely driven by ATP or a related compound. The evidence is that BCECF extrusion (i) occurs against a BCECF gradient, (ii) is strictly correlated with ATP concentration and not with the proton motive force, and (iii) is inhibited by vanadate and to a lesser extent by N,N’-dicyclohexylcarbodiimide. Most convincingly, a UV mutant with a strongly reduced efflux rate was isolated. Such a mutant was isolated from a BCECF-loaded and lactose-energized population by selection of highly fluorescent cells in a flow cytometer-cell sorter. The physiological function of this extrusion system is unknown, but its characteristics classify it among the traffic ATPases.

Monfort P, Baleux B (1992) Comparison of flow cytometry and epifluorescence microscopy for counting bacteria in aquatic ecosystems. Cytometry 13 :188-192


Flow cytometry was used to count bacterial cells from diverse origins : one strain of E. coli, one sample of lake water, and 18 samples of estuary water. To verify the accuracy and the precision of this technique, total bacteria counts made by flow cytometry were compared with counts by direct observation using epifluorescence microscopy. The results of this study showed that flow cytometry was a reliable technique for counting a mixture of bacteria in samples from aquatic ecosystems.

Morgan JA, Clarke KJ, Rhodes G, Pickup RW (1992) Non-culturable Aeromonas salmonicida in lake water. Microb Releases 1 :71-78


The survival of Aeromonas salmonicida subsp. salmonicida was investigated in lake water. During a 21-day study A. salmonicida became non-culturable in sterile lake water held at 10 degrees C. The incubation of replicate samples between 5 degrees C and 25 degrees C produced similar results. The recovery of colony-forming units of A. salmonicida from different lake water systems indicated that they survived longer in water that was naturally enriched (eutrophic) or enriched with tryptone soya broth. Flow cytometry, fluorescence light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) indicated that non-culturable cells were present. These cells could not be revived in dilutions of tryptone soya broth, whole dead fish or dissected fish tissue. Although viability could not be proven, it was shown that the morphological integrity found in viable cells was also maintained in non-culturable cells.

Novelli MR, MacIver AG (1992) Renal cell carcinoma : comparison of morphological and flow cytometric parameters of primary tumour and invasive tumour lying within the renal vein. J Pathol 167 :229-233


Histological assessment and DNA flow cytometry have been performed on 15 kidneys containing primary adenocarcinomas which had invaded the renal vein. Comparison of morphological variables showed that samples of the intravenous tumour were more commonly composed of granular cells (53 per cent) than were samples from the main tumour mass (16 per cent), and were also of higher nuclear grade. In 7 of 14 kidneys, DNA studies showed either a higher S-phase fraction (five cases) or DNA aneuploidy (two cases) in tumour cells lying within the renal vein. The mean S-phase fraction was also shown to increase in higher nuclear grades. Thus, both morphological and biological differences exist between invasive tumour cells lying within the renal vein and those in the main tumour. This is a useful model for the investigation of venous invasion and may give a better prediction of the metastatic potential of renal cell carcinoma.

Pfeifer CG, Campos M, Beskorwayne T, Babiuk LA, Potter AA (1992) Effect of Haemophilus somnus on phagocytosis and hydrogen peroxide production by bovine polymorphonuclear leukocytes. Microb Pathog 13 :191-202


The interactions between bovine polymorphonuclear leukocytes (PMNs) and the bacterium Haemophilus somnus are known to be complex. In this paper, we evaluated the effect of H. somnus on PMN function using a flow cytometric (FC) technique that simultaneously determined the extent of phagocytosis and hydrogen peroxide production by PMNs, as well as using conventional techniques, such as the nitroblue tetrazolium (NBT) and chemiluminescence assays, to analyse the PMN respiratory burst. Results from the FC and chemiluminescence assays demonstrated that in vitro exposure of PMNs to logarithmically growing H. somnus reduced the respiratory burst of PMNs obtained from healthy calves. However, this reduction was not detected by the NBT assay. A decrease in phagocytosis by PMNs could also be shown using the FC assay. In addition, PMNs from calves with acute Hemophilosis (i.e. exposed to H. somnus in vivo) showed reduced activity when compared to PMNs from healthy calves. These in vitro and in vivo observations indicate that the modulation of bovine PMN function by H. somnus may contribute significantly towards the pathogenesis of the disease.

Reiter M, Borth N, Bluml G, Wimmer K, Harant H, Zach N, Gaida T, Schmatz C, Katinger H (1992) Flow cytometry and two-dimensional electrophoresis (2-DE) for system evaluation of long term continuous perfused animal cell cultures in macroporous beads. Cytotechnology 9 :247-253


Immobilization of r-CHO cells at high density using macroporous polyethylene carriers in a modular fluidized bed reactor is demonstrated. Specific growth rates of the cells are measured by incorporation of BrdU. At a cell density of about 10(8) cells/ml a stable growth rate of 0.004 h-1 was established. Total release of proteins into the culture supernatant during protein-free perfusion was analyzed by 2-DE in various phases of the long-term culture showing very similar patterns indicating a constant pattern of gene expression.

Rundegren J, Simonsson T, Petersson L, Hansson E (1992) Effect of delmopinol on the cohesion of glucan-containing plaque formed by Streptococcus mutans in a flow cell system. J Dent Res 71 :1792-1796


Glucan-containing plaque was formed by Streptococcus mutans adhering to saliva-coated glass slides in flow cells thermostated at 37 degrees C. The substrate was Brain Heart Infusion broth containing 1% sucrose and 10% sterile saliva. During the build-up of the plaque, which lasted for 29 h, the plaque was subjected to three two-minute exposures to either 0.1 mol/L sodium acetate buffer, pH 6.0, or the same buffer containing 6.4 mmol/L (0.2%) of the surface-active anti-plaque substance delmopinol hydro-chloride. The glass slides carrying the plaque were weighed, and plaques subjected to delmopinol treatment weighed only seven percent of the control plaques. The glass slides were then mounted in a beaker containing buffer, subjected to ultrasonication, and re-weighed. The delmopinol-treated plaques lost 59% of their wet weight upon sonication, while the controls lost only 19%. Control plaques having the same weight as delmopinol-treated plaques were not different from the control plaques grown for 29 h with regard to reduction of plaque weight after sonication. Transmission electron micrographs (TEM) showed a plaque dominated by globular or fibrillar matrix components in controls, while the delmopinol-treated plaque showed empty or unordered matrix areas between more densely packed cells. The TEM results were confirmed by scanning electron micrographs, which showed amorphous material associated with the bacterial cells in the control but not in the delmopinol-treated plaque. In conclusion, delmopinol reduced surface-associated glucan synthesis and lowered the cohesion of the plaque, indicating that glucan-containing plaque formed during repeated rinsings with delmopinol may be easier to remove by mechanical means than a non-treated plaque of this type.

Sachsenmeier KF, Schell K, Morrissey LW, Pennell DR, West RM, Callister SM, Schell RF (1992) Detection of borreliacidal antibodies in hamsters by using flow cytometry. J Clin Microbiol 30 :1457-1461


Flow cytometry can be used to detect antibody that kills Borrelia burgdorferi. Borreliacidal activity was detected within 3 h of incubating B. burgdorferi with immune serum and complement. Right-angle light scatter and propidium iodide fluorescence were the cytometric parameters which correlated best with in vitro killing of B. burgdorferi. Flow cytometry is a rapid method for determining the presence of borreliacidal activity and may lead to a better serodiagnostic test for the detection of Lyme disease.

Scholz M, Blaheta RA, Hamann A, Encke A, Markus BH (1992) Infection of human vascular endothelial cells with cytomegalovirus : effects on the expression of human leukocyte antigens. Transplant Proc 24 :2565-2566


Steed LL, Akporiaye ET, Friedman RL (1992) Bordetella pertussis induces respiratory burst activity in human polymorphonuclear leukocytes. Infect Immun 60 :2101-2105


Virulent Bordetella pertussis strains survive intracellularly within human polymorphonuclear leukocytes (PMN), at least in part because of inhibition of phagosome-lysosome fusion (L. L. Steed, M. Setareh, and R. L. Friedman, J. Leukocyte Biol. 50:321-330, 1991). Further investigations were done to determine if B. pertussis also inhibited respiratory burst activity of PMN as an additional mechanism of intracellular survival. Chemiluminescence and flow cytometry assays showed that B. pertussis induced significant levels of hydrogen peroxide production. In contrast, ferricytochrome c reduction showed that B. pertussis suppressed extracellular release of superoxide. PMN intracellular reduction of nitroblue tetrazolium verified that superoxide was indeed produced intracellularly during B. pertussis phagocytosis. Therefore, B. pertussis does not inhibit production of superoxide but inhibits only its release. Thus, while phagosome-lysosome fusion is inhibited by B. pertussis, respiratory burst activity of PMN occurs at normal levels.

Telford WG, King LE, Fraker PJ (1992) Comparative evaluation of several DNA binding dyes in the detection of apoptosis-associated chromatin degradation by flow cytometry. Cytometry 13 :137-143


Mouse thymocytes readily undergo apoptosis-associated DNA degradation upon exposure to glucocorticoids or ionizing radiation. It has been previously shown that flow cytometric cell cycle analysis of propidium iodide-stained apoptotic thymocytes results in the appearance of a distinct cell cycle region (the A0 region) below the G0/G1 region. Cells in this region were shown to be undergoing apoptosis, and determination of apoptosis by flow cytometric analysis was proposed as a superior method for evaluating thymocyte apoptosis. In this study, a variety of DNA binding dyes with diverse primary binding mechanisms were evaluated for their ability to detect glucocorticoid and ionizing radiation-induced apoptosis in mouse thymocytes. Apoptotic thymocytes stained with DNA binding dyes from the phenanthridinium, acridine, actinomycin, chromomycinone, anthracycline, and bisbenzimidazole groups all demonstrated clearly defined A0 regions with percentages comparable to those obtained for propidium iodide. These results indicate that the appearance of the A0 region is not dependent on a particular dye binding characteristic and may be the consequence of extensive changes in chromatin structure resulting in a significant degree of dye exclusion.

Thorsen BK, Enger O, Norland S, Hoff KA (1992) Long-term starvation survival of Yersinia ruckeri at different salinities studied by microscopical and flow cytometric methods. Appl Environ Microbiol 58 :1624-1628


Cultures of three strains of the fish pathogenic bacterium Yersinia ruckeri survived starvation in unsupplemented water for at least 4 months. At salinities of 0 to 20/1000 there were no detectable changes in CFU during the first 3 days of starvation and only a small decrease during the following 4 months, whereas at 35/1000 salinity, the survival potential of the cultures was markedly reduced. These results suggest that Y. ruckeri may survive for long periods in freshwater and brackish environments after an outbreak of enteric redmouth disease. Survival was also examined by use of the direct viable count method, and we show that this method can be combined with flow cytometry for automatic counting of viable bacteria. By flow cytometry, it was shown that genome replication initiated before the onset of starvation was completed, during the initial phase of starvation, and that starved cells could contain up to six genomes per cell.

von Freiesleben U, Rasmussen KV (1992) The level of supercoiling affects the regulation of DNA replication in Escherichia coli. Res Microbiol 143 :655-663


The chromosome of Escherichia coli is negatively supercoiled. This favours processes that unwind the two DNA strands, such as DNA replication. In this paper, we have investigated the effect of changed levels of overall chromosomal supercoiling on the initiation of DNA replication. Specifically, we have used flow cytometry to reveal effects on the synchrony of initiations of DNA replication in single cells. An increase in the level of supercoiling moderately reduced initiation synchrony. In contrast, decreased supercoiling led to pronounced asynchrony. We have excluded the possibility that this asynchrony is caused by changes in the level of the Dam methyltransferase or the DnaA protein. We suggest that the global level of supercoiling influences the topology of oriC and thereby the sequence of events leading to initiation of DNA replication in E. coli.

Woods GL (1992) Automation in clinical microbiology. Am J Clin Pathol 98 :S22-30


Automation was introduced into the clinical microbiology laboratory in the 1960s but initially met with limited success. Today, instruments are an integral part of many clinical laboratories and are used for microbial detection, identification, and susceptibility testing ; detection of positive blood cultures ; screening urine samples for potential pathogens ; and assaying levels of antimicrobial agents in body fluids. Automation has allowed more rapid diagnosis and elimination of the subjective interpretation of many manual tests. In addition, in some cases, automated tests are more sensitive and specific than manual techniques. However, automated testing often is more expensive than manual testing and is associated with the possibility of mechanical failure. Automation will continue to be an important part of the clinical microbiology laboratory and in the future will include more molecular biology technologies, such as the polymerase chain reaction. Perhaps practical applications of flow cytometry will be identified.

Yeager CL, Ashmun RA, Williams RK, Cardellichio CB, Shapiro LH, Look AT, Holmes KV (1992) Human aminopeptidase N is a receptor for human coronavirus 229E. Nature 357 :420-422


Human coronaviruses (HCV) in two serogroups represented by HCV-229E and HCV-OC43 are an important cause of upper respiratory tract infections. Here we report that human aminopeptidase N, a cell-surface metalloprotease on intestinal, lung and kidney epithelial cells, is a receptor for human coronavirus strain HCV-229E, but not for HCV-OC43. A monoclonal antibody, RBS, blocked HCV-229E virus infection of human lung fibroblasts, immunoprecipitated aminopeptidase N and inhibited its enzymatic activity. HCV-229E-resistant murine fibroblasts became susceptible after transfection with complementary DNA encoding human aminopeptidase N. By contrast, infection of human cells with HCV-OC43 was not inhibited by antibody RBS and expression of aminopeptidase N did not enhance HCV-OC43 replication in mouse cells. A mutant aminopeptidase lacking the catalytic site of the enzyme did not bind HCV-229E or RBS and did not render murine cells susceptible to HCV-229E infection, suggesting that the virus-binding site may lie at or near the active site of the human aminopeptidase molecule.

Yeaman MR, Sullam PM, Dazin PF, Norman DC, Bayer AS (1992) Characterization of Staphylococcus aureus-platelet binding by quantitative flow cytometric analysis. J Infect Dis 166 :65-73


Quantitative analyses of Staphylococcus aureus binding to platelets were done using flow cytometry after bacterial exposure to the following treatments : proteases (trypsin, protease K), antibiotics (oxacillin, gentamicin), surface carbohydrate modifiers (sodium periodate, anticapsular antibody), or platelet microbicidal protein. In separate studies, platelets were exposed to a monoclonal antibody to their Fc receptor (Fc gamma RII) before binding was quantified. The percentage of bacteria bound to platelets varied significantly among strains (22.1% +/- 3.8% to 76.4 +/- 3.2%). For all isolates, binding to platelets was rapid, saturable, and reversible, suggesting a receptor-ligand interaction. The following modifiers significantly reduced binding : platelet microbicidal protein (by 32.1% +/- 5.2% ; P less than .001), homologous (but not heterologous) anticapsular antibody (by 17.7% +/- 1.9% ; P less than .05), sodium periodate (by 36.3% +/- 4.3% ; P less than .005), and anti-platelet Fc monoclonal antibody (by 41.5% +/- 4.4% ; P less than .002). Collectively, these data suggest that the mechanism(s) involved in S. aureus-platelet binding are complex and multimodal, involving carbohydrate-rich and platelet microbicidal protein-susceptible S. aureus surface ligands as well as the platelet Fc receptor.

Zemtsov A, Koss W, Dixon L, Tyring S, Rady P (1992) Anal verrucous carcinoma associated with human papilloma virus type 11 : magnetic resonance imaging and flow cytometry evaluation. Arch Dermatol 128 :564-565