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Biomedical subjects

G Roy

Publications and source records attributed to G Roy.

At least 109 records · Page 6Linked to original sources
PubMed1991

Adherence of Actinobacillus pleuropneumoniae to porcine tracheal epithelial cells and frozen lung sections.

The ability of 23 different Actinobacillus pleuropneumoniae isolates to adhere in vitro to porcine tracheal epithelial cells and to porcine frozen lung sections was examined. It was found that A. pleuropneumoniae adhered poorly to isolated tracheal epithelial cells. On the other hand, A. pleuropneumoniae adhered to frozen lung sections and marked variations were observed between and within serotypes. Adherence to lung sections did not seem related to the hemagglutinating activity of the isolate. Two noncapsulated variants adhered to lung sections in greater numbers than their capsulated parent strains. Adherence to lung sections was not inhibited by the extracellular matrix components tested namely, laminin, fibronectin, and collagen, but was inhibited by homologous serotype-specific antiserum. The data indicated that the A. pleuropneumoniae isolates tested possess the ability to adhere to porcine lung tissue, a property which did not seem to be related to the serotype and did not seem to involve the capsular material or the hemagglutinins of the isolates.

Actinobacillus↗
PubMed1991

Determination of affinity of Pasteurella multocida isolates for porcine respiratory tract mucus, and partial characterization of the receptors.

The ability of 25 Pasteurella multocida isolates to adhere in vitro to porcine respiratory tract mucus was examined. Microplate wells were coated with crude mucus preparation, then bacteria were added. After incubation, unbound bacteria were removed by washing, and the number of mucus-bound bacteria was estimated by quantitation of the adherent colony-forming units and by use of an ELISA. Pasteurella multocida had affinity to respiratory tract mucus, although significant differences were not observed in affinity of capsular type-A and type-D isolates. Preliminary characterization, using ultrafiltration, gel filtration chromatography, electrophoresis, and enzymatic treatments, indicated that the receptors may be a class of protein molecules of low molecular weight (less than 25,000). The origin of these receptors, however, is not known at this time.

Animals↗
PubMed1990

Single-channel and Fura-2 analysis of internal Ca2+ oscillations in HeLa cells: contribution of the receptor-evoked Ca2+ influx and effect of internal pH.

Patch-clamp and Fura-2 experiments were performed in order to investigate the calcium oscillations due to H1 receptor stimulation in HeLa cells. The cytosolic calcium fluctuations occurring directly at the plasma membrane inner face were detected by measuring the activity of calcium-dependent potassium channels. This method also allowed measurement of changes in intracellular potential using as indicator the amplitude of the channel current jump. The average internal calcium concentration was obtained from Fura-2 experiments carried out at either the single-cell level or from a small population of cells in monolayer. The results indicate that the internal calcium oscillations in HeLa cells arise from a biphasic process with an initial phase independent of the presence of external calcium. External calcium was found, however, to become essential once the regular oscillatory process has been established. Removing external calcium after this initial phase produced a rapid decay in the burst frequency and eventually a complete abolition of the oscillations. In addition, the calcium oscillations occurring during the external-calcium-dependent phase could be blocked by calcium entry blockers such as Co2+ or La3+, or abolished by perfusing the external medium with a high-K+ solution. Experiments were also performed in which the cell internal pH (pHi) was changed by removing the external bicarbonate or by adding NH4Cl to the bathing solution. The results obtained under these conditions indicate that an increase in internal pH abolishes selectively the appearance of calcium spikes without increasing the basal calcium level, while a cellular acidification maintains or stimulates the calcium oscillatory process. It was also observed that the inhibitory effect of alkaline pH was independent of external calcium, and that calcium oscillations could always be seen at alkaline pH during the initial phase of histamine stimulation. On the basis of these results, it is proposed that the internal calcium oscillations in HeLa cells depend on the release of calcium from internal pools, which are reloaded via a pH-dependent mechanism. Part of the calcium sequestration occurring during the oscillatory process would be carried out, however, by pH-insensitive calcium compartments.

Benzofurans↗
PubMed1990

Presteady-state kinetics and carrier-mediated transport: a theoretical analysis.

Kinetic studies of cotransport mechanisms have so far been limited to the conventional steady-state approach which does not allow in general to resolve either isomerization or rate-limiting steps and to determine the values of the individual rate constants for the elementary reactions involved along a given transport pathway. Such questions can only be answered using presteady-state or relaxation experiments which, for technical reasons, have not yet been introduced into the field of cotransport kinetics. However, since two recent reports seem compatible with the observation of such transient kinetics, it would appear that theoretical studies are needed to evaluate the validity of such claims and to critically evaluate the expectations from a presteady-state approach. We thus report such a study which was performed on a simple four-state mechanism of carrier-mediated transport. The time-dependent equation for zero-trans substrate uptake was thus derived and then extended to models with p intermediary steps. It is concluded that (p-1) exponential terms will describe the approach to the steady state but that such equations have low analytical value since the parameters of the flux equation cannot be expressed in terms of the individual rate constants of the elementary reactions for models with p greater than 5. We thus propose realistic simplifications based on the time-scale separation hypothesis which allows replacement of the rate constants of the rapid steps by their equilibrium constants, thereby reducing the complexity of the kinetic system. Assuming that only one relaxation can be observed, this treatment generates approximate models for which analytical expressions can easily be derived and simulated through computer modeling. When performed on the four-state mechanism of carrier-mediated transport, the simulations demonstrate the validity of the approximate solutions derived according to this hypothesis. Moreover, our approach clearly shows that presteady-state kinetics, should they become applicable to (co)transport kinetics, could be invaluable in determining more precise transport mechanisms.

Animals↗
PubMed1990

Intrathecal synthesis of soluble class I antigens (sHLA) in patients with HIV infection and tuberculous meningitis.

sHLA are soluble class I antigens produced by lymphocytes on early activation. We have studied the sHLA index IH = (CSF sHLA/serum sHLA)/(CSF albumin/serum albumin), which reflects the intrathecal synthesis (ITS) of sHLA in 23 intravenous drug abusers with central nervous system (CNS) HIV infection. Their mean IH value was increased and directly correlated with ITS of IgG against HIV when the total group of patients was studied; however, 8 of them, who suffered from concomitant tuberculous meningitis, had a decreased IH. The relationship between this index, blood-brain barrier (BBB) function, and HIV and tuberculous infection was also studied. We consider IH an index of lymphocyte activation within the CNS. Its decrease in patients with CNS HIV infection may reflect the presence of a meningeal opportunistic infection due to Mycobacterium tuberculosis.

AIDS Dementia Complex↗
PubMed1989

Detection of soluble class I molecules (non HLA-A or HLA-B) in serum, spleen membranes and lymphocytes in culture.

Soluble major histocompatibility complex class I molecules (sHLA) present in human serum can be resolved by gel filtration into two different peaks with an apparent molecular mass of about 200 kDa (30% of the total) and 50-60 kDa (60%-70%). The serological analysis of the peaks shows that A or B specificities can only be detected in the 200 kDa peak while both are recognized by the monomorphic W6/32 monoclonal antibody (mAb) and anti-beta 2-microglobulin mAb. Such sHLA (non HLA-A or -B) molecules are released from human spleen membranes upon incubation at 37 degrees C and have been purified by affinity chromatography with mAb W6/32 bound to Sepharose. The molecular mass analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the sHLA (non HLA-A or -B) and of the classical HLA-A or -B antigens still bound to the membranes and purified from the same membranes after detergent solubilization does not show a significant difference, indicating that sHLA do not represent proteolytic fragments of the classical HLA-A or -B antigens. The presence of sHLA (non HLA-A or -B) has also been detected in the supernatants of lymphocyte cultures and increases dramatically upon stimulation by mitogens. The effect of pokeweed mitogen, phytohemagglutinin, Staphylococcus aureus Cowan strain and phorbol 12-myristate 13-acetate on the secretion of sHLA has been studied. The molecular mass of the secreted sHLA (detected using [14C]leucine) is compared with the classical transmembrane proteins.

Cells, Cultured↗
PubMed1988

External ATP triggers a biphasic activation process of a calcium-dependent K+ channel in cultured bovine aortic endothelial cells.

We have used the patch-clamp method in order to investigate the single-channel events underlying the effect of external ATP on the potassium permeability of bovine aortic endothelial cells (BAE). The results obtained from cell-attached and inside-out experiments led first to conclude that BAE cells possess an inward rectifying potassium channel activated by internal calcium at micromolar concentrations. The channel conductance for inward currents was estimated at 40 pS in symmetrical 200 mM KCl and the open-channel probability was found to be voltage insensitive within the membrane voltage range -50 to -100 mV. Based on results obtained in the cell-attached configuration, it could next be established that external ATP and ADP at micromolar concentrations could trigger, via the stimulation of P2 purinergic receptors, a time variable activation process of the observed calcium-dependent potassium channel. This activation process was found to occur in a biphasic manner with an initial phase independent of the presence of calcium in the cell bathing medium. The second phase which could be blocked by calcium channel blockers such as Co2+ or La3+ required, however, the presence of external calcium and could be abolished by depolarizing the cells using high K+ external solutions. Another important aspect related to this phenomenon was the observation that removing ATP from the external medium during the second phase led to a complete abolition of the associated calcium-dependent potassium channel activation process. It is suggested from these results that the action of ATP on the potassium permeability of BAE cells is related to a second messenger mediated release of calcium from internal calcium stores coupled to an ATP-dependent calcium influx abolished at depolarizing voltages.

Adenosine Triphosphate↗
PubMed1988

Polyoma integrates readily in mouse cellular DNA.

Although the natural host of polyoma virus is the mouse, its integration in cellular DNA has been investigated almost exclusively in rat cells. We report here studies on the integration of polyoma in mouse cells. We introduced the polyoma virus genome in two different mouse cell lines as an unselected genetic marker, by cotransfection with the tk gene of herpes simplex virus or the neo gene of E. coli. The number of TK+ or G418R clones obtained was reduced up to 50 fold by the presence of the polyoma genome. The gene coding for the early protein large T of polyoma was necessary and sufficient to produce this reduction. However, this effect appeared to be independent of polyoma replication. Surprisingly, all of the 33 clones analysed that had survived cotransfection with polyoma contained polyoma DNA integrated in their genome. Furthermore, in over 50% of these clones, the entire polyoma genome had been integrated. We conclude that polyoma integrates readily in mouse cellular DNA.

Animals↗
PubMed1988

Hemagglutinating properties of Actinobacillus pleuropneumoniae.

A total of 26 isolates of Actinobacillus pleuropneumoniae were tested for their ability to agglutinate erythrocytes of different origins. Seven different hemagglutination patterns were found. Ten (38%) isolates did not agglutinate any of the erythrocytes tested. The remaining 16 (62%) isolates agglutinated human erythrocytes, and among these, 12 also agglutinated rat, cat, dog, guinea pig, or bovine erythrocytes. No correlation was found between the seven different hemagglutination patterns observed and the serotypes. Hemagglutination activity was destroyed by heating at 100 degrees C as well as by formaldehyde treatment, but was not affected by heating at 60 degrees C, by treatment with trypsin or pronase, or by homogenization of bacterial cells. No fimbriae were observed on examination of bacterial cells negatively stained with phosphotungstate using electron microscopy. Hydrophobic surface properties of the isolates were evaluated. All the isolates appear to possess a hydrophilic cell surface. The present study provides evidence that certain isolates of A. pleuropneumoniae possess hemagglutinating properties which do not appear to be mediated by fimbriae or to involve hydrophobic interactions.

Actinobacillus↗
PubMed1988

Phenotypic and functional abnormalities in monocytes from patients with haemophilia A treated with factor VIII concentrates.

The phenotype and functions of monocytes in patients with haemophilia A and age-matched controls were studied. Fourteen male haemophiliacs were classified in three categories according to the mean number of units of factor VIII received during the last 5 years. Eleven patients were positive for antibodies to human immunodeficiency virus but none of our patients were homosexuals or drug abusers, nor do they fulfill the criteria of acquired immunodeficiency syndrome. Patients treated with high amounts of factor VIII concentrates (greater than 3 x 10(5) U/year) showed a significantly lower percentage of monocytes expressing HLA-DR, LFA-1 and CR3 antigens as compared with patients receiving lower amounts of factor VIII (less than 2 x 10(6) U/year) or controls. Kinetics of DR, LFA-1 and CR3 in cultured monocytes showed tht they were lost faster by monocytes from haemophiliacs treated with large amounts of factor VIII than by control monocytes. Adherence ability and chemotactic response of monocytes from patients treated with less than 3 x 10(5) U/year of factor VIII were also impaired. Although phagocytic indices were in normal ranges in haemophiliacs, a significant difference was observed between percentages of phagocytic monocytes from haemophiliacs treated with the largest doses of factor VIII and normal controls. Tests for respiratory burst activity, measured by chemiluminescence and superoxide anion generation, and Staphylococcus aureus killing were in normal ranges in haemophiliacs' monocytes.

Adult↗
PubMed1987

Oscillatory activation of calcium-dependent potassium channels in HeLa cells induced by histamine H1 receptor stimulation: a single-channel study.

We have used the patch-clamp method (O.P. Hamill et al., Pfluegers Arch., 391:85-100, 1981) in order to investigate the activation pattern of a calcium-dependent potassium channel following H1 receptor stimulation in HeLa cells. Our results essentially indicate that the stimulation of H1 receptors by exogenous histamine at concentrations greater than 1 microM induces an oscillatory activation pattern of calcium-dependent potassium channels characterized by the occurrence of channel current bursts separated by long silent periods. It was also found that the occurrence of these bursts could be directly correlated with transmembrane potential oscillations, the latter being the resulting effect of the calcium-dependent potassium channel synchronous openings. In addition, the cyclic activation of the calcium-dependent potassium channels could be initiated by the addition of histamine to a calcium-free external medium, indicating that the stimulation of the H1 receptors in HeLa cells is mainly related to the release of calcium from internal stores. Finally, the membrane-permeable cyclic AMP analog dibutyryl cyclic AMP was found to be ineffective in initiating single-channel events such as those triggered by exogenous histamine. It is proposed that the oscillatory activation of the calcium-dependent potassium channels in HeLa cells results from a repetitive transient increase in cytosolic free calcium concentration consequent to the H1 receptor stimulation.

Adenylyl Cyclases↗
PubMed1987

Effect of anisotonic media on volume, ion and amino-acid content and membrane potential of kidney cells (MDCK) in culture.

Effects of anisotonic media on a monolayer of confluent kidney cells in culture (MDCK) were studied by measuring: cell thickness and cross-section changes, ion and amino-acid content and membrane potential. The volume was also determined with cells in suspension. When cells in a monolayer were incubated in hypotonic media, the lateral and the apical membranes were rapidly stretched. Afterwards the lateral membranes returned to their initial state while the apical membranes remained stretched. This partial regulatory volume decrease (RVD) was verified with cells in suspension. RVD was accompanied by a loss of K+, Cl- and amino acids, but there was no loss of inorganic phosphate. Also a transient hyperpolarization of the membrane potential was observed, suggesting an increase of the K+ conductance during RVD. Upon restoring the isotonic medium, a regulatory volume increase (RVI) was observed accompanied by a rapid Na+ and Cl- increase and followed by a slow recovery of the initial K+ and Na+ content while amino acids remained at their reduced content. A transient depolarization of the membrane potential was measured during this RVI, suggesting that Na+ and Cl- conductance could have increased. In hypertonic media, only a small and slow RVI was observed accompanied by an increase in K+ and Cl- content but without any change of membrane potential. Quinine partly inhibited RVD in hypotonic media with cells in a monolayer while inhibiting RVD completely with cells in suspension. Incubation during four hours in a Ca2+ free medium had no effect on RVD. Furosemide and amiloride had no effect on RVD and RVI. Volume regulation, RVD or RVI, was not affected by replacing Cl- by nitrate. When cells in a monolayer were incubated in a hypotonic K2SO4 medium, no RVD was observed. From these results, it seems that MDCK cells in a confluent monolayer regulate their volume by activating specific ion and amino-acid transport pathways. Selective K+ and Na+ conductances are activated during RVD and RVI, while the activated anion conductance has a low selectivity. The controlling mechanism might not be the free intracellular Ca2+ concentration.

Amino Acids↗