Characterization of L-arginine uptake by plasma membrane vesicles isolated from cultured pulmonary artery endothelial cells

We investigated the mechanisms of [3H]-L-arginine transport via System Y+ using plasma membrane vesicles derived from cultured pulmonary artery endothelial cells. [3H]-L-arginine uptake into plasma membrane vesicles was Na-independent, sensitive to trans-stimulation, unaffected by proton-conducting ionophores, and selectively inhibited by cationic amino acids. Kinetic experiments performed over a wide range of substrate concentrations revealed only one population of L-arginine transporters with Km = 130 microM. To elucidate the driving force for L-arginine transport, we measured [3H]-L-arginine uptake by plasma membrane vesicles at different transmembrane ion gradients. Plasma membrane vesicles accumulated [3H]-L-arginine only when a membrane potential was imposed across the vesicles, and the velocity of uptake was linearly related to the magnitude of the created membrane potential. The presence of potassium ions inside the vesicles was not essential for uptake of L-arginine into vesicles, but it was essential for trans-stimulation of L-arginine transport. [3H]-L-arginine accumulated in plasma membrane vesicles can be released by agents that dissipate transmembrane potassium gradients (e.g. saponin, gramicidin, and nigericin). Diazoxide and pinacidil, activators of K(+)-channels, had no significant effect on [3H]-L-arginine uptake, whereas tetraethylammonium chloride, 4-aminopyridine, and glibenclamide, inhibitors of K(+)-channels, caused decreases in [3H]-L-arginine transport by plasma membrane vesicles. This study demonstrates for the first time a specific role for potassium ions in the mechanism of L-arginine transport, particularly in the phenomenon of trans-stimulation.     

Monitoring of rotavirus infection in a paediatric hospital by RNA electrophoresis

During the spring of 1987 and the autumn of 1988, stool specimens were collected from infants and young children in the paediatric unit at H. F. Verwoerd Hospital, Pretoria, and examined for the presence of rotaviruses to assess the potential for hospital-acquired infection in the paediatric wards. Stool samples were also collected from children admitted to the hospital for causes unrelated to gastro-enteritis to investigate the possible asymptomatic carriage of rotavirus in this population. Hospital-acquired rotavirus infection was determined in only 9% of cases. Very little asymptomatic carriage of the virus was identified. Electrophoretic analysis of the rotavirus strains showed that the majority of the infections (20 of 42) were associated with a particular strain with a long RNA profile, while 7 minor strains co-circulated (5 with a long electrophoretype and 2 with a short one). An apparent small outbreak of nosocomial infection with a single strain was observed to occur in one of the paediatric wards during the spring and early summer.     

DNA damage induced by fotemustine in cultured HL60 cells

AIM: To detect DNA damage caused by fotemustine (Fot). METHODS: DNA damage in HL60 cells was evaluated by modified alkaline elution technique. DNA interstrand crosslink (ISC) and DNA-protein crosslink (DPC) were determined. Carmustine (Car) was used as control. Drug treatment time was 1 h. RESULTS: After treatment with Fot 300 mumol.L-1, ISC and DPC index were 5.4 and 6.1 at 6 h, 2.7 and 1.2 at 12 h, respectively. ISC reached the maximum at about 6 h. For Fot 100 mumol.L-1, ISC and DPC index at ISC peak time were 2.1 +/- 0.9 and 3.55 +/- 0.23, 5.0 +/- 0.5 and 7.7 +/- 1.1 for Car 100 mumol.L-1, respectively. Single strand break (SSB) was induced by Fot. CONCLUSION: Fot caused HL60 cell DNA ISC, DPC, and SSB. ISC was formed more quickly by Fot than that by Car.     

Characterization of a rotavirus rearranged gene 11 by gene reassortment

The effect of replacement of gene 11 of rotavirus SA-11 by a gene carrying a head to tail duplication obtained from a swine rotavirus strain was studied. The swine rotavirus strain with a duplicated gene (CC86) exhibits both a phenotype that allows to overgrow other viral strains when coinfected and an increased plaque size when plated in both CV-1 and MA-104 monkey kidney cells. Using reassortment methods the duplicated gene of the swine rotavirus was introduced into the SA-11 virus, replacing the regular gene 11. The reassorted strain was characterized to find out the origin of each of the other viral gene segments. Based on electrophoretic mobilities segments 1, 2, 3, 5, 7, 8 and 10 were identified as of SA-11. The SA-11 origin of the segments 4, 6 and 9 was confirmed by neutralization with polyclonal and monoclonal antibodies and by ELISA. The results suggest that the new reassortant virus was a monoreassortant carrying SA-11 genes except the duplicated gene originated from the swine virus CC86. The ability to in vivo replicate and to synthesize viral proteins was identical in the reassorted virus and the parental strains. Sequence analysis indicates that the new phenotype does not originate in the duplication of gene 11 but possibly from mutations in the coding region of NSP5 gene that may result in different phosphorylation patterns of the protein.     

The effect of the increase in the calcium concentration induced by action of the ionophore A-23187 on mitosis in cultured ESK cells

After addition of 20 mM calcium ionophore A23187 to cultured PK (pig kidney embryo) cells, [Ca++] in cytosol increased by more than 10 times. The maximum [Ca++] concentration was observed 1-2 min after drug introduction. Later on [Ca++] gradually decreased, and after 30 min of incubation with A23187 [Ca++] its concentration was 3-5 times higher than in the norm. 1 min after introduction of the calcium ionophore, mitotic spindles shortened for 1/3 and the angle of divergence of spindle microtubules from the centrosome extended. These changes remained for 5 min of treatment. After nocodazole treatment the length of the mitotic spindle reduced (2 min), then mitotic spindle and the metaphase plate were disrupted. The rate of mitotic spindle shortening after addition of the ionophore is about the same as after addition of nocodazole, but after ionophore treatment the metaphase plate remained for more than 5 min. Based on the results obtained we suggest that the maximum distance between spindle poles at metaphase is in the intact cell, and after any perturbation of normal microtubule dynamics the spindle may rapidly collapse. The collapsed mitotic spindle becomes more stable and its size is determined predominantly by kinetochore fibers. The metaphase spindle is completely and rapidly destroyed when the microtubule growth is prohibited, but it is preserved when this growth is restricted.     

Productive penetration of rotavirus in cultured cells induces coentry of the translation inhibitor alpha-sarcin

Internalization of rotavirus in MA104 cells was found to induce coentry of alpha-sarcin, a toxin that inhibits translation in cell-free systems and to which cells are normally impermeable. Entry of the toxin, measured by inhibition of protein synthesis at early times after infection, correlated with virus penetration leading to expression of infectivity, since toxin entry (1) was induced only by trypsin-treated triple-layered virions, to a degree dependent on the toxin and the virus concentration; (2) correlated with the degree of permissivity of different cell lines to rotavirus infection; (3) was inhibited to a similar extent as infectivity by treatment of cells with neuraminidase; and (4) was inhibited by pre- or postadsorption incubation of the virus with neutralizing monoclonal antibodies to VP7 and VP4 (VP8*). Neither the virus infectivity nor the toxin coentry was significantly affected by treatment of cells with bafilomycin A1, an inhibitor of the vacuolar proton ATPase, indicating that both events are independent of the endosomal acid pH. Virus-like particles (VLP), composed of rotavirus proteins 2/6/7/4, but not 2/6/7 or 2/6, were able to induce toxin entry as efficiently as virions. Use of genetically modified VLP in combination with the toxin coentry assay, which measures entry through a productive pathway, should allow identification of the regions of the outer capsid proteins essential for rotavirus penetration.     

An apical-type trafficking pathway is present in cultured oligodendrocytes but the sphingolipid-enriched myelin membrane is the target of a basolateral-type pathway

The nurse functions as a liaison between the injured employee, the insurance carrier, the employer, and the referral physician. Frequently, the nurse's position allows knowledge of psychosocial stressors and task assignments that is not as readily available to other participants in the process of managing a work-related injury.     

Trypsin activation pathway of rotavirus infectivity

The infectivity of rotaviruses is increased by and most probably is dependent on trypsin treatment of the virus. This proteolytic treatment specifically cleaves VP4, the protein that forms the spikes on the surface of the virions, to polypeptides VP5 and VP8. This cleavage has been reported to occur in rotavirus SA114fM at two conserved, closely spaced arginine residues located at VP4 amino acids 241 and 247. In this work, we have characterized the VP4 cleavage products of rotavirus SA114S generated by in vitro treatment of the virus with increasing concentrations of trypsin and with proteases AspN and alpha-chymotrypsin. The VP8 and VP5 polypeptides were analyzed by gel electrophoresis and by Western blotting (immunoblotting) with antibodies raised to synthetic peptides that mimic the terminal regions of VP4 generated by the trypsin cleavage. It was shown that in addition to arginine residues 241 and 247, VP4 is cleaved at arginine residue 231. These three sites were found to have different susceptibilities to trypsin, Arg-241 > Arg-231 > Arg-247, with the enhancement of infectivity correlating with cleavage at Arg-247 rather than at Arg-231 or Arg-241. Proteases AspN and alpha-chymotrypsin cleaved VP4 at Asp-242 and Tyr-246, respectively, with no significant enhancement of infectivity, although this enhancement could be achieved by further treatment of the virus with trypsin. The VP4 end products of trypsin treatment were a homogeneous VP8 polypeptide comprising VP4 amino acids 1 to 231 and a heterogeneous VP5, which is formed by two polypeptide species (present at a ratio of approximately 1:5) as a result of cleavage at either Arg-241 or Arg-247. A pathway for the trypsin activation of rotavirus infectivity is proposed.     

Protective immunity induced by oral immunization with a rotavirus DNA vaccine encapsulated in microparticles

DNA vaccines are usually given by intramuscular injection or by gene gun delivery of DNA-coated particles into the epidermis. Induction of mucosal immunity by targeting DNA vaccines to mucosal surfaces may offer advantages, and an oral vaccine could be effective for controlling infections of the gut mucosa. In a murine model, we obtained protective immune responses after oral immunization with a rotavirus VP6 DNA vaccine encapsulated in poly(lactide-coglycolide) (PLG) microparticles. One dose of vaccine given to BALB/c mice elicited both rotavirus-specific serum antibodies and intestinal immunoglobulin A (IgA). After challenge at 12 weeks postimmunization with homologous rotavirus, fecal rotavirus antigen was significantly reduced compared with controls. Earlier and higher fecal rotavirus-specific IgA responses were noted during the peak period of viral shedding, suggesting that protection was due to specific mucosal immune responses. The results that we obtained with PLG-encapsulated rotavirus VP6 DNA are the first to demonstrate protection against an infectious agent elicited after oral administration of a DNA vaccine.     

High glucose inhibits cytosolic calcium signaling in cultured rat mesangial cells

Glomerular vasodilatation in the early stages of type I diabetes mellitus apparently results from arteriolar insensitivity to vasoconstrictors. Since cytosolic free calcium ([Ca2+]i) is a major signaling mechanism for smooth muscle contraction, we studied whether growth of smooth muscle-like rat glomerular mesangial cells in media with high glucose concentration affects [Ca2+]i responses to vasoconstrictors. In cells grown for five days in 22 mM glucose, we observed blunted responsiveness to three structurally unrelated vasoconstrictors that elevate [Ca2+]i via a phospholipase C-dependent mechanism, angiotensin II, prostaglandin F2 alpha, and arginine vasopressin. Inhibition of [Ca2+]i responses was not due to an osmotic effect of high glucose, since it was not mimicked by hypertonic mannitol. While the size of intracellular Ca2+ pools was unaffected by elevated glucose, Na+/Ca2+ exchange was markedly inhibited, thus ruling out both impaired filling of Ca2+ stores and enhanced counter-regulatory mechanisms. Impaired myoinositol transport or intracellular sorbitol accumulation were not responsible for the effects of high glucose, since supplementation of media with myo-inositol or with the aldose reductase inhibitor. Alcon 1576, failed to reverse insensitivity to vasoconstrictors. On the other hand, down-regulation or pharmacological inhibition of protein kinase C completely reversed the effects of high glucose, thus indicating involvement of this signal transduction pathway. These data suggest a possible intracellular mechanism for the impaired vascular sensitivity underlying early renal hemodynamic changes in diabetes mellitus.     

Mast cell histamine-induced calcium transients in cultured human peritoneal mesothelial cells

OBJECTIVE: Peritoneal inflammation results from a complex interplay of events initiated by macrophage activity in response to infection, with the stimulation of mesothelial cell cytokine release amplifying the recruitment of blood-borne defense cells to the site of injury. Resident peritoneal mast cells may add to this complexity with mast cell derived cytokines released during this cascade. This study examined the influence of histamine, a mast cell-derived inflammatory mediator, on the initial activation of human peritoneal mesothelial cells (HPMC) by intracellular free calcium (Ca2+(i)) mobilization, and changes to the actin cytoskeleton. DESIGN: HPMC signal transduction was examined in response to histamine (1.0 mmol/L) compared to fetal bovine serum (FBS) (0.1%) and 4-br-A23187 (1.0 micromol/L). Intracellular free calcium was measured in fura-2 loaded cells with and without external calcium (Ca2+(ext)), or Ca2+(ext) with verapamil (100 micromol/L). Following treatment with agonists, HPMC actin cytoskeleton was stained using direct immunocytochemistry. RESULTS: HPMC responded to histamine with a twofold transient rise in Ca2+(i) which returned to the baseline, in contrast with FBS- and A23187-induced Ca2+(i) transients, which returned to elevated resting values. In the absence of Ca2+(ext), all agents produced a calcium transient indicative of calcium release from intracellular stores. Histamine induced calcium-dependent changes to the cytoskeleton and cellular organization, including increased actin stress fibers. CONCLUSION: Histamine produced large specific receptor-mediated calcium transients in HPMC, which included components of calcium release from intracellular stores and receptor-mediated calcium influx processes. The observed response to histamine raises the possibility that histamine derived from resident mast cells may modulate mesothelial cell function, in part by calcium-dependent pathways, and influence the performance of the peritoneal membrane during peritoneal dialysis.     

Antiproliferative effects of yogurt fractions obtained by membrane dialysis on cultured mammalian intestinal cells

The consumption of yogurt has been associated with a reduced incidence of colon cancer in population groups. Bioactive peptides produced during bacterial fermentation may alter the risk of colon cancer via modification of cell proliferation in the colon. Using our previously described cell culture model system, we have isolated a yogurt fraction that decreases cell proliferation. Yogurt was fractionated using 10,000- and 500-Da membrane dialysis. When the yogurt fraction was incubated with IEC-6 or Caco-2 cells, cell division was decreased compared with control treatments, as determined by thymidine incorporation. Cell division was not inhibited in response to a similarly produced milk fraction or in response to solutions of lactic acid. The determination of cell kinetics by flow cytometry revealed a decrease in the number of cells in the initial growth phase in response to the yogurt fraction for the IEC-6 cells, but not the Caco-2 cells. Alpha-Lactalbumin inhibited cell division of both cell lines, but beta-casein did not.     

A study on neonatal calf diarrhea induced by rotavirus

This review summarizes the results of a study on rotaviruses isolated from calves affected by neonatal diarrhea. The results indicated that rotavirus infection is widespread and supported the evidence for an etiologic role of these viruses in neonatal diarrhea. Differences in virulence among bovine rotaviruses appeared also to be confirmed. Conventionally reared calves were fully susceptible to the experimental infection induced by rotaviruses originating from heterologous hosts, i.e. monkeys, pigs and rabbits. When rotavirus strains of bovine, simian and rabbit origin were compared by cross neutralization tests, it was found the simian and porcine strains were indistinguishable and both appeared to relate antigenically to the bovine strain. Finally, it was proven that feeding newborn calves with colostrum and first milk of their dams, previously vaccinated with an inactivated adjuvanted rotavirus vaccine, could prevent the neonatal diarrhea from occurring.     

Inhibition of rotavirus infection in vitro and in vivo by a synthetic peptide from VP4

A synthetic peptide corresponding to bovine rotavirus C486 (BRV) VP4 amino acid sequence 232-255 (VP4-peptide) was studied with the objective of defining the origin of the protective immune response reported previously by Ijaz et al. (J. Virol. 1991, 65, 3106-3113). Pretreatment of MA-104 cells with the VP4-peptide before infection with rotavirus prevented both the attachment of 35S-labelled virus and plaque formation in vitro. In vivo studies using a murine rotavirus model demonstrated that intragastric administration of VP4-peptide protected subjects from challenge with virulent rotavirus. These results clearly indicate the importance of this epitope in virus-cell interactions and their potential as a rotavirus vaccine candidate.     

Release of interleukin-2 induced by a major antigenic outer membrane protein of Shigella dysenteriae type 1 in natural infection

An antigen specific modulation of peripheral blood lymphocyte function was examined in a patient-based study of Shigella dysenteriae type 1 infection. Interleukin-2 (IL-2) production by the peripheral blood mononuclear cells (PBMC) from Shigella-infected patients was correlated with the expression of host cellular immune responses. To evaluate the role of a 57 kD major antigenic outer membrane protein of S. dysenteriae 1 in the proliferation of PBMC and the production of IL-2, the in vitro blastogenic transformation assay was employed. The magnitude of the response was monitored morphologically as well as by the proliferation of the IL-2 dependent CTLL-2 cell line. The proliferation of the IL-2 dependent CTLL-2 cell line against PBMC culture fluids after exposure to the major antigen reflected the participation of functionally active T-lymphocytes in shigellosis patients. The precise quantitation of IL-2 concentration in such lymphocyte culture supernatants by immunoassay showed substantial production of IL-2.     

The modification of potential-dependent calcium channels in the membrane of secretory cells in a calcium-free medium

Effect of modification of potential-dependent calcium channels in cells of the upper lobe of the salivary gland in Chironomus larva has been ascertained in the Ca-free medium containing Ca-binding substances (EGTA or EDTA). It is shown that the inward current depends on the sodium transmembrane gradient. The amplitude of the inward sodium current decreases under the influence of verapamil as well as with an increase of external concentration of Ca2+ ions. The metabolic dependence of modified calcium channels as well as the pharmacological sensitivity are unchanged. Tau n of the sodium current through modified calcium channels increases insignificantly comparing with that of the calcium current. Maximum of the current-voltage relationship of the sodium current through modified calcium channels is shifted to a negative side by 10-15 mV as compared with the calcium current through native channels.     

The rotavirus enterotoxin NSP4 mobilizes intracellular calcium in human intestinal cells by stimulating phospholipase C-mediated inositol 1,4,5-trisphosphate production

Rotavirus infection is the leading cause of severe diarrhea in infants and young children worldwide. The rotavirus nonstructural protein NSP4 acts as a viral enterotoxin to induce diarrhea and causes Ca2+-dependent transepithelial Cl- secretion in young mice. The cellular basis of this phenomenon was investigated in an in vitro cell line model for the human intestine. Intracellular calcium concentration ([Ca2+]i) was monitored in fura-2-loaded HT-29 cells using microscope-based fluorescence imaging. NSP4 (1 nM to 5 microM) induced both Ca2+ release from intracellular stores and plasmalemma Ca2+ influx. During NSP4-induced [Ca2+]i mobilization, [Na+]i homeostasis was not disrupted, demonstrating that NSP4 selectively regulated extracellular Ca2+ entry into these cells. The ED50 of the NSP4 effect on peak [Ca2+]i mobilization was 4.6 +/- 0.8 nM. Pretreatment of cells with either 2.3 x 10(-3) units/ml trypsin or 4.4 x 10(-2) units/ml chymotrypsin for 1-10 min abolished the NSP4-induced [Ca2+]i mobilization. Superfusing cells with U-73122, an inhibitor of phospholipase C, ablated the NSP4 response. NSP4 induced a rapid onset and transient stimulation of inositol 1,4,5-trisphosphate (IP3) production in an IP3-specific radioreceptor assay. Taken together, these results suggest that NSP4 mobilizes [Ca2+]i in human intestinal cells through receptor-mediated phospholipase C activation and IP3 production.     

Spontaneous calcium oscillations and mechanically and chemically induced calcium responses in mammary epithelial cells

Binding data point to the coexistence of three endothelin receptors (ET-R) in rat heart myocytes. Induction of phosphoinositide hydrolysis in this preparation by endothelins (ET-1 and ET-3) or sarafotoxins (SRTX-b and SRTX-c) was demonstrated by measurement of labeled inositol phosphate generation. Pertussis toxin (PT) enhanced the induction of phosphoinositide hydrolysis by all four peptides. The process seems to be mediated by at least two heterotrimeric G-proteins, the one sensitive and the other insensitive to PT. Measurement of GTPase activity induced in rat myocytes clearly indicates for the first time the direct functional coupling between ET-R and a G-protein. These GTPase activity experiments provide evidence that phosphoinositide hydrolysis is stimulated via functional coupling between the endothelin receptor of the ETA-R subtype and a PT-insensitive G-protein, Gq/11. The involvement of PT-sensitive G-proteins, i.e. Gi-like and/or Go-like proteins, in the signal transduction pathways of ETs and SRTXs is discussed.     

Characterization of membrane surface proteins of Mycoplasma agalactiae during natural infection

We have analyzed antigenic variation of seven M. agalactiae wild strains using different sera from naturally infected sheep. Only 30 day sera recognized all surface proteins and inhibited the growth of mycoplasmas. Furthermore, we have observed that two strongly immunogenic proteins: 55 and 35 kDa were digested using 500 micrograms/ml of trypsin. These two bands are immunoprecipitated together with four other proteins but only the 35 kDa protein is recognized by eluted antibodies.