Genetic Analysis of Pestiviruses at the 3′ End of the Genome

Specific PCR primers were selected for each pestivirus genotype which flanked the 3-part of the NS5B gene and more than three quarters of the 3-UTR. PCR products were sequenced in both directions using an automatic sequencing device and analyzed by computer package program DNASTAR. A comparative analysis of the 3 untranslated region (3-UTR) of 82 viruses, representing the four genotypes of the Pestivirus genus, provided a similar phylogenetic grouping as other genomic regions. Intertypic recombination was not observed, but Border disease virus (BDV) and Bovine viral diarrhoea virus type 1 (BVDV I) showed great intragenotypic variability. In most pestiviruses the stop codon is TGA, but BDV isolates were found to have either a TAG or a TAA stop codon. Various deletions and insertions were observed in the 3-UTR region. Viruses of the BVDV Ib group contained a characteristic deletion of 41 nucleotides. Compared to the 5-UTR, the 3-UTR was less conserved. The first 50–60 nucleotides were particularly variable, whilst the most conserved part was found at the 3 end of the studied region. All 82 viruses contained AT-rich stretches, the positions and sizes of which differed between the genotypes.     

Cyclische Nucleotide als intracelluläre Überträgerstoffe bei Hormonwirkungen

Zusammenfassung Hormone dienen als extracelluläre Informationsüberträger zwischen ihrem Bildungsort, einer endokrinen Drüse, und den Zellen, deren Funktion sie regulieren. Durch die Reaktion des Hormons mit den an der Zellmembran gelegenen Receptoren wird die Aktivität der mit diesen eng verknüpften Adenyl-Cyclase beeinflußt. Die meisten Hormone erhöhen in ihrem Zielorgan die Aktivität dieses Enzyms und führen hierdurch zu einem raschen Anstieg der intracellulären Konzentration von Adenosin-3:5-monophosphat (Ado-3:5-P). Dieses cyclische Nucleotid wird durch eine spezifische Phosphodiesterase zu Adenosin-5-monophosphat abgebaut. Auch die Aktivität dieses Enzyms bestimmt die intracelluläre Ado-3:5-P-Konzentration, die im Vergleich zu der anderer Nucleotide sehr gering ist.Ado-3:5-P beeinflußt als zweiter, intracellulärer Überträgerstoff die Aktivität zahlreicher Schlüsselenzyme. Die Ado-3:5-P-Konzentration bestimmt hierdurch das Gleichgewicht verschiedener Stoffwechselwege zueinander und damit die Reaktion einer Zelle auf eine hormonale Stimulierung. An einer Reihe von Enzymen wird die durch Ado-3:5-P bedingte Aktivitäts-Änderung durch einen gleichartigen Mechanismus bewirkt. Das cyclische Nucleotid stimuliert Proteinkinasen, die eine Phosphatgruppe des ATP auf verschiedene Proteine übertragen und hierdurch deren Eigenschaften verändern können. So steigt bei Phosphorylierung durch eine Ado-3:5-P-stimulierbare Proteinkinase die Aktivität der Triglyceridlipase und der Glykogen-Phosphorylase-b-kinase an, dagegen nimmt die Aktivität der Glykogen-Synthetase ab; durch Phosphorylierung von Histonen kann deren Repressorcigenschaft vermindert und die Synthese von Enzymen gesteigert werden.In manchen tierischen Geweben wurde auch eine spezifisch durch Guanosin-3:5-monophosphat (Guo-3:5-P) stimulierbare Proteinkinase nachgewiesen. Dieses cyclische Nucleotid kommt wie Ado-3:5-P in allen Säugerorganen vor. Die Bildung von Guo-3:5-P aus GTP wird durch die Guanyl-Cyclase katalysiert, ein Ferment, das im Gegensatz zur Adenyl-Cyclase zum großen Teil nicht an die Zellmembranen gebunden ist. Die Konzentration von Guo-3:5-P in verschiedenen Geweben, im Blutplasma und im Urin wird durch Hormone beeinflußt. Es ist noch nicht bekannt, welche hormonalen Regulationen durch Guo-3:5-P vermittelt werden; dagegen ist bei vielen, rasch einsetzenden Hormonwirkungen die Beteiligung von Ado-3:5-P nachgewiesen worden.

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Abkürzungen Ado-3:5-P Adenosin-3:5-monophosphat - dAdo-3:5-P Desoxy-adenosin-3:5-monophosphat - Guo-3:5-P Guanosin-3 : 5-monophosphate - Nuc-3:5-P Nucleosid-3:5-monophosphat - NTP Nucleosidtriphosphat - NMP Nuclcosid-5-monophosphat - dATP Desoxyadenosintriphosphat - P_i anorganisches Phosphat - PP_i anorganisches Pyrophosphat - DNS Desoxyribonucleinsäure - RNS Ribonucleinsäure - r-RNS ribosomale RNS - m-RNS Boten-RNS - Glykogen-Synthetase UDP-Glucose--1,4-glucan--4-glucosyltransferase - ICSH interstitial-cell-stimulating hormone     

Genetic characterization of an α-specific gene responsible for sexual agglutinability in Saccharomyces cerevisiae: mapping and gene dose effect

Summary A recessive ag1 mutation leads to specific defect in sexual agglutinability specifically in cells of the yeast Saccharomyces cerevisiae. The cryptopleurine resistance gene cry^R 1, closely linked to the mating type locus, was used to select / strains which emerged from / strains by mitotic nonreciprocal recombination, to genetically analyse ag1, since ag1 is expressed only in mating type. The ag1 gene was found to be linked to the centromere tightly, to met3 at 4.4 cM, and to ilv3 at 12 cM on chromosome X. Sexual agglutinability of cells was shown to be dependent on the dose of the AG1 gene, using / isogenic strains carrying AG1/AG1, AG1/ag1 or ag1/ag1. The sst2-1 mutation did not suppress the ag1 mutation. Based on these results, function of the AG1 gene is discussed.Abbreviations cM centimorgan - FDS first division segregation - NPD nonparental ditype - PD parental ditype - SDS second division segregation - TT tetratype     

A point mutation in the 5′-untranslated leader that affects translational activation of the mitochondrial COX 3 mRNA

The 613-base 5-untranslated leader (5-UTL) of the Saccharomyces cerevisiae mitochondrial COX 3 mRNA contains the target of an mRNA-specific translational activator complex composed of at least three nuclearly encoded proteins. We have genetically mapped a collection of cox 3 point mutations, using a set of defined COX 3 deletions, and found one to be located in the region coding the 5-UTL. The strain carrying this allele was specifically defective in translation of the COX 3 mRNA. Nucleotide-sequence analysis showed that the allele was in fact a double mutation comprised of a single-base insertion in the 5-UTL (T inserted between bases-428 and-427 with respect to the start of translation) and a G to A substitution at+3 that changed the ATG initiation codon to ATA. Both mutations were required to block translation completely. The effects of the ATG to ATA mutation alone (cox 3-1) had previously been analyzed in this laboratory: it reduces, but does not eliminate, translation, causing a slow respiratory growth phenotype. The T insertion in the 5-UTL had no detectable respiratory growth phenotype as a single mutation. However, the 5-UTL insertion mutation enhanced the respiratory defective phenotype of missense mutations in pet 54, one of the COX 3-specific translational-activator genes. This phenotypic enhancement suggests that the-400 region of the 5-UTL, where the mutation is located, is important for Pet54p-COX 3 mRNA interaction.     

Expression and ligand-binding function of the integrin α4β1 (VLA-4) on neural-crest-derived tumor cell lines

Human neural-crest-derived tumor cell lines, including three neuroblastomas, an astrocytoma, a glioblastoma, a rhabdomyosarcoma and a melanoma were screened for the expression of the integrin ₄₁ (VLA-4). The neuroblastomas IMR-32 and SK-N-SH, the astrocytoma 131-INI, the glioblastoma Fogerty and the rhabdomyosarcoma TE-671 expressed ₄₁ as determined by cytofluorometry and immunoprecipitation. Another neuroblastoma line, LA-N-1, did not express ₄₁. Analysis of immunoprecipitated ₄₁ showed that the ₄ subunit from the various cell types differed in relative molecular weight (M _r ). The variability in the observed M _r could be accounted for by differences in the levels of N-linked glycosylation. The observed variability in M _r did not appear to affect function since intact cells and solubilized ₄₁ bound to a synthetic peptide identical in sequence to the CS-1 region of the alternatively spliced IIICS domain of fibronectin, a known ₄₁, ligand.     

Pharmacological characterization of P2X7 receptors in rat peritoneal cells

Objective and design: P2X₇ receptor activation by ATP results in the release of IL-1 and IL-18. Prolonged stimulation can lead to pore formation and cell death. In this study we pharmacologically characterized P2X₇ receptors on rat peritoneal cells (RPC) and on 1321N1 cells transfected with rat P2X₇ receptor (1321rP2X₇-11).Materials and methods: RPC were isolated from rats by lavage. P2X₇ agonist induced pore formation in RPC was measured by EtBr uptake. P2X₇-stimulated pore formation and Ca⁺⁺ influx in 1321rP2X₇-11 cells were measured by a fluorometric imaging plate reader. The effects of pyridoxal phosphate-6-azo phenyl –2-4-disulfonic acid (PPADS) on pore formation and Ca⁺⁺ influx were examined in both RPC and 1321rP2X₇-11. P2X₇-mediated IL-1 release in RPC and the effect of PPADS were determined.Results: RPC express functional P2X₇ receptors that were activated by ATP analogs with a rank order of potency of 2- 3-O-(4-Benzoylbenzoyl) adenosine 5-triphosphate (BzATP) > ATP > ,-methylene ATP. Activation of P2X₇ receptors by BzATP was inhibited by PPADS. Similar results were also obtained in 1321rP2X₇-11 cells. Activation of P2X₇ receptors on RPC resulted in IL-1 secretion, which was inhibited by PPADS.Conclusions: RPC express functional P2X₇ receptors that form pores and mediate the release of IL-1.Received 20 July 2004; returned for revision 9 September 2004; accepted by N. Boughton-Smith 5 November 2004     

Adenosine-5′-triphosphate-induced sinus tachycardia mediated by prostaglandin synthesis via phospholipase C in the rabbit heart

Effects of adenosine 5-triphosphate (ATP) and adenosine on cardiac sinus pacemaker activity were examined in the rabbit heart. Electrocardiograms of hearts were recorded while using the Langendorff perfusion method. Both adenosine and ATP, added to the perfusate, slowed the sinus pacemaker activity in a concentration-dependent manner. But in about 40% of the cases, ATP higher than 300 M initially accelerated and then slowed the heart. The sinus slowing caused by adenosine and ATP was blocked by theophylline (a p₁ receptor antagonist) and disappeared in the hearts pre-treated with islet-activating protein (IAP). In contrast, the ATP-induced sinus acceleration was not affected by either theophylline or IAP. In about 75% of the IAP-treated hearts, ATP persistently accelerated the sinus pacemaker. In the remaining 25% of the hearts, ATP caused junctional tachycardia, which may have masked the ATP-induced sinus acceleration. Apamin specifically blocked the ATP-induced sinus acceleration, suggesting that P₂ receptors are involved. Among various adenine nucleotide analogues, the order of potency in inducing tachycardia in IAP-treated hearts is adenosine-5-[-thio]triphosphate > adenylyl imidodiphosphate > adenosine 5-[, -methylene]triphosphate = ATP > adenosine diphosphate = adenosine 5-[, -methylene]triphosphate. ATP-induced acceleration was partially blocked by indomethacin and aspirin (cyclooxygenase inhibitors), but not by nordihydroguaiaretic acid (a lipoxygenase inhibitor). These results suggest that cyclooxygenase and not lipoxygenase metabolites of arachidonic acid, e.g. prostaglandins, may be involved in the generation of tachycardia. Consistent with this notion, exogenously applied cyclooxygenase metabolites, prostaglandin E₂ and 6-keto-prostaglandin F₁, which are known to be produced by extracellular ATP in the rabbit heart [Schwartzman et al. (1981) Eur J Pharmacol 74: 167–173], accelerated the sinus rate. We also observed that the ATP-induced tachycardia was almost completely blocked by neomycin (a phospholipase C inhibitor). We suggest, therefore, that cardiac P₂ receptors may be coupled to prostaglandin synthesis via an IAP-insenstive stimulation of phospholipase C.     

High Level of Sequence Variation in the 3′ Noncoding Region of Japanese Encephalitis Viruses Isolated in Korea

The 3 noncoding region (NCR) of Japanese encephalitis (JE) viruses isolated in Korea and Nakayama-NIH strain have been sequenced and compared with the 3 NCR sequences of other JE isolates reported previously. Sequence alignment of about 60 nucleotides (based on consensus sequence number) immediately downstream of the open reading frame (ORF) stop codon in the 3 NCR of the Korean isolates showed high degree of sequence variation and deletion; thus, this region was termed as the variable region. However, in the predicted RNA secondary structures, a similar type loop exists at the 5-terminus of the 3 NCR of JE viruses, despite low level of sequence homology (22%) and deletion in the variable region. The phylogenetic tree based on the 3 NCR sequences of JE viruses including the variable region showed a similar pattern to that based on envelope genes; in that, there are two genetically different types of JE viruses in Korea. Therefore, the variable region would be a useful genetic marker for JE viruses.     

Aspects of the oxidative metabolism of nicotine

Conclusions The balance of the excreted sum of nicotine and its known metabolites from confirmed results reflects great progress in recent years and today accounts for about 40–60% of the estimated or known intake of the alkaloid, calculating on the basis of parts of well-defined and reliably determined species. The main part of this balance is accounted for by products of the -oxidation of nicotine in the 5-position. A further 40% has recently been attributed to those parts of metabolites which are additionally found after the treatment of urine samples with enzymes generally known to act on phase II metabolites. Conjugates of nicotine, cotinine, and trans-3-hydroxycotinine are held responsible for this. Recently, some of these products have been definitely characterized and determined by direct measurements.Quantitative data are missing on the 1,2-iminium ion, the very recently identified tautomer of 2-hydroxynicotine in urine, and the nature of its prevailing tautomeric form which both are biologically high importance, have there been reports so far on the mechanism for their metabolic formation. Further, the metabolic fate of nornicotine, the first metabolite of the intermediary methylene iminium species, is still awaiting elucidation. Nornicotine is excreted in only a very small ratio. This may be caused by demythylation or by oxidation. Further conclusions require knowledge of the actual structures of the nicotine metabolites under different analytical pH values or in organic solvents. The respective proportions of the tautomeric structures in the biological media in general and especially at the active sites of enzymes determine the metabolic pathways of nicotine and its subsequent metabolites and should be the aim of intense investigations.     

Modulation of Nav1.5 by β1- and β3-subunit co-expression in mammalian cells

Cardiac sodium channels (Na_v1.5) comprise a pore-forming -subunit and auxiliary -subunits that modulate channel function. In the heart, 1 is expressed throughout the atria and ventricles, whilst 3 is present only in the ventricles and Purkinje fibers. In view of this expression pattern, we determined the effects of 3 and 1 co-expression alone, and in combination, on Na_v1.5 stably expressed in Chinese hamster ovary cells. The current/voltage relationship was shifted –5 mV with either 1 or 3 co-expression alone and –10 mV with co-expression of both 1 and 3. In addition, 3 and 1/3 co-expression accelerated macroscopic current decay. There were significant hyperpolarizing shifts in equilibrium gating relationships with co-expression of 1 and 3 alone and in combination. Co-expression of 1/3 together resulted in a greater hyperpolarizing shift in channel availability, and an increase in the slopes of equilibrium gating relationships. Co-expression of 3 and 1/3, but not 1, slowed recovery from inactivation at –90 mV. Development of inactivation at –70 and –50 mV was accelerated by -subunit co-expression alone and in combination. -Subunit co-expression also reduced the late Na current measured at 200 ms. In conclusion, -subunits modulate Na_v1.5 gating with important differences between co-expression of 1 and 3 alone and 1/3 together.     

Synoviocytes in chronic synovitis in situ and cytokine stimulated synovial cells in vitro neo-express α1, α3 and α5 chains of β1 integrins

The expression of the 1 integrins was examined immunohistochemically in synoviocytes from normal synovial membrane and from chronic synovitis of different aetiology and intensity. Normal synoviocytes were 61-positive but lacked 1 through 5. In mild inflammation type A synoviocytes neo-expressed 1, 3, and 5 chains. In severe inflammation both type A and B synoviocytes expressed 3, 4, 5, and 6 chains. The effects of inflammatory cytokines, as single agents or in combination, on the 1 integrin expression in cultured normal synoviocytes was determined by immunocytochemistry and flow cytometry. The 1 chain, while absent in unstimulated synoviocytes, was induced by interleukin-1 (IL-1), tumour necrosis factor- (TNF-), and interferon- (INF-). This effect was enhanced by combining IL-1 and TNF-. Expression of the 3 chain was up-regulated by IL-1 and, more intensely, by IFN-. Transforming growth factor (TGF-) inhibited the up-regulating effect of IL-1 and antagonized the effect of IFN- on 3 chain expression. Expression of the 5 chain was up-regulated significantly by co-stimulation through IL-1 together with TGF- or TNF-. Thus, the 1 integrin profile of cytokine activated synoviocytes in vitro resembled that of synoviocytes in synovitis in situ. These data suggest that IL-1, TNF-, IFN-, and TGF- are likely to be among the effectors regulating 1 integrin expression in synoviocytes in vivo.     

The nonselective cation channel in the basolateral membrane of rat exocrine pancreas

Nonselective Ca²⁺-sensitive cation channels in the basolateral membrane of isolated cells of the rat exocrine pancreas were investigated with the patch clamp technique. With 1.3 mmol/l Ca²⁺ on the cytosolic side, the mean openstate probabilityP _o of one channel was about 0.5. In insideout oriented cell-excised membrane patches the substances diphenylamine-2-carboxylic acid (DPC), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and 3,5-dichlorodiphenylamine-2-carboxylic acid (DCDPC) were applied to the cytosolic side. These compounds inhibited the nonselective cation channels by increasing the mean channel closed time (slow block). 100 mol/l of NPPB or DPC decreasedP _o from 0.5 (control conditions) to 0.2 and 0.04, respectively, whereas 100 mol/l of DCDPC blocked the channel completely. All effects were reversible. 1 mmol/l quinine also reducedP _o, but in contrast to the abov mentioned substances, it induced fast flickering. Ba²⁺ (70 mmol/l) and tetraethylammonium (TEA⁺; 20 mmol/l) had no effects. We investigated also the stilbene disulfonates 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS), 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) and 4,4-dinitro-2,2-stilbenedisulfonate (DNDS). 10 mol/l SITS applied to the cytosolic side increasedP _o from 0.5 to 0.7 and with 100 mol/l SITS the channels remained nearly permanently in its open state (P _o1). A similar activation of the channels was also observed with DIDS and DNDS. These effects were poorly reversible. The stilbene disulfonates acted by increasing the channel mean open time. When the channel was inactivated by decreasing bath Ca²⁺ concentration to 0.1 mol/l, addition of 100 mol/l of SITS had no effect. Similarly, reducing bath Ca²⁺ concentration from 1.3 mmol/l in presence of 100 mol/l SITS (channels are maximally activated) to 0.1 mol/l, inactivated the channels completely. These results demonstrate, that SITS can only activate the channels in the presence of Ca²⁺. SITS had no effects, when applied to the extracellular side in outside out patches. In summary, the substances DPC, NPPB and DCDPC inhibit nonselective cation channels, where DCDPC has the most potent and NPPB the smallest effect; whereas SITS, DIDS and DNDS activate the channel when applied from the cytosolic side in the presence of Ca²⁺ ions.     

Synergistic effects of tumour necrosis factor-α and interferon-γ on feline haemopoietic progenitors

Pathogenic mechanisms that underlie feline leukaemia virus subgroup-C (FeLV-C) induced erythroid aplasia are unknown. FeLV-C infection is associated with higher serum levels of interferon- (IFN-) and tumour necrosis factor- (TNF-), which may act synergistically to cause haemopoietic suppression. In the present studies, the synergistic effects of TNF- and IFN- on feline bone marrow progenitors in vitro were evaluated. Bone marrow mononuclear cells from specific-pathogen-free cats were exposed to TNF- (100 and 200 pg/ml) and IFN- (100 or 200 units/ml), alone or in combination, for 2 h before plating for clonal assays of colony forming units. Our results show that TNF- and IFN- in combination caused marked suppression of feline colony forming units-erythroid (CFU-E), burst forming units-erythroid (BFU-E), and colony forming units-fibroblasts (CFU-F), whereas colony forming units-granulocyte/macrophage (CFU-GM) were minimally affected. The same concentrations of TNF- and IFN- alone had minimal effects on CFU-E, BFU-E and CFU-F. These results suggest that TNF- and IFN- may play a significant role in regulating haemopoiesis in cats and may be involved in the pathogenesis of erythroid aplasia in cats infected with feline leukaemia virus.     

Analysis of γδ+ T cells in peripheral blood of children with perinatal human immunodeficiency virus (HIV) infection

The present study examined CD8 antigen expression and variable (V) gene segment usage by T cell receptor (TCR)-⁺ lymphocytes in peripheral blood of symptomatic children with perinatal HIV infection. The relative number of ⁺, CD8⁺ T cells in most of the infected children was higher than that in uninfected children from HIV⁺ or HIV^– mothers and correlated with the immunodeficiency status of the patients. Infected infants and children over 1 year old also showed an increased proportion of V1-J1⁺ T lymphocytes. CD8 expression on those cells was higher in infected than in uninfected infants and children. Sequence analysis of the gene rearrangement of the predominant V1 family in peripheral blood of three HIV⁺ donors revealed extensive junctional diversity. These results suggest that the V skewing in the majority of HIV⁺ children reflects peripheral expansion of V1-J1⁺ T lymphocytes early in life, which might be involved in the mechanisms of HIV-induced immunodeficiency.     

Transient Exposure of Human Bronchial Epithelial Cells to Cytokines Leads to Persistent Increased Expression of ICAM-1

Effects of several cytokines on kinetics of Intercellular Adhesion Molecule-1 (ICAM-1) and Vascular Cell Adhesion Molecule-1 (VCAM-1) expression were studied on a bronchial epithelial cell line (BEAS-2B). VCAM-1 was neither constitutively expressed on BEAS-2B cells nor induced by Interferon-gamma (IFN-), Tumor Necrosis Factor-alpha (TNF-), Interleukin-1beta (IL-1), IFN-, IL-4, IL-6, IL-8 or Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF). ICAM-1 was constitutively expressed on BEAS-2B cells. IFN- and TNF- upregulated ICAM-1 expression on these cells. The functional importance of IFN- plus TNF- upregulation of ICAM-1 expression on BEAS-2B cells was demonstrated by neutrophil-BEAS-2B cell adhesion assays. Cytokines are rapidly released and cleared in animals. Therefore, transient cytokine(s) exposure might occur on the bronchial mucosa. Brief incubation of BEAS-2B cells with IFN- plus TNF- led initial upregulation of ICAM-1 expression followed by a protracted downregulation. Our findings stress the importance of studying the mechanism(s) controlling the persistent increased expression of ICAM-1 after brief cytokine(s) exposure.     

Differential Regulation of IL-8 by IL-1β and TNFα in Hyaline Membrane Disease

Mechanisms that regulate cytokine-mediated inflammation in the lungs of preterm infants, including factors which regulate production of the chemokine IL-8, remain poorly defined. Sequential bronchoalveolar lavage samples were obtained from preterm newborns with hyaline membrane disease over a 28-day period. Bronchoalveolar lavage cell cytokine relationships were evaluated and the differential regulation of IL-8 by IL-1 and TNF was studied in a short-term culture system. In vivo, IL-8 and IL-l protein levels correlated closely with each other and with macrophage counts. In cell culture, exogenous anti-IL-1 antibody led to a 40% maximum inhibition (approximately) of IL-8 production by lipopolysaccharide stimulated lung inflammatory cells. Comparable amounts of exogenous anti-TNF antibodies achieved a 15% maximum inhibition (approximately) of IL-8 production. Anti-IL-1 and anti-TNF antibodies in combination did not inhibit IL-8 production beyond that achieved by anti-IL-l antibody alone. These results, in preterm newborns, support the concept of lung inflammation mediated in part by a macrophage, IL-1, and IL-8 cell cytokine pathway. The results also suggest that factors other than IL-1 and TNF regulate IL-8 expression in the lungs of preterm infants.     

Block of non-L-, non-N-type Ca2+ channels in rat insulinoma RINm5F cells by ω-agatoxin IVA and ω-conotoxin MVIIC

The high-voltage-activated (HVA) Ba²⁺ currents of rat insulinoma RINm5F cells insensitive to dihydropyridines (DHP) and -conotoxin GVIA (-CTx-GVIA) have been studied for their sensitivity to -agatoxin-IVA (-Aga-IVA) and -CTx-MVIIC. Blockade of HVA currents by -Aga-IVA was partial (mean 24%), reversible and saturated around 350 nM (half block 60 nM). Blockade by -CTx-MVIIC was more potent (mean 45%), partly irreversible and saturated above 3 M. The effects of both toxins were additive with that of nifedipine (5 M) and were more pronounced at positive potentials. -Aga-IVA action was additive with that of -CTx-GVIA (3 M) but was largely prevented by cell pre-treatment with -CTx-MVIIC (3 M). In contrast, -CTx-MVIIC block was attenuated by -CTx-GVIA treatment ( 15%), suggesting that -CTx-MVIIC blocks the N-type ( 15%) and the non-L-, non-N-type channel sensitive to -Aga-IVA ( 30%). Consistent with this, cells deprived of most non-L-type channels by pre-incubation with -CTx-GVIA and -CTx-MVIIC exhibited predominant L-type currents that activated at more negative potentials than in normal cells (-30 mV in 5 mM Ba²⁺) and were effectively depressed by nifedipine (maximal block of 95% from -30mV to +40 mV). Our results suggest that, besides L- and N-type channels, insulin-secreting RINm5F cells possess also a non-L-, non-N-type channel that contributes significantly to the total current ( 30%). Although the pharmacology of this channel is similar to Q-type and ₁ class A channels, its range of activation (>-20 mV) and its slow inactivation time course resemble more that of N- and P-type channels. The channel is therefore referred to as Q-like.     

Synchronous appearance of fibronectin,integrin α5β1, vinculin and actin in epithelial cells and fibroblasts during rat tracheal wound healing

The distribution of integrin 51 (51) and associated components during wound healing was investigated in the rat trachea following mechanical injury. Under anesthesia, the ventral surface of the trachea was scratched, and tissue specimens were obtained from 6 h to 3 weeks after injury and studied using light and electron microscopy and immunohistochemistry. 51, vinculin and actin in regenerating epithelial cells and extracellular fibronectin appear virtually simultaneously after injury (from 12 h to 7 days) as do 51, vinculin and -smooth muscle actin in fibroblasts and cellular fibronectin in granulation tissue (from 3 to 10 days). Immunoelectron microscopy 2 days after injury showed that 51 and vinculin were localized on the basal and lateral surfaces of regenerating epithelial cells and fibroblast surfaces, and fibronectin was localized just under the regenerating epithelial cells, around collagen fibrils and sporadically around fibroblasts. Bromodeoxyuridine labeling showed that the appearance of these components was associated with the period of cell proliferation. The appearances of fibronectin, 51, vinculin and actin in regenerating epithelial cells and fibroblasts during tracheal wound healing are well coordinated. During the initial cell migration phase, plasma fibronectin may stimulate cell migration before cellular fibronectin is produced in situ, and regenerating epithelial cells appear to begin to migrate into the wound before cell proliferation starts.     

Is there a role for the tumor cell integrin αIIbβ3 and cytoskeleton in tumor cell-platelet interaction?

In vitro tumor cell-platelet interaction was examined using B16 amelanotic (B16a) melanoma cells. These tumor cells express the _IIb3-type cytoadhesin. Aggregation studies demonstrated that tumor cell surface _IIb3 mediates the recognition of platelets since pretreatment of tumor cells with antibody against _IIb3 prevents platelet-tumor cell interaction as well as platelet activation measured by aggregometry, platelet eicosanoid metabolism and ultrastructural analysis. In B16a cells, disruption of the microfilaments and intermediate filaments inhibits mobility of _IIb3 on the cell surface. Microtubules do not play a role in receptor mobility, because B16a cells do not possess well-defined microtubules in interphase and colchicine does not affect receptor mobility. Disruption of microfilaments or intermediate filaments results in an inhibition of tumor cell-platelet interaction as evidenced by aggregometry studies and ultrastructural analysis. We suggest that platelet interaction with tumor cells begins with _IIb3-mediated receptor recognition followed by not only platelet activation but also microfilament- and vimentin intermediate filament-dependent tumor cell activation.