Studien über den Mechanismus der Immunhämolyse: Der Bindungsmodus der vierten Komplementkomponente

Zusammenfassung Die Bindung der vierten Komplementkomponente (C₄) an sensibilisierte Hammelblutzellen (EA) ist von der vorhergehenden Bindung der ersten Komplementkomponente abhängig. Das Präparat EAC₁ kann, wenn ihm C₄ in geeigneter Form angeboten wird, in den Komplex EAC_1,4 überführt werden. Hierbei verschwindet der titrierbare Gehalt der flüssigen Phase an C₄; gleichzeitig acquirieren die Zellen die Fähigkeit, mit R₄ zu lysieren. Der zeitliche Ablauf dieser Veränderung wird untersucht. Die Eeaktion zwischen EAC₁ und C₄ verläuft äußerst schnell, und ihre Geschwindigkeit wird durch die Reaktionstemperatur nicht beeinflußt. Während die Bindung von C₁ an EA nur in Gegenwart von Ca⁺⁺ erfolgt, läuft die Bindung von C₄ an EAC₄ auch in Abwesenheit von zweiwertigen Metallionen ab. Die vonLevine u.Mayer als ein Ganzes betrachtete Ca⁺⁺-abhängige Überführung von EA in EAC_1,4 besteht mithin aus zwei aufeinanderfolgenden Teilreaktionen, von denen die erste (C₁-Bindung) calciumabhängig und die zweite (C₄-Bindung) von zweiwertigen Ionen unabhängig ist.Mit Unterstützung der Deutschen Forschungsgemeinschaft sowie der Gesellschaft der Freunde und Förderer der Medizinischen Akademie Düsseldorf.     

Examination of a purified A′ influenza virus preparation by potentiometry

Summary The potentiometric titration of a purified influenza A virus preparation revealed 100.7×10^–4 M base and 68.8 × 10^–4 M acid-binding capacity per g. of virus protein N. The titration curve was characterized by the following fourpK values:pK ₁ = 3.37;pK ₂ = 4.50;pK ₃ = 6.37, andpK ₄ = 9.75. The isoionic point was at pH 5.43.Attempt was made to identify the dissociating groups and it was found that the carboxylic groups (pK ₁ andpK ₂) may either be glutamyl or aspartyl groups, while the cationic groups are probably the imidazolium of histidine (pK ₃) and the -amino residues of lysine (pK ₄).Inaotivation of the hemagglutinating activity of the virus preparation by mild treatment with formaldehyde at pH 8.0 resulted in a simultaneous disappearence of the -amino groups of lysine (pK ₄). The same treatment at pH 9.0 resulted in the loss of all the cationic groups previously demonstrable.The possible role of the stable positive charges on the surface of the virus at physiological pH is discussed from the point of view of the physico-chemistry of the hemagglutination.     

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.     

Repetitive sequences in the ITS1 region of ribosomal DNA in congeneric microphallid species (Trematoda: Digenea)

In searching for species-specific DNA sequences of microphallid species (Digenea, Trematoda) we examined the ribosomal internal transcribed spacer regions (ITS) of three closely related species (Levinseniella group) hosted by mud snails (first intermediate host) and marine crustaceans (second intermediate host). In the ITS1 region we found consistent patterns of repeating sequences of 130 bp. Within each main repeat there was a varying number of subrepeats specific for each of the species. All repeats including subrepeats were identified by a similar starting sequence: 5-CCTGTGG-3. As this sequence has close resemblance to the chi sequence 5-GCTGGTGG-3 found in phage we speculate if it serves the same function as a recombination hotspot. Alternatively but less likely, it could be an inactive, mutational relic of a sequence that once served this purpose.     

Production of AMP and adenosine in the interstitial fluid compartment of the isolated perfused normoxic guinea pig heart

The pathway of production of AMP and adenosine in the myocardial interstitial fluid compartment was studied in the isolated perfused normoxic guinea pig heart by collecting the transmyocardial effluent (t.m.e.) with the method of De Deckere and Ten Hoor (1977). Besides adenosine and inosine, AMP was found in t.m.e. Infusion of ,-methylene adenosine 5-diphosphate (AOPCP), a specific inhibitor of the ecto 5-nucleotidase, resulted in increases in t.m.e. AMP and inosine and a decrease in adenosine. Infusion of acetate producing a nearly twofold increase in myocardial AMP content did not increase the t.m.e. AMP even in the presence of AOPCP. In preparations made from 6-OH dopamine treated animals, the t.m.e. adenosine and inosine were reduced and AOPCP produced smaller increases in AMP and inosine, indicating that most if not all of the t.m.e. AMP originated from the sympathetic nerve terminals. Infusions of ,-imidoadenosine and ,-methylene adenosine 5-triphosphate (AMPPNP and AMPPCP), non-hydrolysable analogs of ATP, resulted in dose-dependent increases in the t.m.e. AMP, which were much augmented in the presence of AOPCP. AMPPNP produced similar effects in 6-OH dopamine-treated preparations. As AMPPNP and AMPPCP are good substrates of ATP pyrophosphohydrolase, these findings indicate the presence of ATP pyrophosphohydrolase in the myocardial interstitial space.     

Effect of analogs of cinanserin on human lymphocytes in vitro

Cinanserin, 2-(3-dimethylaminopropylthio)cinnamanilide, was compared in phytohemagglutinin (PHA)-treated human lymphocytes in vitro to two analogs, 2-(3-dimethylaminopropoxy)-5-methylcinnamanilide (SQ 11,276) and 5-tert.-butyl-2-(3-dimethylaminopropoxy) cinnamanilide (SQ 11,332). These analogs, like cinanserin, exhibit immunosuppressive activity but, unlike cinanserin, possess low antiserotonin activity.Within 1–1.5 hours after addition of the drugs (0.15 mM), incorporation of³H-uridine,¹⁴C-leucine, and³H-thymidine into a macromolecular fraction of PHA-treated cells were inhibited, in an increasing order, by cinanserin, SQ 11,276 and SQ 11,332. At this concentration cellular recovery and viability wre unaltered by cinanserin and SQ 11,276 and substantially decreased by SQ 11,332. SQ 11,332 was less cytotoxic at lower concentrations and 4–7 times more active than cinanserin as an inhibitor of the incorporation of the nucleosides into the macromolecular fraction.     

3′-Deoxy-3′-fluoroinosine as a potent antileishmanial agent

We studied the antileishmanial activity of 3-deoxy-3-fluoroinosine (3-FI) againstLeishmania tropica andL. donovani. In in vitro cultivation, the EC₅₀ values (the concentration of drug necessary to inhibit the growth rate of cells to 50% of the control value) obtained for 3-FI against the promastigotes ofL. tropica andL. donovani were 2.3×10^–7 and 1.0×10^–6 M, respectively. It was less toxic toward mouse mammary-tumor FM3A cells, a model host; the EC₅₀ value was 1.9×10^–4 M. Leishmania promastigote metabolized 3-FI to 3-deoxy-3-fluoroadenosine 5-triphosphate (3-FATP) but FM3A cells did not. 3-FI was effective againstL. donovani amastigotes in J774.1 cells in an in vitro cultivation system under conditions similar to those used in the in vivo assay. 3-FI (50 mg/kg, given i.v.)showed a cytotoxic effect against the amastigotes ofL. donovani in mice.     

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.

---

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     

Translation enhancer in the 3′-untranslated region of rotavirus gene 6 mRNA promotes expression of the major capsid protein VP6

Summary. The eleven rotavirus mRNAs contain 5-cap structures and most end with the 3-consensus sequence 5-UGACC-3. The UGACC functions as a common translation enhancer (3-TE-con) that upregulates viral protein expression through a process mediated by the nonstructural protein NSP3. To address the possibility that gene-specific enhancers are also contained in the untranslated regions (UTRs) of the rotavirus mRNAs, we used rabbit reticulocyte lysates to investigate the translation efficiencies of analog RNAs containing viral-specific 5-and 3-UTRs and the open reading frame for chloramphenicol acetyltransferase. These experiments combined with the analysis of full-length viral RNAs and RNAs containing 3-truncations showed that a highly active enhancer was present near the 5-end of the 139-nucleotide 3-UTR of the gene 6 mRNA (3-TEg6). The 3-TEg6 represents a functionally independent enhancer, as no other portion of the gene 6 mRNA was required for its activity. The 3-TEg6 differs significantly from the 3-TE-con in that the gene 6-specific enhancer does not require viral protein for activity and is formed by a sequence unique to only one of the eleven viral mRNAs. Together, our findings suggest that the 3-UTR of the gene 6 mRNA contains two TEs, one is gene-specific (3-TEg6) and the other is common to nearly all rotavirus genes (3-TE-con). The activity of the 3-TEg6 is likely important for directing the efficient translation of the gene 6 mRNA at levels sufficient to provide the 780 copies of VP6 necessary for the assembly of each progeny virion.     

Chimeric organization of two genes for the soybean mitochondrial ATPase subunit 6

Summary There are two copies of the ATPase subunit 6 (atp6) gene in the soybean mitochondrial genome which differ in their gene organization but share extensive homology with the maize atp6 gene except at their 5 ends. The two soybean genes are chimeric, containing regions with homology to other known mitochondrial genes at their 5 ends. Sequences homologous to the cytochrome oxidase subunit II (coxII) are located in one copy and sequences homologous to the ATPase subunit 9 (atp9) gene are located in the other copy, both of which contain methionine (ATG) codons that are in-frame with the remainder of the atp6 open reading frame. At least the copy of atp6 that contains the coxII sequence at its 5 end is abundantly transcribed to give an RNA of approximately 1,200 nucleotides.     

Rhinovirus detection using probes from the 5′ and 3′ end of the genome

Summary This study investigated the abilities of cDNA probes from the 5 and 3 ends of the genome of human rhinoviruses (HRV-) 14, 9, and 1B to detect RNA from 59 rhinovirus serotypes. The results show that probes from the 5 end of the genomes of HRV-14, 9, and 1B detected a large number of serotypes but the detection rate was variable and depended on the degree of homology with the particular probe. In contrast, all the 3 end probes were specific for the homologous virus. However, along HRV-9 probe detected a large number of serotypes.It was concluded that such cDNA probes would not detect all serotypes with equal efficiency. Synthetic oligonucleotides corresponding to short but highly conserved regions in the 5 non coding region may overcome this problem.     

DNA fingerprinting involving fluorescence-labeled termini of any enzymatically generated fragments of DNA

Summary We have developed a new fluorescence-based method for DNA fingerprinting that does not require a fluorescent linker or a synthetic oligonucleotide primer, both of which are normally used for labeling of DNA. Cosmid DNAs are digested with appropriate restriction enzymes and the 3 termini of DNA fragments are labeled with the corresponding, fluorescent dye-conjugated dideoxynucleotide triphosphate terminator (dye-ddNTP) by the Klenow fragment of DNA polymerase I fromEscherichia coli, which has 35 exonuclease and replacement activities as well as its main 53 polymerase activity. Samples are separated on a DNA-sequencing gel and data are analyzed by application of both the Version 0.3.8a mapper program (Applied Biosystem Inc., Foster City, CA) and our Overlap I program that facilitate rapid analysis of the frequency of overlapping of cosmid DNAs. Using this method we have determined the overlap frequency of DNA fragments of each cosmid clone from the mouse MHC class I gene cluster.     

Two polymorphicAvaI andHhaI sites in a differentially methylated region of the human H19 gene

Summary The H19 gene is paternally imprinted both in the human and mouse (Bartolomeiet al., 1991; Zhang and Tycko, 1992), although its expression pattern seems somewhat different between the two species (Jinno,et al., 1995). DNA-methylation is a promising candidate for a parent-of-origin mark of the gene, and a paternal allele-specific methylation imprint was recently identified at the mouse H19 locus (Tremblayet al., 1995). We found a 50% methylated region in the human H19 gene (Jinno, unpublished data). A search for polymorphisms in this region revealed two novelAvaI andHhaI RFLPs, which contribute to the detection of allele-specific methylation at the human H19 locus.PCR primers for the AvaI-site PANL2 5-GAGCCTGCCAAGCAGAGCG-3 - PANR2 5-CACATAAGTAGGCGTGACTTGA-3 PCR primers for the HhaI-site ASMA 5-CAATGAGGTGTCCCAGTTCCA-3 - PANR2 5-CACATAAGTAGGCGTGACTTGA-3     

Nucleotidstoffwechsel und Magensäuresekretion

Zusammenfassung Es wird die Frage geprüft, inwieweit Hormone und Pharmaka, die in verschiedenen Geweben die Konzentration von Adenosin-3,5-cyclischem-monophosphat (cyclischem AMP) verändern können, die Bildung von Magensäure beim Menschen während maximaler Säurestimulation beeinflussen. Durch intravenöse Injektion von Stoffen, die strukturgebunden in verschiedenen Geweben die Aktivität von Adenylcyclase oder von 3,5-AMP-Phosphodiesterase verändern, kann nur unter bestimmten Bedingungen eine Wirkung auf die Säuresekretion am stimulierten Magen erzielt werden. Vermutlich unterliegt der Abbau von cyclischem AMP nicht nur der Wirkung 3,5-AMP-Phosphodiesterase. Verschiedene experimentelle Befunde werden im Hinblick auf die mögliche Bedeutung von cyclischem AMP bei der Bildung und Sekretion von Magensäure diskutiert. Mit einer Ausnahme stehen direkte Messungen des Gewebsgehaltes von cyclischem AMP in der Magenschleimhaut unter den beschriebenen Versuchsbedingungen noch aus. Die Fragen, ob cyclisches AMP oder eines seiner Stoffwechselprodukte Mittlersubstanz bei der Bildung von Magensäure ist und ob cyclisches AMP letzter Effektor einiger durch Hormone oder Pharmaka induzierten Änderungen der Säuresekretion durch die stimulierte Magenschleimhaut sein kann, bleiben vorläufig ungelöst.

---

Summary The possibility is reviewed in how far hormones and drugs which are known to change the intracellular level of adenosine-3,5-cyclic monophosphate (cyclic AMP) within various tissues may interfere with the formation of gastric acid in humans during maximal stimulation of gastric mucosa. Substances capable of activating or inactivating the enzymes adenyl cyclase and 3,5-AMP-phosphodiesterase in the responsive tissues may involve interference with the formation of gastric acid only in certain conditions. It is suggested that there may exist a second pathway for the metabolism of cyclic AMP except the degradation by 3,5-AMP-phosphodiesterase. Experimental findings are discussed with respect to the possibility that cyclic AMP participates on a molecular basis in the secretory mechanism of gastric acid. In defined conditions direct measurements of the content of cyclic AMP within the cells of gastric mucosa are lacking at present excepting stimulation by methylxanthines. The problem remains unsolved whether cyclic AMP or a metabolite of cyclic AMP is a mediator of gastric acid formation. It is not known whether cyclic AMP is the final effector of some hormone or drug-induced alterations of acid secretion by the stimulated gastric mucosa.

     

Mechanisms underlying facilitation by dopamine of ATP-activated currents in rat pheochromocytoma cells

Mechanisms underlying facilitation by dopamine of extracellular adenosine 5-triphosphate (ATP)-activated current were investigated in rat pheochromocytoma PC12 cells using the whole-cell voltage-clamp techniques. Dopamine (10 and 100 M) augmented the peak amplitude of an inward current elicited by ATP (3–100 M). The activation time course of the ATP-evoked current was accelerated by dopamine; the presence of 10 M dopamine shifted the dependence of activation rate constants on the concentration of ATP toward a lower concentration range two fold. Dopamine also accelerated the inactivation and the deactivation, which was determined from the current decay upon washout of ATP. Intracellular mediators responsible for the dopamine-induced facilitation was estimated by loading various compounds in patch pipettes. Facilitation was not observed when K-252a (1 M), a protein kinase inhibitor, was included in the intracellular solution. In addition, facilitation was also attenuated by intracellular adenosine 5-O-(thiotriphosphate)tetralithium salt (ATPS (1 mM) or --methylene ATP (1 mM). Inclusion of adenosine 3, 5-cyclic monophosphate sodium salt (cAMP, 100 M), guanosine 3,5-cyclic monophosphate sodium salt (cGMP, 100 M), 12-O-tetradecanoylphorbol-13-acetate (TPA, 1 M) or phorbol-12,13-dibutylate (1 M) in the intracellular solution did not affect the facilitation. Guanonsine 5-O-(thiotriphosphate)tetralithium salt (GTPS, 500 M) or guanosine 5-O(2-thiodiphosphate)-trilithium salt (GDPS, 500 M) did not modify the facilitation either. The results suggest that dopamine augments the ATP-activated inward current by facilitating association of ATP to its binding site, and that the augmentation may be mediated through some protein kinase which is different from cyclic-nucleotide-dependent protein kinases or protein kinase C.     

Studien über den Mechanismus der Immunhämolyse: Die Bindung der zweiten Komplementkomponente

Zusammenfassung Es wird die Reaktion EAC_1,4 + C₂ isoliert untersucht. Hierzu bietet man einer Charge von stufenweise aufgebautem EAC_1,4 ein von allen übrigen Komplementkomponenten befreites C₂-Präparat an. Bei der Reaktion kommt es zu einem meßbaren Schwund von C₂ im Überstand; gleichzeitig erwerben die Zellen die Fähigkeit, mit cheliertem Komplement zu lysieren. Die Reaktionsgeschwindigkeit ist bei 37° C hoch und bei 0° C gering. Für die Fähigkeit der Zellen, C₂ zu binden, ist nicht allein das gebundene C₄ maßgebend, sondern ein von C₁ nicht abgrenzbarer Zusatzfaktor. Demnach kommen dem C₁ nach seiner Bindung zwei Funktionen zu: Einmal vermittelt es die Bindung von C₄, zum anderen vermittelt es zusammen mit dem gebundenen C₄ die Bindung von C₂.Mit Unterstützung der Deutschen Forschungsgemeinschaft sowie der Gesellschaft der Freunde und Förderer der Medizinischen Akademie Düsseldorf.     

A Neurospora crassa heat-shocked cell lysate translates homologous and heterologous messenger RNA efficiently,without preference for heat shock messages

Summary Cell-free protein synthesis systems were prepared from normally-grown (N-lysate) and heat-shocked (HS-lysate) Neurospora crassa mycelium. Although both lysates translated homologous mRNA, the HS-lysate was more active, yielding a higher incorporation of [³⁵S]-methionine into hot TCA-insoluble material and a vastly superior protein synthesis profile. The optimal temperature for translation by both lysates was 21 °C; the HS-lysate did not translate heat-shock mRNA preferentially at any temperature tested. Fortuitously, heterologous messenger RNAs from diverse eukaryotic and viral sources — Drosophila, dog pancreas, rabbit globin mRNA, brome mosaic virus, tobacco mosaic virus — were translated by the HS-lysate with an efficiency comparable to that of the commercial rabbit reticulocyte system and superior to the wheat germ system. The cap analogues, m⁷G(5)ppp(5)G and m⁷G(5)ppp(5)Gm, inhibited translation significantly.     

Phosphorylation-regulated low-conductance Cl− channels in a human pancreatic duct cell line

A low-conductance Cl^– channel has been identified in the apical membrane of the human pancreatic duct cell Capan-1 using patch-clamp techniques. Cell-attached channels were activated by the vasoactive intestinal polypeptide (VIP, 0.1 mol/l), dibutyryl-adenosine 3,5-cyclic monophosphate (db-cAMP, 1 mmol/l), 8-bromo adenosine 3,5-cyclic monophosphate (8-BrcAMP, 1 mmol/l), 3-isobutyl-1-methyl-xanthine (IBMX, 100 mol/l) and forskolin (10 mol/l). No channel activity was observed in non-stimulated control cells. In both cell-attached and excised inside-out patches, the channel had a linear current/voltage relationship and a unitary conductance of 9 pS at 23°C and 12 pS at 37°C. Its opening probability was not voltage dependent although pronounced flickering was induced at negative potentials. Anionic substitution led to the selectivity sequence Cl^–>I^–>HCO₃ ^–>gluconate. In insideout excised patches, the channel activity declined spontaneously within a few minutes. Reactivation of silent excised channels was achieved by adding protein kinase A (PKA, in the presence of ATP, cAMP and Mg²⁺). Conversely, active channels were silenced in the presence of alkaline phosphatase. The PKA-activated Cl^– channel was 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS, 100 mol/l) and 4-acetamido-4-isothiocyanatostilbene-2, 2-disulphonic acid (SITS, 100 mol/l) insensitive, but was blocked by diphenylamine-2-carboxylic acid (DPC, 100 mol/l). These results demonstrate that the apical low-conductance Cl^– channel in Capan-1 is regulated on-cell by VIP receptors via cAMP and off-cell by PKA and phosphatases. They provide evidence that this channel is closely related to the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– channel.     

Vasoactive hormones and cAMP affect pericyte contraction and stress fibresin vitro

Summary Pericytes are contractile cells of the microvascular wall that may influence capillary haemodynamics and permeability. We examined the contractile responses of cultured pericytes to selected vasoactive agents and cAMP agonists. Morphological and biochemical changes associated with these responses were also studied. Pericytes seeded onto silicone rubber contracted when stimulated with histamine or serotonin, relaxed in response to the beta-adrenergic agonist isoproterenol and did not respond to epinephrine. Since hormonal-induced relaxation of vascular smooth muscle involves cAMP, we investigated the ability of cAMP, to modulate pericyte contraction. Dibutyryl cAMP and forskolin (an adenylate cyclase activator) both induced pericyte relaxation and elevated intracellular cAMP levels. Isoproterenol increased cAMP levels but epinephrine had no effect. However, when epiniphrine and isoproterenol were co-incubated with the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (IBMX), cAMP was increased to levels above those elicited by these agonists alone. Serotonin and histamine in the presence of IBMX did not affect cAMP levels. These results suggest that certain vasoactive agents may relax pericytes by cAMP-dependent processes. We have shown previously that stress fibres are also involved in pericyte contraction. Hence, changes in the staining patterns of stress fibres in response to these selected agonists were studied. Histamine, serotonin and epinephrine had no apparent effect on stress fibre staining. Dibutyryl cAMP, forskolin, and isoproterenol, which relax pericytes and increase cAMP, disassembled fibres. In summary, the results demonstrate that the contractile activity of cultured pericytesin vitro can be regulated by vasoactive agonists and that changes in cAMP and stress fibres may mediate the regulation.Abbreviations cAMP cyclic adenosine 3,5-monophosphate - dBcAMP dibutyryl cyclic adenosine 35-monophosphate - DiL-Ac-LDL 1,1-dioctadecyl 1-3,3,3,3 tetramethylindo-carbocyanine perchlorate - DME Dulbecco's Modified Eagles Medium - FCS foetal calf serum - HBSS Hanks balanced salt solution - IBMX 3-isobutyl-1-methylxanthine - PBS phosphate buffered saline - R020-1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone - TCA trichloroacetic acid     

Characterization of the hog cholera virus 5′ terminus

Hog cholera virus (HoCV) 5 terminus of the ALD and GPE(–) strains were analyzed by using rapid amplification of cDNA end method (5RACE). An additional nine nucleotides were found at the 5 termini of genomic RNA in the ALD and GPE(–) strains of HoCV. These nine nucleotides were also conserved in BVDV and were suggested to form a hairpin structure at the 5 terminus by computer-assisted analysis. It seems possible that the secondary structure and/or the 5 terminus sequence has a significant role in the HoCV virus genome.