Formation of prostanoids and hydroxy fatty acids by stimulated peritoneal mast cells: role of the dietary fat type in rat.

To study the influence of membrane fatty acid composition on the formation of prostanoids and hydroxy fatty acids by rat peritoneal mast cells (MC), animals were fed three different types of fatty acids: mackerel oil (MO), abundant in n-3 fatty acids; sunflower seed oil (SO), rich in linoleic acid; and hydrogenated coconut oil (HCO), mainly containing saturated fatty acids. The presence of n-3 fatty acids in the diet resulted in the incorporation of 20:5(n-3), 22:5(n-3) and 22:6(n-3) in MC phospholipids. A decrease of arachidonic acid, 20:4(n-6), was observed in MC-phospholipids of the MO-fed animals. Furthermore, increasing the relative amounts of 18:2(n-6) in the diet (SO group) led to an increased incorporation of linoleic acid, 18:2(n-6) in MC phospholipids when compared to both other dietary groups. The changes in MC phospholipid fatty acid composition were (partly) reflected in the formation of prostanoids and hydroxy fatty acids upon stimulation with the calcium ionophore A23187. The decrease in arachidonic acid content in MC phospholipids of MO-fed rats resulted in a decreased formation of PGD2 when compared to both other groups. Also, the increased amounts of 18:2(n-6) in MC phospholipids of SO-fed rats resulted in an increased formation of 9- and 13-HODE upon stimulation. The results show that modifications in the fatty acid composition of the diet influences MC membrane fatty acid composition which ultimately results in changes in prostanoid and hydroxy fatty acid synthesis by MC upon stimulation with the calcium ionophore A23187.

Differential release of histamine and prostaglandin D2 in rat peritoneal mast cells: roles of cytosolic calcium and protein tyrosine kinases.

We studied how the release of histamine and prostaglandin D2 (PGD2) were connected in stimulated rat peritoneal mast cells, and to what extent these processes were controlled by the cytosolic Ca2+ concentration, [Ca2+]i, and protein tyrosine kinases. In the presence of 1 mM CaCl2, the G-protein activating compound 48/80 (10 micrograms/ml) evoked a transient rise in [Ca2+]i and a relatively high secretion of histamine, but only a low release of PGD2. In contrast, 5 microM thapsigargin (an inhibitor of endomembrane Ca(2+)-ATPases) and 5 microM ionomycin evoked high and prolonged rises in [Ca2+]i, and stimulated the cells to release relatively small amounts of histamine and high amounts of PGD2. Stimulation of the cells with CaCl2 and 10 microM ATP4- gave only minor quantities of histamine and PGD2, despite of the micromolar level of [Ca2+]i reached. When CaCl2 was replaced by EGTA, rises in [Ca2+]i as well as release of histamine and PGD2 were reduced with each agonist, but the preference of agonists to release more histamine or PGD2 remained unchanged. In mast cells with depleted Ca2+ stores, the addition of CaCl2 stimulated the store-regulated Ca2+ entry resulting in a prolonged rise in [Ca2+]i. However, simultaneous addition of compound 48/80 and CaCl2 was required for release of histamine and PGD2. In cells with full stores, PGD2 release evoked by compound 48/80 was greatly reduced by genistein and methyl-2,5-dihydroxycinnamate, two structurally unrelated inhibitors of protein tyrosine kinases, whereas histamine secretion was not influenced by these inhibitors. Similarly, with thapsigargin or ionomycin as agonist, PGD2 release was more sensitive to the tyrosine kinase inhibitors than histamine secretion. We conclude that in activated rat peritoneal mast cells: (i) the influx of extracellular Ca2+ potentiates agonist-evoked rises in [Ca2+]i as well as histamine secretion and PGD2 release; (ii) the amplitude of the [Ca2+]i rise does not determine the preferential effect of agonists to release more histamine or more PGD2; (iii) the relatively high PGD2 release evoked by thapsigargin and ionomycin is probably due to their potency to evoke a prolonged rise in [Ca2+]i and to activate protein tyrosine kinases.

Radioimmune assays and molecular studies that place Anopheles B and Turlock serogroup viruses in the Bunyavirus genus (Bunyaviridae).

Molecular analyses indicate that Turlock virus (TUR, Turlock serogroup) and Boraceia virus (BOR, Anopheles B serogroup) have virion RNA species and polypeptides comparable in size to those of members of the Bunyavirus genus and unlike those of members of the newly defined Phlebovirus, Nairovirus, or Uukuvirus genera (Bunyaviridae). The 11 terminal 3' end nucleotides of the three virion RNA species of both BOR and TUR viruses (HOUCAUCACAUG...) are identical in sequence to the 3' end sequences of the viral RNA species of snowshoe hare (SSH) and La Crosse bunyaviruses (LAC, California serogroup, Bunyavirus genus). Competition radioimmune assays (RIA), using iodinated LAC nucleocapsid polypeptide (N), or LAC glycoproteins (G1, G2), and LAC rabbit hyperimmune antisera, or iodinated Oriboca (ORI, Group C, Bunyavirus genus) N, or G1 and G2 polypeptides and LAC antisera, or iodinated Bunyamwera (BUN, Bunyamwera serogroup, Bunyavirus genus) N, or G1 and G2 polypeptides and BUN or LAC antisera, have indicated that the virion polypeptides of BOR virus share antigenic determinants with these other bunyaviruses. Competition RIA analyses also have shown that TUR virus shares antigenic determinants with LAC virus. The competition RIA analyses have confirmed the antigenic relationships of LAC, SSH, trivittatus, Bwamba, Aino, Simbu, Mermet, Guaroa, Lumbo, Tahyna, ORI, Anopheles A, BUN, Capim, Guama and Shark river viruses (Bunyavirus genus members), and lack of antigenic relationships between Karimabad, or Chagres, or sandfly fever, Sicilian, Viruses (Phlebovirus genus members), and the bunyaviruses, LAC, ORI, or BUN.

Genomic and biologic analyses of snowshoe hare virus field and laboratory strains.

Low-passage field strains of snowshoe hare (SSH) virus (Bunyaviridae), the prototype SSH virus (originally isolated in Montana), and La Crosse (LAC) virus were compared serologically by plaque-reduction neutralization (PRNT) and molecularly by oligonucleotide fingerprinting (ONF). The PRNT and ONF results confirmed the identity of the field strains, although some differences in the fingerprints were observed. We have examined the RNA genome variability in the two field and three laboratory strains of SSH virus, using direct sequence analysis of selected RNase T1 oligonucleotides. Few changes were observed among three Montana prototype-derived laboratory isolates, although they have different passage histories. In contrast, the field isolates differed greatly from the laboratory strains. In addition, we have located several of the larger T1 oligonucleotides within the known sequence of the small and large RNA genome segments. We then compared the viruses for their ability to replicate in and be transmitted by Aedes triseriatus mosquitoes. The oral infection rates for LAC, the field isolates, and the SSH prototype, as determined by immunofluorescent examination of midgut tissues, were 100%, 82%, and 47%, respectively. All viruses were also transmissible from mosquitoes to mice.

3'-Terminal sequences of influenza C virion RNA. Brief report.

The 3'-terminal nucleotides of the genome segments of influenza C/Taylor/47. C/Bavaria/79 and C/Johannesburg/1/66 were identified by two RNA sequencing techniques. These comprised 11 nucleotides (3' UCGUCUUCGUC) which were found to be conserved among the genome segments of each virus.

Oligonucleotide sequence analyses indicate that vesicular stomatitis virus large defective interfering virus particle RNA is made by internal deletion: evidence for similar transcription polyadenylation signals for the synthesis of all vesicular stomatitis virus mRNA species.

RNase T(1) oligonucleotide fingerprint analyses of three vesicular stomatitis virus Indiana serotype small defective interfering (DI) particle RNA species indicate that they only have oligonucleotides derived from the 5' region of the viral genome. These studies also indicate that these three DI RNAs have partial L gene sequences as well as two 5' viral oligonucleotides (59 and 70) that are not transcribed into L (or other) mRNA species (J. P. Clewley and D. H. L. Bishop, J. Virol. 30:116-123, 1979). Analyses of the large DI RNA (LT DI) reveal a different origin. The LT DI RNA has oligonucleotides derived from both the 3' end of the genome (including all the large oligonucleotides identified for N, NS, M, and G genes), in addition to at least one of the 5'-proximal L gene oligonucleotides (47), as well as all seven oligonucleotides (3, 38, 42, 43, 44B, 59, and 70) that are not protected from nuclease digestion after the formation of mRNA-viral RNA duplexes (Clewley and Bishop). It appears therefore that the genesis of LT RNA involves a deletion of internal L gene sequences from the viral RNA. Oligonucleotide sequence analyses have been undertaken on several of the vesicular stomatitis viral RNA oligonucleotides, including all seven (3, 38, 42, 43, 44B, 59, and 70) that are not transcribed into mRNA. The analyses confirm that oligonucleotides 59 [3'...GAACACCAAAAAUAAAAAAUA(G)...5'] and 70 [3'...GACCAAAACACCA(G)...5'] are at the 5'-end region of the viral genome. Oligonucleotide 38 [3'...GAAAUUCAUACUUUUUU(U)(G)...5'] may represent the termination signal for L mRNA synthesis (R. A. Lazzarini, personal communication). Oligonucleotide 43 [3'...GUAUACUUUUUUU(G)...5'] corresponds to the sequence shown to be the N gene mRNA polyadenylation signal (D. J. McGeoch, Cell 17:673-681, 1979). The other three oligonucleotides share a common feature with oligonucleotides 43 and 38, viz., a stretch of 6 or 7 U residues preceded by an AUAC sequence. Thus the sequence of oligonucleotide 3 is 3'...GAAUUAAUAUAAAAUUAAAAAUUAAAAAUACUUUUUU(U)(G)...5', whereas that of oligonucleotide 42 is 3'...GAUACUUUUUUUCAU(U)(G)...5', and that of oligonucleotide 44B is 3'...G(U)AUACUUUUUU(G)...5'. These sequence analyses suggest a common polyadenylation signal for the synthesis of all vesicular stomatitis virus mRNA species, i.e., the sequence (3')...AUACUUUUUU(U)...(5').

Antigen-evoked mast cell degranulation in the isolated rat heart: no effect on subsequent ischemia-reperfusion induced damage.

In the present study, the possible role of mast cells in ischemia/reperfusion-induced myocardial injury was evaluated in the isolated 'mast cell depleted' rat heart. Hearts isolated from sensitized and non-sensitized rats were perfused according to Langendorff. After 30 min of normoxic perfusion, hearts were challenged with antigen, a procedure which is known to result in a massive mast cell degranulation in sensitized hearts. After another 20 min, both 'mast cell depleted' and control hearts were subjected to 30 min of ischemia followed by 30 min of reperfusion. The release of lactate dehydrogenase (LDH) was determined, to quantitate the extent of irreversible injury of cardiomyocytes. Histamine release was measured to establish mast cell degranulation. Coronary flow (CF) and left ventricular developed pressure (LVDP) were monitored to study the consequences of the procedures on hemodynamic recovery. It was found that both CF and LVDP significantly increased during the first min after antigen challenge. These changes were accompanied by an almost complete degranulation of cardiac mast cells. The increase in CF and LVDP values were rapidly followed by a decrease, reaching minimal values of 159 +/- 4% and 85 +/- 4% of those before administration of antigen, respectively, at 2-3 min after antigen challenge. No effect of antigen challenge on LDH release were found indicating that mast cell degranulation did not compromise myocyte integrity. During reperfusion following 30 min of ischemia both the increase in CF and LVDP in 'mast cell-depleted' hearts were not significantly different from those in control (non-sensitized) hearts. Similarly, at the end of the reperfusion-phase, CF and LVDP values in sensitized hearts were comparable to those in control hearts. Reperfusion results in increased LDH release, which at no point in time was significantly different between sensitized and non-sensitized hearts. In non-sensitized hearts histamine release during the reperfusion phase was not detectable. Therefore, the results indicate that in the isolated rat heart, mast cells are most likely not involved in acute ischemia/reperfusion-induced myocardial injury.

Host and virus specific RNA polymerases in alfalfa mosaic virus infected tobacco.

The particulate fraction of a homogenate of alfalfa mosaic virus infected tobacco was found to contain the viral replicase, and one or more host-specific RNA-polymerases. Using an endogeneous template, the membrane-bound replicase incorporates 3H-CTP in virion-type RNA. The synthesized RNA is part of a replicative intermediate; after RN-ase-treatment the product comigrates with viral double-stranded RNA in polyacrylamide gels. A preliminary characterization of this double-stranded RNA was made by RNA-RNA hybridization. Treatment of the particulate fraction with lubrol releases a host-specific RNA-polymerase. The activity of this enzyme is completely dependent on exogeneous template-RNA. Probably only a very small region of the template is transcribed. After washing with lubrol, the particulate fraction still contains the viral replicase. When this fraction is resuspended in a Mg++-deficient buffer, about 60% of the enzyme activity is released into the supernatant. No such activity is found in a comparable extract of healthy leaves.

The complete sequence and coding content of snowshoe hare bunyavirus small (S) viral RNA species.

The complete sequence of the small (S) viral RNA species of snowshoe hare (SSH) bunyavirus has been determined, principally from a DNA copy of the RNA cloned in the E.coli plasmid pBr322. The viral S RNA (negative sense strand) is 982 nucleotides long (3.3 x 10(5) daltons) with complementary 5' and 3' end sequences. It has a base composition of 30.5%U, 25.8%A, 24.9%C and 18.7%G. In the viral complementary (plus sense) strand there are two overlapping open reading frames initiated by methionine codons. One reading frame codes for a 26.8 x 10(3) dalton protein, the other for a 10.5 x 10(3) dalton protein. The larger gene product is presumably related to the viral nucleoprotein (N) that is coded by the S RNA (Gentsch and Bishop (1978) J. Virol. 28, 417-419). The smaller gene product is probably related to the recently identified S RNA coded nonstructural protein (NSS) induced in virus infected cells (Fuller and Bishop (1982) J. Virol. 41, 643-648).

Nucleotide sequence analyses and predicted coding of bunyavirus genome RNA species.

We performed 3' RNA sequence analyses of [(32)P]pCp-end-labeled La Crosse (LAC) virus, alternate LAC virus isolate L74, and snowshoe hare bunyavirus large (L), medium (M), and small (S) negative-stranded viral RNA species to determine the coding capabilities of these species. These analyses were confirmed by dideoxy primer extension studies in which we used a synthetic oligodeoxynucleotide primer complementary to the conserved 3'-terminal decanucleotide of the three viral RNA species (Clerx-van Haaster and Bishop, Virology 105:564-574, 1980). The deduced sequences predicted translation of two S-RNA gene products that were read in overlapping reading frames. So far, only single contiguous open reading frames have been identified for the viral M- and L-RNA species. For the negative-stranded M-RNA species of all three viruses, the single reading frame developed from the first 3'-proximal UAC triplet. Likewise, for the L-RNA of the alternate LAC isolate, a single open reading frame developed from the first 3'-proximal UAC triplet. The corresponding L-RNA sequences of prototype LAC and snowshoe hare viruses initiated open reading frames; however, for both viral L-RNA species there was a preceding 3'-proximal UAC triplet in another reading frame that was followed shortly afterward by a termination codon. A comparison of the sequence data obtained for snowshoe hare virus, LAC virus, and the alternate LAC virus isolate showed that the identified nucleotide substitutions were sufficient to account for some of the fingerprint differences in the L-, M-, and S-RNA species of the three viruses. Unlike the distribution of the L- and M-RNA substitutions, significantly fewer nucleotide substitutions occurred after the initial UAC triplet of the S-RNA species than before this triplet, implying that the overlapping genes of the S RNA provided a constraint against evolution by point mutation. The comparative sequence analyses predicted amino acid differences among the corresponding L-, M-, and S-RNA gene products of snowshoe hare virus and the two LAC virus isolates.

Analyses of Patois group bunyaviruses: evidence for naturally occurring recombinant bunyaviruses and existence of immune precipitable and nonprecipitable nonvirion proteins induced in bunyavirus-infected cells.

Shark River (SR) and Pahayokee (PAH) bunyaviruses (Patois serogroup, Bunyavirus genus, family Bunyaviridae) have almost identical L and S RNA oligonucleotide fingerprints, but M RNA fingerprints that are different, suggesting that the two viruses may represent naturally occurring reassortant viruses. These observations are in agreement with serological studies (B. N. Fields, B. E. Henderson, P. H. Coleman, and T. H. Work, 1969, Amer. J. Epidemiol., 89, 222-226) which have distinguished these two viruses by neutralization of infectivity tests (presumably reflecting differences in M RNA gene products, J. R. Gentsch, E. J. Rozhon, R. A. Klimas, L. H. El Said, R. E. Shope, and D. H. L. Bishop, 1980, Virology102, 190-204), but not by complement fixation tests (which probably relate to the viral N polypeptide coded by the S RNA, J. Gentsch, L. R. Wynne, J. P. Clewley, R. E. Shope, and D. H. L. Bishop, 1977b, J. Virol. 24,893-902). The 3' terminal 11 nucleotides of PAH S RNA (3' (HO)OUCAUCAAAUGA ... 5') are identical in sequence to those of the S RNA species of snowshoe hare (SSH) and La Crosse (LAC) viruses, except for a position 7A residue which is a C residue in the SSH and LAC sequences. The major virion polypeptides of SR and PAH viruses include a nucleocapsid polypeptide (N, 22 x 10(3)) and two glycoproteins (PAH: G1 118 x 10(3), G2, 35 x 10(3); SR: G1 113 x 10(3), G2, 35 x 10(3)). In SR-infected cells several immune precipitable polypeptides have been detected. These include 11-, 54-, 64-, 93-, and 104 x 10(3)-dalton polypeptides. In addition, both SR and PAH viruses induce a 74 x 10(3)-dalton polypeptide (p74) that has not been detected in actinomycin D-treated infected cells, and is not immune precipitated from infected cell extracts.

Dietary modulation of fatty acid composition of mast cell phospholipids does not affect histamine release induced by compound 48/80.

OBJECTIVE AND DESIGN: In the present study we determined the extent to which the degranulation process in mast cells was related to the fatty acid composition of membrane phospholipids. MATERIAL: Peritoneal mast cells were isolated from Wistar rats (3 groups of 18 animals each), fed for 6 weeks diets which differed in their fatty acid compositions: (i) genuine salmon oil, abundant in (n-3) fatty acids, (ii) sunflower seed oil, rich in (n-6) fatty acids, particularly linoleic acid, and (iii) hydrogenated coconut oil, rich in saturated fatty acids. METHODS: Mast cells (10(6)/ml) were stimulated with various concentrations of the mast cell-degranulating agent, compound 48/80 (0.1-10 micrograms/ml). The extent of mast cell degranulation was quantified by determination of histamine in the supernatants using HPLC techniques. RESULTS: No differences in compound 48/80-induced histamine release between the three dietary groups for any of the concentrations of compound 48/80 tested were found. Analysis of variance followed by Tukey's method for multiple comparisons was used to evaluate the effect of changes in the dietary fat type. CONCLUSION: These findings strongly suggest that in contrast to the formation of eicosanoids, the process of mast cell degranulation by a receptor-independent pathway is not controlled by the fatty acid composition of membrane phospholipids.

The 3' terminal RNA sequences of bunyaviruses and nairoviruses (Bunyaviridae): evidence of end sequence generic differences within the virus family.

The 3' terminal nucleotide sequences of the three virus RNA species of viruses representing eight serogroups of bunyaviruses (genus Bunyavirus, Bunyaviridae) and six serogroups of nairoviruses (genus Nairovirus, Bunyaviridae) have been characterized. Members of the Bunyavirus genus have conserved 3' end sequences (generally, 3' UCAUCACAUGA...) that differ from the conserved 3' end sequences of members of the Nairovirus genus (generally, 3' AGAGUUUCU...).

Rapid and highly sensitive high-performance liquid chromatographic method for the determination of histamine and 3-methylhistamine in biological samples using fluorescamine as the derivatizing agent.

A highly sensitive and rapid high-performance liquid chromatographic assay for the determination of histamine and 3-methylhistamine in biological samples using 1-methylhistamine as the internal standard is described. Samples were purified and concentrated on cation-exchange columns and derivatized with fluorescamine. The lower detection limit was 20 pg on-column. Linearity was demonstrated up to 20 ng on-column. The samples could be derivatized simultaneously before injection and were stable for 7 days. The method was used for the determination of histamine and related compounds in coronary perfusates, extracts of homogenized rat hearts, and supernatants of stimulated peritoneal mast cells.

Mast cell-mediated induction of ICAM-1, VCAM-1 and E-selectin in endothelial cells in vitro: constitutive release of inducing mediators but no effect of degranulation.

Mast cell (MC)-mediated induction of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) and of E-selectin was studied in cultures of rat heart endothelial cells (EC) and human umbilical vein EC (HUVEC) respectively. MC induced VCAM-1 and E-selectin, but hardly any ICAM-1 in EC. Induction was not dependent on MC degranulation, but seemed to be provoked by constitutively released substances, other than histamine, from MC. Co-incubation of MC and EC, allowing for direct contact between the two cell types, was more potent in induction than MC co-incubated separately from EC using a permeable membrane. MC were less potent in induction than exogenous added cytokines or LPS. Induction of cell adhesion molecules in rat heart EC was MC-specific, since EC incubations with either rat cardiomyocytes or heart fibroblasts had no effect. The data show that rat MC, independent of degranulation, secrete mediators relevant for the induction of a specific set of EC adhesion molecules in vitro. This suggests a (supportive) role for MC in cell-adhesion molecule induction in the endothelium in settings of early or mild inflammation. The results are discussed in the context of inflammatory processes in the heart in vivo.

Lack of evidence for a role of mast cell degranulation in acute hypoxia/reoxygenation-induced injury in the isolated rat heart.

In the present study, we evaluated the potential role of mast cell degranulation in acute hypoxia/reoxygenation-induced injury to cardiomyocytes in the isolated rat heart. Histamine release was determined to delineate the extent of mast cell degranulation, whereas the release of creatine kinase (CK) and lactate dehydrogenase (LDH) was assessed to quantitate the extent of irreversible injury to cardiomyocytes. The suitability of peroxidase (PO) as a marker for mast cell degranulation was also evaluated. Reoxygenation resulted in a release of histamine corresponding with 6.5% +/- 0.6% of total tissue content, whereas LDH, CK and PO release amounted to 30% +/- 2%, 28% +/- 2% and 32% +/- 3% of their respective tissue contents. Identical perfusion in the presence of the mast cell stabilizer lodoxamide tromethamine resulted in a reduced histamine release (2.8% +/- 0.1%) of total tissue content upon reoxygenation, but the release of LDH, CK or PO was not influenced. Cumulative histamine release did not correlate with the amount of LDH, CK or PO released. Treatment with consecutive bolus injections of the mast cell degranulating compound 48/80 during normoxic perfusion resulted in an almost complete histamine release, whereas PO release remained below detection limit. When the compound 48/80-treated hearts were subjected to hypoxia/reoxygenation, the release of LDH, CK or PO during reoxygenation again remained unchanged, whereas histamine release was negligible. Determination of PO activity of freshly isolated cardiomyocytes demonstrated that the bulk of PO in rat hearts was located in this particular cell type. Therefore we conclude that in the isolated rat heart, PO release is not a specific marker of mast cell degranulation. In addition, our data provide no firm evidence that in this experimental model, mast cell degranulation contributes to a significant extent to acute hypoxia/reoxygenation-induced injury to cardiomyocytes.