Urinary excretion of and recent N-6-3 fatty acid intake

Epidemiological studies were performed in a Japanese fishing village when catches of fish were highest and in a Japanese farming village with usual fish consumption. Intake of eicosapentaenoic, docosahexaenoic and also arachidonic acid were significantly higher in the fishing village during the 3 days of the study than in the farming village. The correlation between eicosapentaenoic acid intake on the day when urine was collected and excreion of Δ 17-2,3-dinor-6-keto-prostaglandin F_1α, the main urinary metabolite of prostaglandin I₃, was highly significant, whereas there was no correlation between arachidonic or linoleic acid intake and excretion of 2,3-dinor-6-keto-prostaglandin F_1α, the main urinary metabolite of prostaglandin I₂. We suggest that the arachidonic acid pool for prostaglandin I₂ production is not quickly influenced by dietary linoleic or arachidonic acid because of a large pool size of arachidonic acid and a slow conversion of linoleic acid to arachidonic acid, while prostaglandin I₃ formation is directly related to the intake of eicosapentaenoic acid.     

Cyproheptadine metabolites inhibit proinsulin and insulin biosynthesis and insulin release in isolated rat pancreatic islets

The contribution of drug metabolites to cyproheptadine (CPH)-induced alterations in endocrine pancreatic -cells was investigated by examining the inhibitory activity of CPH and its biotransformation products, desmethylcyproheptadine (DMCPH), CPH-epoxide and DMCPH-epoxide, on hormone biosynthesis and secretion in pancreatic islets isolated from 50-day-old rats. Measurement of (pro)insulin (proinsulin and insulin) synthesis using incorporation of ³H-leucine showed that DMCPH-epoxide, DMCPH and CPH-epoxide were 22, 10 and 4 times, respectively, more potent than CPH in inhibiting hormone synthesis. The biosynthesis of (pro)insulin was also inhibited by CPH and DMCPH-epoxide in islets isolated from 21-day-old rat fetuses. The inhibitory action of CPH and its metabolites was apparently specific for (pro)insulin, and the synthesis of other islet proteins was not affected. Other experiments showed the metabolites of CPH were active in inhibiting glucose-stimulated insulin secretion but were less potent than the parent drug in producing this effect. CPH and its structurally related metabolites, therefore, have differential inhibitory activities on insulin synthesis and release. The observation that CPH metabolites have higher potency than CPH to inhibit (pro)insulin synthesis, when considered with published reports on the disposition of the drug in rats, indicate that CPH metabolites, particularly DMCPH-epoxide, are primarily responsible for the insulin depletion observed when the parent compound is given to fetal and adult animals.Abbreviations CPH cyproheptadine - CPH-epoxide cyproheptadine-10-11-epoxide - DMCPH desmethylcyproheptadine - DMCPH-epoxide desmethylcyproheptadine-10,11-epoxide - HPLC high-performance liquid chromatography - KBB Krebs biocarbonate buffer Recipient of a Society of Toxicology Predoctoral Research Fellowship.Present address: Department of Biochemistry, The University of Hong Kong, Hong Kong.     

Ultrastructure of calcitonin gene-related peptide-and substance P-like immunoreactive nerve fibres in the carotid body and carotid sinus of the guinea pig

Summary Previous studies have demonstrated that substance P-(SP) and calcitonin gene-related peptide-like immunoreactivities (CGRP-LI) coexist in sensory nerve fibres in the guinea-pig carotid body and carotid sinus. In the present study the ultrastructure of these nerve fibres was investigated by means of single-and double-labelling immunocytochemistry. In both, carotid body and carotid sinus immunoreactive fibres were unmyelinated axons of small dianeter (0.12–0.56 m). At the subcellular level, SP-and CGRP-LI were colocalized in intra-axonal dense core vesicles, suggesting corelease and simultaneous action of these two compounds. SP/CGRP-LI nerve fibres within the carotid body were mainly found in the interparenchymal connective tissue, but also occurred in relationship to blood vesslesl and nests of glomus cells. Neither in the carotid body not in the carotid sinus, SP/CGRP-LI axons corresponded to the large terminals which are generally considered to represent the main chemoreceptor and baroreceptor endings, respectively. Thus, SP/CGRP-LI fibres either belong to the chemo-and baroreceptors of the C-fibre class or constitute a fibre population not directly involved in conduction of baro-and chemoreflexes.This study was supported by the DFG, grant He 919/6-2     

CD45, an integral membrane protein tyrosine phosphatase. Characterization of enzyme activity

Although CD45 resembles the low Mr protein tyrosine phosphatases (PTPases) from human placenta in its specificity for phosphotyrosyl residues and absolute dependence on sulfhydryl compounds for activity, it also exhibits a number of distinguishing features. Most notably, it displayed substrate specificity in vitro, preferentially dephosphorylating myelin basic protein, over the other substrates tested, with high specific activity. Limited trypsinization of CD45 generated active fragments of approximately 65 kDa that were apparently derived exclusively from the intracellular segment of the molecule. These retained high activity against myelin basic protein, suggesting that this is an intrinsic feature of the PTPase domains and not the result of secondary interactions between the substrate and the putative ligand binding structure. With reduced carboxamidomethylated and maleylated lysozyme as substrate, CD45 was stimulated up to 12-fold by basic compounds such as spermine; divalent metal ions were also stimulatory, most notably Zn2+, which was previously identified as a potent inhibitor of the low Mr PTPases. CD45 was phosphorylated to high stoichiometry by casein kinase-2 (up to 1.5 mol/mol) and also by glycogen synthase kinase 3 (approximately 0.3 mol/mol) and protein kinase C (approximately 0.1 mol/mol); in all cases, no alteration in enzyme activity was detected following these modifications. Autophosphorylated preparations of epidermal growth factor receptor, insulin receptor, and p56lck protein tyrosine kinases were also substrates for CD45 in vitro.     

Production of TNF-alpha and TNF-beta by staphylococcal enterotoxin A activated human T cells

Staphylococcal enterotoxin at concentrations of less than 1 pg/ml induces significant TNF activity in human peripheral blood T cells and monocytes. Maximal TNF activity is routinely detected after 48 to 72 h of culture. IL-2 and IL-4 were both growth promoting for human T cells but only IL-2 could efficiently induce TNF production. The production of TNF-alpha and TNF-beta differed greatly in kinetics. An early intracytoplasmatic production of TNF-alpha after 6 h was detected in both monocytes and T cells whereas a late production of TNF-beta (lymphotoxin) after 48 h, occurred in the T cell population. Induction of TNF-alpha and TNF-beta production by Staphylococcal enterotoxin requires the presence of both monocytes and T cells. The CD4+45R- but not CD4+45R+ and CD8+ cells supported TNF-alpha production in monocytes. The main lytic component from Staphylococcal enterotoxin-activated mononuclear cells is TNF-beta. CD4+ and CD8+ T cells produced about equal amounts of biologically active TNF into the culture supernatants but a fourfold higher frequency of TNF-beta producing cells was demonstrated among CD4+ vs CD8+ cells. The CD4+45R- T cell subset was an efficient producer of TNF-beta and IFN-gamma whereas the CD4+45R+ T cell subset produced significant amounts of TNF-beta but only marginal amounts of IFN-gamma.     

Expression and biochemical characterization of human immunodeficiency virus type 1 nef gene product.

nef genes from human immunodeficiency virus type 1 isolates BH10 and LAV1 (lymphadenopathy-associated virus type 1) were expressed in Escherichia coli under the deo operon promoter. The two proteins found in the soluble compartment of the bacterial lysate were purified by ion-exchange column chromatography to apparent homogeneity. Determination of the amino-terminal sequence revealed glycine as the first amino acid in the Nef protein, indicating removal of the initiator methionine during expression in E. coli. Under native conditions, the recombinant Nef protein is a monomer of 23 kilodaltons. In denaturing polyacrylamide gels, however, BH10 and LAV1 Nef proteins migrate as 28 and 26 kilodaltons, respectively. GTP binding and GTPase activity were monitored during Nef protein purification. These activities did not copurify with the recombinant Nef protein from either the BH10 or the LAV1 isolate. Purified recombinant BH10 Nef protein was used as an immunogen to elicit mouse monoclonal antibodies. A series of monoclonal antibodies were obtained which reacted with sequences at either the amino or carboxy terminus of Nef. In addition, a conformational epitope reacting with native BH10, but not LAV1, Nef was isolated.     

Cloning, sequencing and overexpression of the mannitol-specific enzyme-III-encoding gene of Staphylococcus carnosus

The gene which encodes the mannitol-specific enzyme III (EIIImtl) of the phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus carnosus, has been cloned. Genomic libraries of S. carnosus DNA were constructed using the expression vector pUC19 and EIIImtl-producing clones were identified using rabbit polyclonal antiserum. A 700-bp Dde I fragment, containing the complete gene encoding EIIImtl, was sequenced by the dideoxy chain-termination technique. Upstream from the ORF for EIIImtl one can find a sequence analogous to that of the Escherichia coli promoter. This region acts as a strong promoter when subcloned into the promoter test vector M13HDL17. EIIImtl was overproduced using the inducible T7 polymerase system and purified to homogeneity. Amino acid sequence comparison confirmed a 38% similarity to the hydrophilic enzyme-III-like portion of enzyme IImtl of E. coli. There is also a 36% similarity to the N terminus of the fructose-specific phospho-carrier protein from E. coli.     

Polygalacturonase Isozymes and Pectin Depolymerization in Transgenic rin Tomato Fruit

We have previously described the construction and expression of a chimeric gene that allows developmentally regulated expression of tomato (Lycopersicon esculentum) polygalacturonase in ripening-impaired, mutant (rin) tomato fruit (JJ Giovannoni, D DellaPenna, AB Bennett, RL Fischer [1989] The Plant Cell 1: 53-63). We now show that expression of the chimeric polygalacturonase gene in rin tomato fruit resulted in the accumulation of all three polygalacturonase isozymes (PG1, PG2A, and PG2B). Polyuronide solubilization and polyuronide depolymerization both reached their maximal levels in transgenic rin fruit prior to the appearance of PG2 isozymes. These results demonstrate that PG1, PG2A, and PG2B all arise by differential processing of a single gene product and further suggest that the PG1 isozyme is sufficient to carry out both polyuronide solubilization and depolymerization in vivo.     

Influence of dietary fish on eicosanoid metabolism in man

Two groups of 40 volunteers were given a dietary supplement consisting of 135 g of mackerel or meat (control) paste per day for 6 weeks. Compliance was about 80% in both groups and the daily intake of 20:5(n-3) and 22:6(n-3) from the mackerel supplement was about 1.3 and 2.3 g, respectively. In collagen-activated platelet rich plasma, the potency of blood platelet to produce HHT from arachidonic acid (AA) clearly reduced in the mackerel group, whereas the formation of HHTE from timnodonic acid (TA) increased slightly. Changes in the formation of HHT and HHTE, measured by HPLC, correlated significantly with those of TxB2 and TxB3, respectively, measured by GC/MS. Changes in the formation of the lipoxygenase products HETE (ex AA) and HEPE (ex TA) were qualitatively similar to that seen for the cyclo-oxygenase products, but quantitatively the responses were smaller. Formation of ir TxB2 in clotting blood significantly reduced in the mackerel group. In collagen-activated, citrated whole blood, TxB2 formation tended to be reduced in the mackerel-supplemented volunteers. Mackerel consumption was associated with the formation of considerable amounts of PGI3, as judged from the appearance of 2,3-dinor-delta 17-6-keto-PGF1 alpha in urine. The amount of the major metabolite of PGI2, 2,3-dinor-6-keto-PGF1 alpha was not reduced, or even increased. The daily amount of tetranor prostaglandin metabolites in the urine did not change significantly, which indicates that mackerel supplementation did not alter the formation of prostaglandins E and F.