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Query: UNIPROT:P06889 (Mol)
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Studies of gonococcal pilus biogenesis are fundamental to understanding organelle structure/function relationships and identifying new approaches to controlling disease. This area of research is also relevant to elucidating the basic mechanisms of outer membrane translocation of macromolecules, which requires components highly related to those involved in type IV pilus expression. Previous studies have shown that products of several ancillary pil genes are required for organelle biogenesis but of these only PilQ, a member of the GspD protein family, is a component of the outer membrane. DNA sequencing of the region upstream of pilQ revealed the presence of two open reading frames (ORFs) whose deduced polypeptides shared significant identities with proteins required for pilus expression in Pseudomonas aeruginosa and Pseudomonas syringae, the genes for which are arrayed upstream of a gene encoding a PilQ homologue. Gonococcal mutants bearing transposon insertions in these ORFs were non-piliated and failed to express pilus-associated phenotypes, and the corresponding genes were designated PilO and pilP. The piliation defects in the mutants could not be ascribed to polarity on distal pilQ expression as shown by direct measurement of PilQ antigen in those backgrounds and the use of a novel technique to create tandem duplications in the gonococcus (Gc) genome. As predicted by the presence of a consensus lipoprotein signal sequence, PilP expressed in both Escherichia coli and Gc could be labelled with [3H]-palmitic acid. PilP- as well as PilQ- mutants shed PilC, a protein which facilitates pilus assembly and is implicated in epithelial cell adherence, in a soluble form. Combined with the finding that levels of multimerized PiIQ were greatly reduced in PilP- mutants, the results suggest that PilP is required for PilQ function and that PilQ and PilC may interact during the terminal stages of pilus biogenesis. The findings also support the hypothesis that the Gc PilQ multimer corresponds to a physiologically relevant form of the protein required for pilus biogenesis.
Mol Microbiol 1997 Feb
PMID:PilP, a pilus biogenesis lipoprotein in Neisseria gonorrhoeae, affects expression of PilQ as a high-molecular-mass multimer. 915 38

We previously identified HpuB, an 85 kDa Fe-repressible protein required for utilization of Fe from, and binding to, haemoglobin and the haemoglobin-haptoglobin complex. The gene for hpuB was cloned from Neisseria meningitidis strain DNM2 and the predicted amino acid sequence indicates that HpuB is an outer membrane receptor belonging to the TonB family of high-affinity transport proteins. A second open reading frame, predicted to encode a 34.8 kDa lipoprotein, was discovered 5' to hpuB, and was designated hpuA. HpuA was identified in a total-membrane-protein preparation by construction of a mutant lacking HpuA. Acylation of HpuA was confirmed by [3H]-palmitic acid labelling of meningococci. Consensus promoter sequences were not apparent 5' to hpuB. The hpuA insertion mutation exerted a polar effect, abolishing expression of hpuB, suggesting that hpuA and hpuB are co-transcribed. The 3.5 kb polycistronic hpuAB mRNA was identified and shown to be transcriptionally repressed by iron. The transcriptional start site was identified 33 nucleotides 5' to the hpuA translational start site, appropriately positioned around consensus promoter and ferric uptake regulator (Fur)-box sequences. The structure of this operon suggests that HpuA-HpuB is a two-component receptor analogous to the bipartite transferrin receptor TbpB-TbpA.
Mol Microbiol 1997 Feb
PMID:Molecular characterization of hpuAB, the haemoglobin-haptoglobin-utilization operon of Neisseria meningitidis. 915 45

Sphingomyelinase activity against pure sphingomyelin monolayers is constant up to a surface pressure of 18 mN/m and falls above it. Sphingomyelinase- and phospholipase A2-mediated phosphohydrolytic pathways are mutually modulated by the presence of their respective substrates and products. At 15 mN/m non-substrate lipids such as ceramide at a mole fraction of 0.1 in mixed films with the pure substrate, inhibit the sphingomyelinase activity. Ganglioside GM1, another ceramide-containing complex sphingolipid, also inhibits sphingomyelinase activity, while a chemically related glycosphingolipid such as asialo-GM1 has no effect. The activity is unaltered by dipalmitoylphosphatidylcholine and by an equimolar mixture of its products of hydrolysis by phospholipase A2, fatty acid and lysoderivative, but it is inhibited by only one of them or by dilauroylphosphatidylcholine. Phospholipase A2 is inhibited by sphingomyelin, and activated by ceramide and by palmitic acid, one of the products of its own phosphohydrolytic reaction.
Mol Membr Biol
PMID:Mutual modulation of sphingomyelinase and phospholipase A2 activities against mixed lipid monolayers by their lipid intermediates and glycosphingolipids. 916 Mar 38

Evidence from use of pertussis and cholera toxins and from NaF suggested the involvement of G proteins in GnRH regulation of gonadotrope function. We have used three different methods to assess GnRH receptor regulation of G(q/11)alpha subunits (G(q/11)alpha). First, we used GnRH-stimulated palmitoylation of G(q/11)alpha to identify their involvement in GnRH receptor-mediated signal transduction. Dispersed rat pituitary cell cultures were labeled with [9,10-(3)H(N)]-palmitic acid and immunoprecipitated with rabbit polyclonal antiserum made against the C-terminal sequence of G(q/11)alpha. The immunoprecipitates were resolved by 10% SDS-PAGE and quantified. Treatment with GnRH resulted in time-dependent (0-120 min) labeling of G(q/11)alpha. GnRH (10(-12), 10(-10), 10(-8), or 10(-6) g/ml) for 40 min resulted in dose-dependent labeling of G(q/11)alpha compared with controls. Cholera toxin (5 microg/ml; activator of G(i)alpha), pertussis toxin (100 ng/ml; inhibitor of G(i)alpha actions) and Antide (50 nM; GnRH antagonist) did not stimulate palmitoylation of G(q/11)alpha above basal levels. However, phorbol myristic acid (100 ng/ml; protein kinase C activator) stimulated the palmitoylation of G(q/11)alpha above basal levels, but not to the same extent as 10(-6) g/ml GnRH. Second, we used the ability of the third intracellular loop (3i) of other seven-transmembrane segment receptors that couple to specific G proteins to antagonize GnRH receptor-stimulated signal transduction and therefore act as an intracellular inhibitor. Because the third intracellular loop of alpha1B-adrenergic receptor (alpha1B 3i) couples to G(q/11)alpha, it can inhibit G(q/11)alpha-mediated stimulation of inositol phosphate (IP) turnover by interfering with receptor coupling to G(q/11)alpha. Transfection (efficiency 5-7%) with alpha1B 3i cDNA, but not the third intracellular loop of M1-acetylcholine receptor (which also couples to G(q/11)alpha), resulted in 10-12% inhibition of maximal GnRH-evoked IP turnover, as compared with vector-transfected GnRH-stimulated IP turnover. The third intracellular loop of alpha2A adrenergic receptor, M2-acetylcholine receptor (both couple to G(i)alpha), and D1A-receptor (couples to G(s)alpha) did not inhibit IP turnover significantly compared with control values. GnRH-stimulated LH release was not affected by the expression of these peptides. Third, we assessed GnRH receptor regulation of G(q/11)alpha in a PRL-secreting adenoma cell line (GGH(3)1') expressing the GnRH receptor. Stimulation of GGH(3)1' cells with 0.1 microg/ml Buserelin (a metabolically stable GnRH agonist) resulted in a 15-20% decrease in total G(q/11)alpha at 24 h following agonist treatment compared with control levels; this action of the agonist was blocked by GnRH antagonist, Antide (10(-6) g/ml). Neither Antide (10(-6) g/ml, 24 h) alone nor phorbol myristic acid (0.33-100 ng/ml, 24 h) mimicked the action of GnRH agonist on the loss of G(q/11)alpha immunoreactivity. The loss of G(q/11)alpha immunoreactivity was not due to an effect of Buserelin on cell-doubling times. These studies provide the first direct evidence for regulation of G(q/11)alpha by the GnRH receptor in primary pituitary cultures and in GGH3 cells.
Mol Endocrinol 1997 Jun
PMID:Regulation of G(q/11)alpha by the gonadotropin-releasing hormone receptor. 917 Dec 37

Changes in red blood cell shape and membrane properties in response to the interaction with free fatty acids and their derivatives were studied by light scattering at small and large angles, light microscopy and fluorescence anisotropy. The influence of these agents depended on the end groups and increased with increasing chain length. The fatty acids exerted a biphasic effect on the cell size, shape and surface properties, and induced erythrocyte aggregation. After transient size alteration with a reduction in diameter, caused by low free fatty acid concentrations (up to 5-10 microM in the case of palmitic acid), fatty acids increased the erythrocyte diameter at higher concentrations (20-60 microM in the case of palmitic acid). The aliphatic aldehydes and methyl esters of fatty acids significantly decreased the cell diameter at the concentrations used. Changes in erythrocyte shape and size were accompanied by changes in membrane microviscosity. Palmitic acid decreased the rotational diffusion of the fluorescence probe incorporated into the membrane whereas methyl ester of palmitic acid and lauric aldehyde increased probe mobility. Also the erythrocyte modification by malondialdehyde influenced cell morphology and highly decreased membrane fluidity.
Biochem Mol Biol Int 1997 Jun
PMID:Effect of free fatty acids on erythrocyte morphology and membrane fluidity. 919 92

Palmitoylation of the V2 vasopressin receptor (V2R) and its functional role were investigated in transfected cells. Palmitoylation was assessed by incubating transfected cells with [3H]palmitic acid and immunoprecipitating the receptor with an antibody raised against a portion of the third intracellular loop of V2R. Wild-type and nonglycosylated V2R yielded tritium signals at 45-55 and 40 kDa, respectively, demonstrating that the V2R is palmitoylated and that receptor palmitoylation is independent of glycosylation. Substitution of CC341/342 for serines eliminated receptor palmitoylation, whereas replacement of a single amino acid, C341S or C342S, restored partial palmitoylation. Saturation binding assays revealed decreased cell surface expression of the nonpalmitoylated receptor compared with the wild-type; this effect was more pronounced when a truncated form of V2R (G345ter) was studied. The presence of either cysteine residue (C341S or C342S) elevated receptor expression to normal levels, most likely due to the partial restoration of palmitoylation. Ligand binding affinity, hormone-induced stimulation of adenylyl cyclase activity, receptor internalization, and desensitization were not affected by the absence of palmitoylation. No increase but rather a slight decrease in the extent of receptor palmitoylation was detected after exposure to vasopressin. It was concluded that the V2R is palmitoylated in both cysteines, each cysteine is palmitoylated independently from the other, and palmitoylation enhances cell surface expression of the V2R.
Mol Pharmacol 1997 Jul
PMID:Palmitoylation of the V2 vasopressin receptor. 922 8

We have investigated pathways of lipid metabolism in spermatozoa and generation of various metabolites with potential messenger functions during exocytosis stimulated with A23187/Ca2+. Stimulation of boar spermatozoa resulted in a considerable rapid increase in saturated/unsaturated 1,2-diacylglycerol (1,2-SU-DAG) and, concomitantly, a substantial reduction in disaturated 1,2-diacylglycerol (1,2-DS-DAG), and in phosphatidylcholine (PC). These changes preceded the onset of exocytosis. Phosphatidic acid was sometimes generated in parallel, but usually rose later, suggesting that 1,2-SU-DAG may be formed directly by phospholipase C action. Lipid changes observed in stimulated spermatozoa that have been prelabelled with several lipid precursors ([14C]palmitic acid, [14C]glycerol, [14C]choline, or [14C]arachidonic acid) suggested the existence of a unique process involving the utilization of the high basal levels of 1,2-DS-DAG to form 1,2-SU-DAG, with the latter being subsequently employed to replenish the PC pool. An ensuing generation of lysoPC and arachidonic acid, which paralleled the occurrence of exocytosis, revealed that the newly synthesized PC was hydrolyzed by phospholipase A2. The highest levels of 1,2-SU-DAG, minimum levels of 1,2-DS-DAG, and the regeneration of the PC pool were tightly coupled to the beginning of visible exocytosis. These results suggest that changes in these lipid metabolites may be fundamental processes during acrosomal exocytosis occurring in response to physiological agonists.
Mol Reprod Dev 1997 Sep
PMID:Diacylglycerol species as messengers and substrates for phosphatidylcholine re-synthesis during Ca2+-dependent exocytosis in boar spermatozoa. 926 66

Babesia bovis, an intraerythrocytic, protozoal parasite of cattle, undergoes clonal antigenic variation (Allred DR, Cinque RM, Lane TJ, Ahrens KP. Infect Immun 1994;62:91-98). This ability could provide a mechanism by which the parasite escapes host immune defenses to establish chronic infection. Previous work identified two parasite-derived antigens of Mr 128,000 and 113,000 that were present on the surface of the infected erythrocyte and appeared to be associated with clonal antigenic variation (Allred DR, Cinque RM, Lane TJ, Ahrens KP. Infect Immun 1994;62:91 98). Two monoclonal antibodies (mAbs), 3F7.1H11 and 4D9.1G1, which recognize the variant erythrocyte surface antigen (VESA1) have been identified. These mAbs react only with the surface of erythrocytes infected with the B. bovis C9.1 clone in live-cell immunofluorescence assays. In both conventional and surface immunoprecipitations, the mAbs precipitate a variant antigen doublet that matches in mass the infected red blood cell (IRBC) surface antigens precipitated with bovine serum. In contrast, Western blot analysis revealed that only the Mr 128,000 polypeptide is recognized by the mAbs. Neither mAb recognizes antigenically variant progenitor or progeny parasite clones in any of the immunoassays, confirming the involvement of this antigen in rapid clonal antigenic variation. Failure to label this antigen with [9,10(n)-3H]myristic acid, [9,10(n)-3H]palmitic acid or D-[6-3H]glucosamine indicates that these polypeptides are neither N-glycosylated nor fatty acylated. Identity of the variant antigen recognized by the mAbs with that putatively identified with immune serum was confirmed by comparison of partial proteolytic digestion products. Unambiguous identification of the VESA1 antigen as a component of antigenic variation will facilitate characterization of the events leading to antigenic variation on the B. bovis-infected erythrocyte surface and its significance to parasite survival during chronic infection.
Mol Biochem Parasitol 1997 Nov
PMID:Characterization of a variant erythrocyte surface antigen (VESA1) expressed by Babesia bovis during antigenic variation. 936 70

Chronic opioid treatment of stably mu-opioid receptor transfected human mammary epidermoid A431 carcinoma cells (clone A431/mu 13) results in sensitization of adenylyl cyclase (AC), a cellular adaptation associated with drug dependence. Up-regulation of AC is characterized by significantly increased levels of both basal and post-receptor-stimulated effector activities, which develop without any apparent change in the quantity of stimulatory G proteins and the maximum catalytic activity of AC. Here, we report that detergent extracts from membranes of chronically morphine-treated (10 microM; 2 days) A431/mu 13 cells display higher stimulatory AC activities as assessed in the S49cyc- reconstitution assay. This finding is most likely due to an increased functional activity of Gs alpha because the addition of exogenous G beta gamma subunits, which per se stimulate AC in S49cyc- membranes, failed to affect the difference in reconstitutive AC activity. Moreover, both chemical depalmitoylation by hydroxylamine and inhibition of palmitoyl-CoA transferase in vivo by tunicamycin treatment incresed the reconstitutive activity of detergent extracts and eliminated the differences between native and opioid-dependent cells, indicating that the increase in stimulatory activity is due to depalmitoylation of Gs alpha. Indeed, metabolic labelling studies with [3H]palmitic acid revealed that chronic opioid treatment reduces considerably the fraction of palmitoylated Gs alpha in the plasma membrane. Furthermore, high affinity [3H]forskolin binding experiments demonstrated that depalmitoylated Gs alpha is able to associated directly with AC during the state of opioid dependence even without preceding receptor activation. These results suggest that post-translational palmitoylation of Gs alpha provides a potential regulator of transmembrane signaling. Moreover, accumulation of the depalmitoylated form of Gs alpha in the plasma membrane as reported herein may contribute to the increase in stimulatory AC signaling, as is characteristic for the state of opioid dependence.
Mol Pharmacol 1997 Dec
PMID:Enhanced stimulatory adenylyl cyclase signaling during opioid dependence is associated with a reduction in palmitoylated Gs alpha. 941 9

The mechanism of the hypolipidemic effect of n-3 fatty acids was studied using isolated rat hepatocytes maintained in culture. EPA and DHA caused a significant reduction in the incorporation of 3[H]-leucine into apoB associated with the VLDL produced by hepatocytes in culture when compared to that in presence of palmitic acid. Presence of indomethacin, an inhibitor of cyclo-oxygenase reversed the effect of EPA on VLDL synthesis while diethyl carbamazine an inhibitor of lipoxygenase did not show any effect suggesting that the effect of EPA may be mediated through prostaglandins. This was further tested by invivo experiments where animals were fed fish oil containing diet with and without aspirin, which inhibits formation of prostaglandins. The incorporation of 3[H]-leucine into apo B and 14[C]-acetate into cholesterol of VLDL produced by hepatocytes from aspirin treated animals were significantly high. The reversal of the effect of n-3 fatty acids by agents which inhibit the formation of prostaglandin suggests that the n-3 fatty acids may exert their effect on VLDL production by liver cells through prostaglandins.
Biochem Mol Biol Int 1997 Dec
PMID:Effect of n-3 fatty acids on VLDL production by hepatocytes is mediated through prostaglandins. 941 16


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