Epidermal-growth-factor-induced production of phosphatidylalcohols by HeLa cells and A431 cells through activation of phospholipase D. In response to epidermal growth factor (EGF), HeLa cells and A431 cells rapidly accumulate substantial amounts of phosphatidic acid (up to 0.16 and 0.2 micrograms/10(6) cells respectively), which represents approx. 0.17% of total phospholipid. Phosphatidic acid may be a potential product of diacylglycerol kinase and/or of phospholipase D. To evaluate the contribution of phospholipase D, the phosphatidyl-transfer reaction to a primary alcohol (mostly butan-1-ol; 0.2%) was measured; this reaction is known to be mediated exclusively by phospholipase D in intact cells. In HeLa and in A431 cells prelabelled with [1-14C]oleic acid, EGF (10 and 100 nM respectively) caused a 3-fold increase in radioactive phosphatidylbutanol within 5 min at the expense of labelled phosphatidic acid. Dose-response relationships showed 10 nM- and 100 nM-EGF to be maximally effective in HeLa cells and A431 cells respectively. Mass determinations showed that the phosphatidylbutanol formed within 5 min represented only part of the phosphatidic acid. Depletion of protein kinase C by pretreatment of A431 cells for 17 h with the phorbol ester phorbol 12-myristate 13-acetate (0.1 microM) did not impair EGF-induced formation of phosphatidylbutanol, thus indicating that the reaction was independent of this enzyme. Since phosphatidic acid is suggested to exert second-messenger functions as well as to induce biophysical changes in cellular membranes, its formation, including that via the phospholipase D pathway, may represent an important link between extracellular signals and intracellular targets.


Human neutrophil phospholipase D activation by N-formylmethionyl-leucylphenylalanine reveals a two-step process for the control of phosphatidylcholine breakdown and oxidative burst. A comparative study of real-time kinetics of respiratory burst, monitored by H2O2-dependent chemiluminescence, and phospholipase D (PLD)-mediated phosphatidylcholine breakdown has been undertaken on human neutrophils stimulated by N-formylmethionyl-leucylphenylalanine in the absence of cytochalasin B. The fungal metabolite 17-hydroxywortmannin (HWT), an inhibitor of NADPH oxidase activation, decreases phosphatidic acid (PA) production by 30% at a concentration of 1 nM. Higher concentrations (10 nM-1 microM) inhibit PA formation maximally by 50% as compared with control. In all cases, the inhibition is delayed by 20-30 s after addition of the agonist. Thus the full PA generation is actually the result of an early (HWT-insensitive) and a late (HWT-sensitive) phosphatidylcholine breakdown. However, under all conditions, alkylacylglycerol remains at the basal level. PLD activity is dependent on Ca2+ influx, but is fully inhibited in cells depleted of Ca2+ with EGTA and Quin 2. The effect of HWT on the respiratory burst was investigated by measuring the kinetics of H2O2-induced chemiluminescence. This method allows to distinguish various phases of superoxide ion production: a lag, an increase in H2O2 formation (early phase), the duration of H2O2 production (late phase) and the termination of the oxidative burst. The lag remains constant for all HWT concentrations. A concentration of 10 nM-HWT, which fully inhibits the HWT-sensitive part of PA production, decreases superoxide ion production with a delay of about 20 s after addition of the agonist. Higher HWT concentrations, which have no additional effect on PLD inhibition, equally affect an early and a late phase of the burst. Thus high doses of HWT have a site of action which decreases the whole burst but does not affect the PLD any more. Therefore HWT and Ca2+ provide evidence for a two-step process for PLD activation. Only the delayed PA generation is functionally linked to a late phase of the oxidative burst.


Intracellular pH and the stimulus-secretion coupling in insulin-producing RINm5F cells. The regulation of intracellular pH (pHi) and its role in the insulin-secretory process were evaluated, by using the clonal insulin-secreting cell line RINm5F. Glyceraldehyde, lactate and dihydroxyacetone decreased pHi, but only the first two released insulin. In the presence of extracellular Na+ the cells counteracted the acidification. Blocking the Na+/H+ exchange in acidic cells resulted in a drastic further lowering of pHi, an effect not obtained under basal conditions. Whereas glyceraldehyde depolarized the cells, lactate was without effect. Dihydroxyacetone hyperpolarized the cells in the presence of extracellular Na+, but this effect disappeared when Na+ was excluded from the medium. Stimulation with glyceraldehyde resulted in increased free cytoplasmic Ca2+ concentration ([Ca2+]i). Dihydroxyacetone and lactate had no effect on [Ca2+]i in the presence of Na+, but lactate induced a decrease in [Ca2+]i in Na(+)-deficient medium. In RINm5F cells the activity of the Na+/H+ antiport could not be augmented by activation of protein kinase C (PKC). Hence, in insulin-secreting cells a PKC-insensitive Na+/H+ antiport is the major mechanism restoring a decrease in pHi. Acidification itself does not affect membrane potential, but may directly interact with the mechanisms regulating exocytosis.


The dependence of lipid monolayer lipolysis on surface pressure. Brönsted-Bjerrum theory [Brönsted (1922) Z. Phys. Chem. 102, 169-207; (1925) Z. Phys. Chem. 115, 337-364; Bjerrum (1924) Z. Phys. Chem. 108, 82-100] as applied to reactions at interfaces is used to interpret published data on the lipolysis of dinonanoyl phosphatidylcholine monolayers by pancreatic phospholipase A2. Reasonable quantitative agreement between theoretical and experimental results occurs when the reported effects of surface pressure on the amount of adsorbed enzyme are used together with the assumption that the Langmuir-Syskowski [Lange (1967) Nonionic Surfactants (Schick, M., ed.), chap. 4, Marcel Dekker, London and New York] isotherm describes the adsorption of lipid. The equations of Mass Action kinetics are less successful. Equivalent data on the lipolysis of didodecanoyl phosphatidylglycerol by pancreatic lipase can also be interpreted by arguing that the adsorbed enzyme forms significant amounts of enzyme substrate complex which reacts to give products in accordance with the well-known Briggs-Haldane [Lehninger (1975) Biochemistry, 2nd edn., chap. 8, Worth, New York] mechanism. The positions of the observed maxima in rate versus surface pressure plots suggests that the adsorption of an enzyme molecule displaces less lipid than one expects from the pronounced differences in molecular sizes of the two adsorbed species.


Different triggers for calcium oscillations in mouse eggs involve a ryanodine-sensitive calcium store. Relative intracellular free Ca2+ concentrations ([Ca2+]i) were monitored in mature unfertilized mouse eggs by measuring fluorescence of intracellular fluo3. A number of different agents were found to cause sustained repetitive transient [Ca2+]i oscillations. These were microinjection of a cytosolic sperm factor, sustained injection of Ins-(1,4,5)P1, or extracellular addition of the thiol reagent thimerosal. Stimulating G-protein activity by injection of guanosine 5'-[gamma-thio]triphosphate plus application of carbachol also caused [Ca2+]i oscillations, but less reliably than other stimuli. A role for Ca(2+)-induced Ca2+ release and a ryanodine-sensitive Ca2+ channel in mouse eggs was suggested by the finding that microinjection, or external addition, of ryanodine also caused [Ca2+]i increases. Furthermore, ryanodine, along with thimerosal, increased the sensitivity of eggs to Ca(2+)-induced [Ca2+]i oscillations. When ryanodine was added to eggs oscillating in response to the sperm factor, InsP3 or thimerosal, it caused a decrease in amplitude of oscillations and eventually a block of [Ca2+]i oscillations associated with a sustained elevation of [Ca2+]i. These data suggest that a ryanodine-sensitive Ca(2+)-release mechanism exists in mouse eggs and that a ryanodine-sensitive Ca2+ store plays a role in generating intracellular [Ca2+]i oscillations.


Immunological detection of phenylalanine hydroxylase protein in Drosophila melanogaster. A monoclonal antibody raised against monkey liver phenylalanine hydroxylase (PAH) has been used to detect this protein in Drosophila melanogaster. A cross-reacting material (CRM) band of apparent molecular mass 50-52 kDa, equivalent to that deduced for the Drosophila melanogaster PAH protein based on the pah gene cDNA sequence, has been detected. This CRM was analysed throughout development and showed an equivalent pattern to that reported for PAH activity in this insect, with maxima at pupariation and at pharate adult formation. Distribution of this CRM in larval tissues, the haemolymph and the adult body is mainly restricted to the larval fat body and the adult head. Demonstration of this CRM as the PAH protein comes from the correlation between the decreased PAH enzyme activities of two mutant strains and their decreased amounts of CRM by Western blotting.


Evidence that beta-hydroxyacyl-CoA dehydrase purified from rat liver microsomes is of peroxisomal origin. The present study provides strong evidence that the previously isolated hepatic microsomal beta-hydroxyacyl-CoA dehydrase (EC 4.2.1.17), believed to be a component of the fatty acid chain-elongation system, is derived, not from the endoplasmic reticulum, but rather from the peroxisomes. The isolated dehydrase was purified over 3000-fold and showed optimal enzymic activity toward beta-hydroxyacyl-CoAs or trans-2-enoyl-CoAs with carbon chain lengths of 8-10. The purified preparation (VDH) displayed a pH optimum at 7.5 with beta-hydroxydecanoyl-CoA, and at 6.0 with beta-hydroxystearoyl-CoA. Competitive-inhibition studies suggested that VDH contained dehydrase isoforms, and SDS/PAGE showed three major bands at 47, 71 and 78 kDa, all of which reacted to antibody raised to the purified preparation. Immunocytochemical studies with anti-rabbit IgG to VDH unequivocally demonstrated gold particles randomly distributed throughout the peroxisomal matrix of liver sections from both untreated and di-(2-ethylhexyl) phthalate-treated rats. No labelling was associated with endoplasmic reticulum or with the microsomal fraction. Substrate-specificity studies and the use of antibodies to VDH and to the peroxisomal trifunctional protein indicated that VDH and the latter are separate enzymes. On the other hand, the VDH possesses biochemical characteristics similar to those of the D-beta-hydroxyacyl-CoA dehydrase recently isolated from rat liver peroxisomes [Li, Smeland & Schulz (1990) J. Biol. Chem. 265, 13629-13634; Hiltunen, Palosaari & Kunau (1989) J. Biol. Chem. 264, 13536-13540]. Neither enzyme utilizes crotonoyl-CoA or cis-2-enoyl-CoA as substrates, but both enzymes convert trans-2-enoyl substrates into the D-isomer only. In addition, the VDH also contained beta-oxoacyl-CoA reductase (beta-hydroxyacyl-CoA dehydrogenase) activity, which co-purified with the dehydrase.


The human IGF-I gene contains two cell type-specifically regulated promoters. The human insulin-like growth factor-I (IGF-I) gene contains two alternative leader exons: exons 1 and 2. We have identified, by transient transfection experiments, the putative promoters P1 and P2 upstream of these leader exons. The promoter regions were cloned in front of the luciferase reporter gene and their promoter activities were measured in transfected SK-N-MC (human neuroepithelioma) and OVCAR-3 (human ovarian carcinoma) cells. Both of these cell lines express the IGF-I gene endogenously, resulting in normally sized IGF-I mRNAs of 7.6, 1.3 and 1.1 kb. In SK-N-MC cells, in which P1 is the most active IGF-I promoter, P2 displayed a three times lower promoter activity than P1. However, in OVCAR-3 cells, P2 is four times more active than P1, resulting in an overall 12-fold difference in the relative promoter activities of the two IGF-I gene promoters in these two cell types. This indicates that the IGF-I promoters show a cell type-specific expression pattern.


Reverse transcription with nested polymerase chain reaction shows expression of basic fibroblast growth factor transcripts in human granulosa and cumulus cells from in vitro fertilisation patients. Basic fibroblast growth factor (bFGF) is a potent angiogenic factor that has also been implicated in granulosa cell and oocyte maturation. We now report the expression of messenger ribonucleic acid (mRNA) encoding bFGF in human granulosa and cumulus cells obtained at oocyte recovery in in vitro fertilisation patients. It was necessary to use the sensitive technique of a nested polymerase chain reaction (PCR) after reverse transcription (RT) to detect transcripts. This finding in conjunction with a recent report showing the presence of transcripts for transforming growth factor beta (TGF beta) in the same type of cells by PCR indicates that mechanisms are in place for controlling extracellular proteolysis and cell differentiation.


Production of a low molecular weight growth inhibitory factor by adenovirus 12-transformed cells. Malignant rodent cells transformed by human adenovirus 12 produce a potent cell growth inhibitory factor. The cell growth inhibitory factor inhibits the growth of and DNA synthesis in normal fibroblasts in vitro. Extent of the production of the cell growth inhibitory factor appears to be proportional to that of the malignancy of the transformed cells. C57AT1-AB cells, an adenovirus 12-transformant of C57BL/6 mouse origin, are highly tumorigenic in the syngeneic and allogeneic mice. The cell growth inhibitory factor produced by these cells was characterized for the physicochemical properties; the cell growth-inhibitory activity was quantitatively recovered in the filtrates of YM-2 membrane (M(r) less than 1,000), resistant to the heat treatments at 56 degrees C for 30 min and 100 degrees C for 5 min, and extractable by ethyl acetate under acid-condition. These results suggest that the cell growth inhibitory factor may be lipid or oligopeptides.


Truncation of N-terminal extracellular or C-terminal intracellular domains of human ETA receptor abrogated the binding activity to ET-1. We have investigated the function of N-terminal and C-terminal domains of the human ETA receptor by expressing truncated mutants in COS-7 cells. Three kinds of ETA receptors truncated in the N-terminal extracellular or C-terminal intracellular domains were produced. Deletion of the entire extracellular N-terminal or intracellular C-terminal domain completely inactivated the ET-1 binding activity. However, the deletion of one half of the N-terminal extracellular domain of the ETA receptor, missing one of two N-linked glycosylation sites, maintained complete binding activity. Specific monoclonal antibodies detected all the truncated ETA receptors in the cell membrane fraction of transfected COS-7 cells. The size of the ETA receptor was heterogeneous due to differential glycosylation and distributed in 48K, 45K and 42K dalton bands in Western blot analysis. These results demonstrated that a part of the N-terminal domain in close proximity to the first transmembrane region is required for the ligand binding activity of the ETA receptor, and the C-terminal domain is perhaps necessary as an anchor for maintenance of the binding site.


Protease inhibitors generate cytotoxic fragments from Alzheimer amyloid protein precursor in cDNA-transfected glioma cells. A human glioma cell line (Bu-17) was stably transfected with full-length cDNA encoding beta/A4 amyloid protein precursor (APP). When the transfectants were treated with protease inhibitors (leupeptin, E-64, and antipain) and the lysosomotropic agent chloroquine, aberrantly processed fragments of APP having molecular sizes of 8-30 kDa were detected with an antibody against the carboxyl-terminal sequence of APP. Immunocytochemistry revealed that these fragments were localized in the lysosome-like organelles. Treatment of the APP cDNA transfectants with chloroquine for 3 days caused cellular degeneration, and leupeptin and E-64 enhanced chloroquine-induced cytotoxicity. These results suggest that inhibition of lysosomal hydrolases impairs intracellular APP metabolism to generate aberrantly processed fragments that induce cytotoxicity.


Inhibition of apoptosis by zinc: a reappraisal. Apoptosis--or programmed cell death--is an active type of cell death, occurring in several pathophysiological conditions. One of the most important characteristics of apoptosis is that cell death is preceded by DNA fragmentation, consequent to the activation of nuclear calcium- and magnesium-dependent endonuclease(s). DNA fragmentation can be inhibited by zinc ions. By using several techniques, such as DNA agarose gel electrophoresis, cytofluorimetric analysis of DNA content and of cell cycle, 3H-thymidine incorporation and trypan blue dye exclusion test, we show that zinc, despite completely inhibiting DNA fragmentation and the consequent loss of nuclear DNA content, does not protect rat thymocytes from spontaneous or dexamethasone-induced death. Our data also suggest that DNA fragmentation, although characteristic, is not a critical event for thymocyte death of apoptotic type.


Isolation and characterization of conglutinin as an influenza A virus inhibitor. Normal horse and guinea pig sera contain alpha 2-macroglobulin which inhibits the infectivity and hemagglutinating activity of influenza A viruses of the H2 and H3 subtypes. On the other hand, normal bovine serum contains a component termed beta inhibitor that inhibits the infectivity and hemagglutinating activity of influenza A viruses of the H1 and H3 subtypes. To investigate the nature of the beta inhibitor of influenza A virus, we purified the conglutinin and examined its characteristics. First, we found a high correlation between the hemagglutination inhibition(HI) titer and conglutinin titer in several bovine sera (r = 0.906, p less than 0.005). The HI of bovine serum was mainly dependent on conglutinin because the HI activity was abrogated by N-acetylglucosamine but not by D-mannose. The conglutinin, purified from bovine serum, had neutralizing-activity as well as HI activity on influenza A viruses of the H1 and H3 subtypes. The HI activity of conglutinin was heat stable (56 degrees C, 30 min), Ca(++)-dependent, and resistant to both neuraminidase and periodate treatments. The HI activity of purified conglutinin was blocked by N-acetylglucosamine but not by D-mannose. The conglutinin was bound to hemagglutinin which had high mannose and complex sugar chains and its binding was inhibited by N-acetylglucosamine and dependent on divalent cations. These data indicate that the beta-like inhibitor activity of bovine serum is mainly dependent on conglutinin which inhibits hemagglutination and neutralizes the virus infectivity by its binding to a carbohydrate site at the HA.


Peptide analogs of the pseudosubstrate domain of smooth muscle myosin light chain kinase inhibit actomyosin ATPase activity at concentrations that do not inhibit superprecipitation. The inhibitory effect of calmodulin antagonists, synthetic peptide analogs of the pseudosubstrate domain of smooth muscle MLC kinase, and an inhibitor based on the sequence of MLC were examined using bovine aortic actomyosin and isolated chicken gizzard MLC. Much lower concentrations of the peptides were necessary to inhibit actomyosin ATPase activity than to inhibit superprecipitation. In contrast, calmodulin antagonists inhibited both ATPase activity and superprecipitation at similar concentrations. The peptide analogs were competitive with isolated MLC, but not calmodulin, for inhibition of MLC kinase. These results suggest that in addition to the calmodulin dependence of MLC phosphorylation, a second calmodulin-like protein may be important in actin-myosin interactions. The data also suggest that the pseudosubstrate hypothesis may not completely account for regulation of MLC kinase activity.


Secretory processing of the Alzheimer amyloid beta/A4 protein precursor is increased by protein phosphorylation. The 39-43 residue polypeptide (amyloid beta protein, beta A4) deposited as amyloid in Alzheimer's disease (AD) is derived from a set of 695-770 residue precursors referred to as the amyloid beta A4 protein precursor (beta APP). In each of the 695, 751, and 770 residue precursors, the 43 residue beta A4 is an internal peptide that begins 99 residues from the COOH-terminus of the beta APP. Each holoform is normally cleaved within the beta A4 to produce a large secreted derivative as well as a small membrane associated fragment. Neither of these derivatives can produce amyloid because neither contains the entire beta A4 peptide. In this study, we employ cells stably transfected with full length beta APP695, beta APP751, or beta APP770 expression constructs to show that phorbol ester activation of protein kinase C substantially increases the production of secreted forms from each isoform. By increasing processing of beta APP in the secretory pathway, PKC phosphorylation may help to prevent amyloid deposition.


Multiple growth factor mRNAs are expressed in chicken adipocyte precursor cells. We have examined the expression of growth factor genes in primary cultures of chicken adipocyte precursors. RNA was extracted from proliferating and differentiated cells, reversed transcribed and amplified by PCR using gene specific primers. The identity of the PCR products was confirmed by restriction mapping. We show, for the first time, constitutive expression of TGF-beta 2, TGF-beta 3, TGF-beta 4 and bFGF genes in chicken adipocyte precursors. We also detect GH-independent, but differentiation-dependent IGF-I gene expression. The synthesis and action of these growth factors supports the hypothesis that they act as autocrine and/or paracrine regulators of adipocyte precursor cell proliferation and differentiation.


Essential role of collagens for terminal differentiation of preadipocytes. In order to study the role of collagens in the differentiation of TA1 preadipose cells in vitro, ethyl-3,4-dihydroxybenzoate (EDHB) was used as a specific inhibitor of collagen synthesis. The secretion of collagenous proteins only was severely decreased after exposure to EDHB, and this was accompanied by a decrease of differentiation as indicated by low activity levels of glycerophosphate dehydrogenase. The effect of EDHB was dose-dependent and also dependent upon the stage of cell differentiation. Northern-blot analysis show that EDHB addition to undifferentiated cells did not prevent the induction of A2COL6 gene, a marker of the preadipose state, but prevented the induction of the gene encoding for the adipocyte lipid binding protein and the modulation of the expression of the lipoprotein lipase gene which are both indicators of the adipose state. These results demonstrate that differentiation of preadipose cells into adipose cells requires active synthesis of collagens during the preadipose state.


The primary structure of TE-6: a novel neuropeptide from the nematode Ascaris suum. Extensive immunoreactivity (IR) towards a hexapeptide (sequence KGQELE), which flanks the C-terminus of the pancreastatin sequence in rat chromogranin A (CGA), is found throughout the nervous system of the nematode parasite Ascaris suum. The peptide IR was purified from the gonoduct of the parasite and found to have the sequence TKQELE. This peptide, designated TE-6, has some C-terminal homology with several regions of the CGA molecule. However, TE-6 was the only peptide isolated suggesting that either the nematode does not possess CGA, or that the -ELE regions of parasite CGA-like peptides which would be larger than TE-6 are not accessible to the antiserum in RIA, or are not being successfully extracted from the parasite. The N-terminus of TE-6 has little homology with any of the sequences preceding -ELE regions in CGA. This, and the fact that the tissue from which TE-6 was isolated does not contain IR towards another, highly conserved, region of the CGA molecule (WE-14) suggests that TE-6 may belong to a new class of regulatory peptide unrelated to CGA.


Cleavage of farnesylated COOH-terminal heptapeptide of mouse N-ras by brain microsomal membranes: evidence for a carboxypeptidase which specifically removes the COOH-terminal methionine. Brain microsomal membranes are capable of sequentially removing Met, Leu and Val from a chemically synthesized COOH-terminal heptapeptide (propionyl-Gly-Ser-Pro-(farnesyl-Cys)-Val-Leu-Met) of mouse N-ras protein. The carboxypeptidase generating Met displays maximum activity at neutral pH and shows high affinity for the farnesylated substrate (Km = 73 microM) as compared to its non farnesylated precursor (Km = 600 microM). The results of inhibitor action suggest that the membrane carboxypeptidase is a novel, probably thiol-dependent, serine type peptidase.


CDw 60 antibodies bind to acetylated forms of ganglioside GD3. Four monoclonal antibodies, M-T21, M-T32, M-T41 and UM4D4, which belong to the new CDw 60 cluster of antibodies specific for a subpopulation of human T-lymphocytes, were found to bind mainly to acetylated forms of ganglioside GD3. After O-deacetylation of the antigen, binding was reduced ("M-T"-antibodies) or abolished (UM4D4).


Chloroacetates of 2- and 3-demethylthiocolchicine: specific covalent interactions with tubulin with preferential labeling of the beta-subunit. We synthesized two chemically reactive A ring modified analogs of colchicine, 2-chloroacetyl-2-demethylthiocolchicine (2-CTC) and 3-chloroacetyl-3-demethylthiocolchicine (3-CTC). Both are similar to colchicine as inhibitors of tubulin polymerization and act as competitive inhibitors of colchicine binding (apparent Ki values, 3 microM). [14C]-labeled 2-CTC and 3-CTC bound to tubulin at 37 degrees C but not at 0 degree C, and bound drug formed covalent bond(s) with tubulin. The binding and covalent reactions were inhibited by podophyllotoxin. About 60% of the bound 3-CTC rapidly formed a covalent bond with tubulin. With 2-CTC the covalent reaction was slower than the binding reaction, and only one-third of the bound 2-CTC reacted covalently with tubulin. The ratio of radiolabel in beta-tubulin to that in alpha-tubulin was about 4:1 with both 2-CTC and 3-CTC.


N,O-diacylated-N-hydroxyarylsulfonamides: nitroxyl precursors with potent smooth muscle relaxant properties. N,O-Diacylated-N-hydroxyarylsulfonamides are capable of slowly releasing nitroxyl (HNO) by simple, non-enzymatic hydrolysis in Krebs solution at 37 degrees C. Release of nitric oxide (NO) was not seen. These compounds were also found to elicit vasorelaxation in rabbit thoracic aorta in vitro, presumably as a result of their ability to release HNO. This effect was enhanced by the addition of superoxide dismutase (SOD). Thus, these results are consistent with previous work indicating that HNO is a potent vasorelaxant.


Tissue-specific response of estrogen receptor gene expression to estrogen in chick. We have developed a sensitive assay system by RT (reverse transcription)-PCR(polymerase chain reaction) to detect the low level of chicken estrogen receptor (cER) transcript. Using this system, the differential expression of cER gene in chick tissues was observed. Moreover, we found that the oviduct cER transcript levels in chick was not affected, albeit the remarkable growth of this tissue, by exogenous estrogen. In contrast, estrogen enhanced the hepatic cER transcript level several folds. Thus, the present study clearly showed a novel tissue-specific response of cER gene expression to estrogen in chick.


A recombinant rat vascular AT1 receptor confers growth properties to angiotensin II in Chinese hamster ovary cells. A rat vascular AT1 receptor cDNA has been stably expressed into Chinese Hamster Ovary cells and the resulting recombinant AT1a receptor has been functionally characterized. This receptor binds 125I Sar1-angiotensin II with an affinity of 0.9 nM and the displacement of this ligand by a series of peptidic and nonpeptidic analogs is shown. Binding of angiotensin II to this receptor causes a rapid increase in inositol phosphate production, whereas this effect is not observed in nontransfected cells. Des-aspartyl1 angiotensin II and at a lesser extent angiotensin I are also able to produce an increase in inositol phosphates. More importantly, the actions of angiotensin II on cell division were clearly demonstrated in this model, since angiotensin II is able to stimulate DNA synthesis by 400% and double the cell population of the transfected cells in 36 hours in the absence of any other growth factor, whereas no effect is observed in nontransfected cells.


A single-stranded DNA binding protein which interacts with sequences within the bovine preprotachykinin promoter: regulation by nerve growth factor. The production of substance P (SP) and the mRNA encoding its precursor preprotachykinin (PPT) is regulated by nerve growth factor (NGF) in dorsal root ganglion neurons. We have shown previously that two regions of the bovine PPT promoter are capable of mediating the induction by NGF of the downstream structural gene in transfected PC12 cells. Both regions contained a sequence element, similar to a known transcription factor binding site, which is present in several other NGF-regulated genes. We show here that PC12 cells contain a single-stranded DNA binding protein (SSBP-PC12) which can interact specifically with this site. Binding activity was increased by NGF treatment of PC12 cells.


Tissue- and cell-specific control of guinea pig cathepsin E gene expression. Northern blotting of RNAs from normal guinea pig tissues revealed that the tissue distribution of cathepsin E mRNA was relatively limited and the highest level of the mRNA was observed in the stomach mucosa. Expression of the mRNA was also observed in the spleen, although the level was very low. These results were in good agreement with the distribution of the cathepsin E-producing cells as revealed by immunohistochemistry. In the separated fractions of dispersed mucosal cells prepared from the stomach by centrifugal elutriation, the extent of cathepsin E mRNA expression was closely correlated with the enrichment of the producing cells. In addition, both CCGG and GCGC sites within the gene region were hypomethylated to a greater extent in the producing tissues than elsewhere, reflecting specific hypomethylation in the producing cells. The observed tissue- and cell-specific transcriptional control of cathepsin E gene, which is correlated with a decreased level of methylation in the gene region, suggests that the enzyme is probably involved in specific functions of particular differentiated cells, especially those of the stomach mucosa.


Quinobene, a new potent anti-HIV agent.

Quinobene, a new potent anti-HIV agent. A simple synthesis of the sulfonated azo dye Quinobene (3) and its derivatives, as well as the results of their evaluation in anti-HIV screening have been described. Thus, reacting the diazonium salt of 4,4'-diaminostilbene-2,2'-disulfonic acid with 8-hydroxyquinoline-5-sulfonic acid yielded the readily isolable title compound. The lithium and tetramethylammonium salts of Quinobene and its complexes with Cu(II), Zn(II), Mg(II) were also prepared. In vitro tests showed considerable activity of these compounds against HIV-1.


Identification of amino acid residues of rat angiotensin II receptor for ligand binding by site directed mutagenesis. To determine the specific mechanism of ligand binding to angiotensin (Ang II) receptor AT1, mutagenized rat receptor cDNAs were expressed transiently in COS-7 cells and the effect of the mutations on the binding to peptidic and non-peptidic ligands was analyzed by Scatchard plots. Mutation of Lys199 to Gln in the intramembrane domain strongly reduced the affinity to both [125I] Ang II and [125I]-1Sar, 8Ile-Ang II whereas mutation of two other Lys had little effect, indicating involvement of Lys199 in binding ligands. Replacement of each of four Cys in the extracellular domain markedly reduced binding affinity, indicating the importance of two putative disulfide bridges in the formation of active receptor conformation. Substitution of Asp for Asn in N-glycosylation had no effect on ligand binding or expression of the receptor. These studies indicate mutated receptors are expressed in the plasma membrane and are amenable for further detailed studies.


Molecular characterization and expression of the cell-associated glucosyltransferase gene from Streptococcus mutans. A gene encoding cell-associated glucosyltransferase (CA-GTase) was cloned from Streptococcus mutans MT8148 into Escherichia coli DH5 alpha by using a low-copy-number plasmid, pMW119. After screening of a gene library with the oligonucleotide probe designed on the basis of a partial amino acid sequence of CA-GTase, a recombinant plasmid, pSK6, that had a 5.6 kb insert carrying the CA-GTase gene was selected. The gene product (recombinant CA-GTase) of pSK6 was expressed by using a lac promoter in pMW119. Western blotting revealed that rCA-GTase reacted with antibody to CA-GTase. rCA-GTase was found to synthesize water-insoluble glucans. Southern blotting indicated that the MT8148 chromosome contained another gene which was homologous to pSK6. A plasmid harboring this gene (pSK16) was also isolated from the gene library, the gene product of pSK16 exhibited GTase activity but ten times lower than that of pSK6.


Oxidation-induced persistent activation of protein kinase C in hippocampal homogenates. Incubation of purified protein kinase C with H2O2 results in the generation of a persistently activated form of the enzyme which is no longer dependent on Ca2+ or lipid cofactors. This oxidative activation of purified protein kinase C requires added Fe2+ in the incubation medium. Treatment of the soluble fraction of hippocampal homogenates with H2O2 also leads to persistent activation of protein kinase C; however, oxidative activation of protein kinase C under these conditions does not require the addition of Fe2+. The persistently activated form of protein kinase C appears as a novel peak of activity on DE52 anion exchange columns, suggesting a modification of the charge character of the enzyme. Thus, oxidative modification of protein kinase C can result in its persistent activation, and this mechanism may constitute a pathway for physiological activation of the enzyme in the hippocampus.


A POU-domain gene of zebrafish, ZFPOU1, specifically expressed in the developing neural tissues. We have isolated a POU domain-containing cDNA (ZFPOU1) from a cDNA library of zebrafish (Brachydanio rerio). The ZFPOU1 cDNA contained an open reading frame encoding a 425 amino acid peptide. The conserved POU domain was located near the carboxy terminus. The deduced amino acid sequence of the reading frame was most similar to that of the mouse class III POU-domain gene, Brain-1. Northern blot analysis revealed that the ZFPOU1 transcripts first appeared at the early neurula stage of embryogenesis and transiently increased thereafter. A significant level of expression, however, was not found in adult tissues except in the brain. In situ hybridization analysis demonstrated that the ZFPOU1 transcripts were localized in the neural tissues of embryos, but not in mesodermal, endodermal or ectodermal tissues. In adult zebrafish, the ZFPOU1 transcripts were detected in the restricted regions of the brain. Spatial and temporal expression patterns suggest that ZFPOU1 has distinct roles in the early neural development of zebrafish.


Diacylglycerol formation from phosphatidylcholine in angiotensin II-stimulated vascular smooth muscle cells. In cultured vascular smooth muscle cells (VSMC), angiotensin II (Ang II) induces a biphasic diacylglycerol (DAG) formation peaking at 15 sec and 5 min. Although it has been well established that the first peak is produced by the hydrolysis of inositol 4,5-bisphosphate (PIP2), the origin of the second DAG peak has never been examined in detail. In the present paper, we provide evidence that the second peak of DAG formation in Ang II-stimulated VSMC originates mainly from PC.


Purification and characterization of a new 120 kDa alkaline proteinase of Trypanosoma cruzi. A new alkaline proteinase activity was identified in cell-free extracts of Trypanosoma cruzi epimastigotes on the basis of its ability to hydrolyze the fluorogenic substrate N-Z-Gly-Gly-Arg-AMC. The optimal activity was at pH 8.0. After a three step-chromatography procedure using two anionic columns (DEAE-Sepharose and Mono Q) and a chromatofocusing column (Mono P), the proteolytic activity was associated with a single 120 kDa protein and was called Tc 120 proteinase. The molecular mass of the proteinase was confirmed by direct visualization of the proteolytic activity using a fluorometric assay on SDS-PAGE. The Tc 120 proteinase which also cleaves N-Z-Arg-AMC, N-Z-Phe-Arg-AMC and N-glutaryl-Gly-Arg-AMC substrates, is a cysteine-type proteinase with an unusual low sensitivity to E-64.


Identification of calcitonin gene related peptide in ovine hypothalamic extract. Calcitonin gene-related polypeptide (CGRP) was purified from ovine hypothalamic extracts. Its amino acid sequence was determined as: Ser-(Cys)-Asn-Thr-Ala-Thr-(Cys)-Val-Thr-His-Arg-Leu-Ala-Gly-Leu-Leu-Ser- Arg-Ser - Gly-Gly-Val-Val-Lys-Ser-Asn-Phe-Val-Pro-Thr-Asn-Val-Gly-Ser-Gln-Ala-Phe- NH2. This sequence differs from rat CGRP by two amino acid substitutions (Ser for Asp25 and Gln for Glu35). Adenylate cyclase stimulating activity in rat pituitary cell cultures was monitored during the isolation. CGRP had adenylate cyclase stimulating activity comparable to corticotropin-releasing hormone, suggesting a hypophysiotropic role for CGRP. This is the first chemical characterization of CGRP in the brain (hypothalamus).


Adenosine modulates cell growth in human epidermoid carcinoma (A431) cells. Adenosine mediates many physiological functions via activation of extracellular receptors. The modulation of cell growth by adenosine was found to be receptor-mediated. In A431 cells adenosine evoked a biphasic response in which a low concentration (approximately 10 microM) produced inhibition of colony formation but at higher concentrations (up to 100 microM) this inhibition was progressively reversed. Evidence for the involvement of A1 (inhibitory) and A2 (stimulatory) adenosine receptors in regulating cell growth of these tumor cells was obtained through plating efficiency studies based on the relative potency of adenosine agonists and antagonists. When both A1 and A2 receptors were blocked, colony formation or growth was not inhibited at low concentrations of adenosine but was inhibited at high adenosine concentrations.


Structure of the human spermidine/spermine N1-acetyltransferase gene (exon/intron gene organization and localization to Xp22.1). The super induction of spermidine/spermine N1-acetyltransferase (SSAT), has been implicated in the cytotoxic response of human solid tumors to the bis(ethyl)polyamines. The SSAT response is a phenotype specific response and is modulated at the level of increased steady-state mRNA levels and enzyme protein. The human genomic region (4,095 bases) containing the coding sequence of SSAT has been cloned and localized to the Xp22.1 region. Primer extension analysis indicates the transcription of SSAT starts 179 bases upstream from the translational start site and appears to be under the control of a "TATA-less" promoter. The availability of this human clone will facilitate the direct functional examination of the SSAT gene.


Neointima formation after vascular injury is angiotensin II mediated. Smooth muscle proliferation of injured blood vessels leads to pathologically significant stenosis in animals and humans. We report here the pharmacological confirmation of an involvement of angiotensin II in this process as a major, necessary mediator of neointima formation. In the rat carotid artery, an animal model of post-angioplastic restenosis, we have obtained by local intraluminal infusion of peptidic angiotensin II antagonist after balloon catheterization, suppression of neointima formation and preservation of the luminal integrity. Sham operated control animals treated without medication and operated control animals treated simultaneously with angiotensin converting enzyme inhibitor and with agonistic angiotensin II, suffered major stenosis through the myoproliferative response of the injured vessel. These results prove that angiotensin II plays a key role as a mediator of vascular neointima formation.


Activation of phospholipase C by the agonist U46619 is inhibited by cromakalim-induced hyperpolarization in porcine coronary artery. We measured inositol 1,4,5-trisphosphate (IP3) production, intracellular calcium concentration ([Ca2+]i) and force of contraction induced by a thromboxane A2 analogue U46619 in porcine coronary artery to elucidate the relaxant effect of a K+ channel opener cromakalim. Cromakalim (10 microM) significantly inhibited the production of IP3, Ca2+ release from intracellular stores and contraction induced by 300 nM U46619. The inhibitory effect of cromakalim on IP3 was blocked by a K+ channel blocker tetrabutylammonium (TBA, 3 mM) and counteracted by 20 mM KCl-induced depolarization. These results suggest that the hyperpolarization of the plasma membrane by cromakalim inhibits the activation of phospholipase via the stimulation of the thromboxane A2 receptor to result in vasodilation.


Synthesis of 6-substituted tetrazolo[1,5-c]pyrimidin-5(6H)ones: new modification of 3'-azido-3'-deoxythymidine. An efficient method for the preparation of 6-methyltetrazolo-[1,5-c]pyrimidin-5(6H)ones using as the precursors 4-chloro-1,2-dihydro-1-methyl-2-oxopyrimidines is described. The method is also extended to the synthesis of novel tetrazole analogue of 3'-azido-3'-deoxythymidine (AZT).


An ND-6 mitochondrial DNA mutation associated with Leber hereditary optic neuropathy. A mitochondrial DNA mutation at nucleotide position 14,484 was found in 14 independent probands with Leber hereditary optic neuropathy and in 0/250 controls. The 14,484 mutation, which changes methionine-64 to valine in a conserved domain of the ND-6 gene, occurred in association with a mitochondrial DNA haplotype that includes the 13,708 secondary mutation in 10/14 probands. An associated mutation at nucleotide position 3,394, which changes conserved tyrosine-30 to histidine in the ND-1 gene, was observed in 5/14 probands positive for the 14,484 mutation, all of whom harbored the same mitochondrial DNA haplotype. Multiple mitochondrial DNA mutations may interact in the pathogenesis of Leber hereditary optic neuropathy and the 13,708 secondary mutation appears to play a central role in this process.


Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor. Vascular endothelial cell growth factor (VEGF), also known as vascular permeability factor, is an endothelial cell mitogen which stimulates angiogenesis. Here we report that a previously identified receptor tyrosine kinase gene, KDR, encodes a receptor for VEGF. Expression of KDR in CMT-3 (cells which do not contain receptors for VEGF) allows for saturable 125I-VEGF binding with high affinity (KD = 75 pM). Affinity cross-linking of 125I-VEGF to KDR-transfected CMT-3 cells results in specific labeling of two proteins of M(r) = 195 and 235 kDa. The KDR receptor tyrosine kinase shares structural similarities with a recently reported receptor for VEGF, flt, in a manner reminiscent of the similarities between the alpha and beta forms of the PDGF receptors.


A novel fluorescent fatty acid, 5-methyl-BDY-3-dodecanoic acid, is a potential probe in lipid transport studies by incorporating selectively to lipid classes of BHK cells. The 5-methyl-BDY-3-dodecanoic acid (B12FA) labelling of BHK cell lipids was analyzed by thin layer and reverse phase column chromatography. Incorporation to phospholipids was selective: over 90% of B12FA label was enriched in phosphatidylcholine. The major molecular species of PC was that containing palmitate as the unlabelled fatty acid. Small amounts of label was also found in other phosphoglycerides, but not in sphingomyelin. Triglycerides and diglycerides constituted the main B12FA-labelled neutral lipid classes; however, no label was found in cholesterol esters. B12FA was degraded to shorter homologues, which had significantly slower lipid incorporation rates. B12FA-labelled cells displayed in a microscope initially green reticular type fluorescence, but later red spherical structures, representing neutral lipid droplets, could also be seen. It is concluded that B12FA does not incorporate indiscriminately to all lipid classes of BHK cells, but is enriched to PC, diglycerides and triglycerides, which could be utilized in studies on lipid transport as well as metabolism.


DNA strand cleavage at 8-hydroxyguanine residues by hot piperidine treatment. The two findings indicate that 8-hydroxyguanine(8-OH-Gua) is a hot piperidine-sensitive lesion in DNA. These are cleavages of DNA containing 8-OH-Gua at the site of this residue and decomposition of 8-hydroxydeoxyguanosine when the DNA and nucleoside were treated in 1 M piperidine for 30 min at 90 degrees C. However, no cleavage was observed in DNA containing 8-hydroxyadenine or O6-methylguanine. 8-OH-Gua was found to be different from apurinic sites that are also alkali-labile lesions since the former was more resistant to alkali treatment. This property of 8-OH-Gua can be used as a check for the incorporation of this base into DNA after the synthesis of DNA containing 8-OH-Gua at a specific position or possibly can be one of the markers for the identification of 8-OH-Gua formed in DNA exposed to reactive oxygen species.


Mouse hepatic microsomal oxidation of aliphatic aldehydes (C8 to C11) to carboxylic acids. Addition of saturated and alpha, beta-unsaturated aliphatic aldehydes (C8 to C11) significantly increased NADPH oxidation with mouse hepatic microsomes, and the aldehydes themselves were oxidized to the corresponding carboxylic acids. When these aldehyde substrates were incubated similarly under oxygen-18 gas and the carboxylic acids formed were analyzed by GC-MS after methylation, it was indicated that oxygen-18 was significantly incorporated into the carboxylic acids formed from alpha, beta-unsaturated aldehydes, but not significantly into the carboxylic acids formed from saturated aldehydes. These results indicate that enzyme and/or mechanism responsible for the oxidation of these two types of aldehydes is different from each other.


Differential regulation of putrescine uptake in Trypanosoma cruzi and other trypanosomatids. Putrescine uptake in Trypanosoma cruzi epimastigotes is 10 to 50-fold higher than in Leishmania mexicana or Crithidia fasciculata. Polyamine transport in all these trypanosomatids is an energy-dependent process strongly inhibited by the presence of 2,4-dinitrophenol or KCN. Putrescine uptake in T. cruzi and L. mexicana was markedly decreased by the proton ionophore carbonylcyanide m-chlorophenylhydrazone but it was not affected by ouabain, a Na(+)-K+ pump inhibitor. The depletion of intracellular polyamines by treatment of parasite cultures with alpha-difluoromethylornithine elicited a marked induction of putrescine uptake in L. mexicana and C. fasciculata by increasing considerably the Vmax of this process. Conversely, the uptake of putrescine in T. cruzi was essentially unchanged by the same treatment. The differential regulation of putrescine transport in T. cruzi might be related to some distinctive features of polyamine metabolism in this parasite.


Identification of heptanal and nonanal in bronchoalveolar lavage from rats exposed to low levels of ozone. Heptanal and nonanal are identified from in vitro studies as potential biomarkers of exposure to ozone, the former resulting from ozonation of palmitoleic acid and the latter from oleic acid. An analytical method is developed based on derivatization using O-pentafluorobenzylhydroxylamine HCl and gas chromatography. These molecules also are present in the lung lavage of Sprague-Dawley rats exposed to 1.3 ppm ozone for 10 hr. These results suggest aldehydes may be useful dosimeters for ozone and indicate that unsaturated fatty acids in the lung lining fluid layer undergo ozonation in vivo.


Hormonal and nutritional stimuli modulate apolipoprotein B mRNA editing in mouse liver. Human livers produce apoB-100, a major protein of VLDL, while intestines produce apoB-48, the major protein of chylomicrons. ApoB-48 is translated from apoB-100 mRNAs that are post-transcriptionally edited at codon 2153, converting CAA (glutamine) to TAA, a stop codon. In contrast to humans, mouse and rat livers contain the apoB-100 mRNA editing mechanism. Because hormones and nutrients affect the metabolism of apoB containing lipoproteins, we studied the effects of sex hormones and diets on apoB mRNA editing. Groups of male and female C3H/HeJ mice were castrated and treated with 17 beta-estradiol at 0.16 (E2L) or at 5 micrograms (E2H), or with testosterone propionate at 1 microgram/g body weight/day for 14 days. Plasma apoB levels and ratios of apoB-100/apoB-48 both increased 2-fold, but only in the E2H group. To determine if the increased apoB-100/apoB-48 ratios were associated with altered levels of apoB-100 and apoB-48 mRNA, both forms of apoB mRNA were quantified. We found that indeed ApoB-100 mRNA increased 1.8-fold (p < 0.025) compared to apoB-48 mRNA only in the E2H group. Next, we studied the individual effects of dietary fatty acids and dietary cholesterol on the relative abundance of apoB-100 and apoB-48 mRNA. Contrary to the estrogen effect, the high fat-combination diet increased apoB-48 mRNA relative to apoB-100 mRNA. Total plasma apoB as well as apoB-48 synthesis in liver also increased. Our studies demonstrate that estrogens and high fat diet both modulate apoB editing in mouse liver, but that estrogens and fat diet affected apoB mRNA editing in opposite directions.


Direct evidence for protein kinase C involvement in insulin-stimulated hexose uptake. Insulin has been reported to translocate protein kinase C (PKC) in rat adipocytes, and activation of PKC by phorbol esters is known to increase hexose uptake in these cells (1.2). To test the hypothesis that PKC may participate in insulin-stimulated hexose uptake, adipocytes were partially depleted of protein kinase C by overnight phorbol ester treatment, thereby impairing insulin effects on hexose uptake. Purified PKC was then introduced into these PKC-depleted adipocytes by electropermeabilization, and this fully restored insulin-stimulated hexose uptake. These findings provide direct evidence that PKC is required for insulin-stimulated hexose uptake.


Distribution of GLUT3 glucose transporter protein in human tissues. To investigate the tissue distribution of the GLUT3 glucose transporter isoform in human tissue we produced affinity purified antibodies to the COOH terminus of the human GLUT3. Both antibodies recognize a specific GLUT3 band in oocytes injected with GLUT3 mRNA but not in those injected with H2O or GLUT1, 2, 4, 5 mRNA. This immunoreactive band in GLUT3 injected oocytes is photolabelled by cytochalasin-B in the presence of L- but not D-glucose indicating that it is a glucose transporter. A high cross reactivity between the human GLUT3 antibodies and a 43 kDa cytoskeletal actin band was identified in all oocyte lysates and many human tissues. However, the specific GLUT3 band could be distinguished from the actin band by carbonate treatment which preferentially solubilized the actin band. Using these antibodies we show that GLUT3 is present as a 45-48 kDa protein in human brain with lower levels detectable in heart, placenta, liver and a barely detectable level in kidney. No GLUT3 was detected in membranes from any of 3 skeletal muscle groups investigated. We conclude that a major role of GLUT3 in humans is as the brain neuronal glucose transporter.


A new synthetic hypoglycaemic polysaccharide. The synthetic (1-->6)-alpha-D-glucopyranan with branching and without branching were tested as a new hypoglycaemic drug. (1-->6)-alpha-D-glucopyranan having an alpha-D-glucopyranosyl branch at the C-3 position (1) showed a remarkable hypoglycaemic activity on i.p. injection to mice. The polysaccharide having both alpha- and beta-glucopyranosyl branches (2) also lowered the blood sugar (glucose) level in mice. On the other hand, the synthetic linear (1-->6)-alpha-D-glucopyranan (3) and alpha-D-glucopyranosyl branched polysaccharide (4) did not have a hypoglycaemic function, indicating that the branching glucose units are essential for the biological activity.


Presence of nuclear factors bound to both cAMP-responsive element and AP1 factor binding site in the porcine anterior pituitary. Factors binding to consensus sequences of the cAMP-responsive element (CRE) and the AP1 factor binding site (AP1) were investigated using porcine anterior pituitary nuclear extracts. Each element showed specific gel mobility shifts. By reciprocal competition for the AP1 and CRE binding, CRE prevented AP1 binding completely. On the other hand, AP1 decreased the CRE binding considerably to 20%, suggesting that approximately 80% of the total CRE binding is due to factors which bind to a common site shared by both CRE and AP1, whereas proteins binding to AP1 alone are absent. Relative binding affinities of AP1 against CRE estimated from the reciprocal competition data were 0.17 for CRE binding and 0.56 for AP1 binding. UV cross-linking experiments showed that CRE and AP1 gave different patterns consisting of different molecular size. Inconsistency of the relative binding affinities and the multiple molecular size of binding factors, cannot be explained simply by the presence of two types of binding factor, common CRE/AP1-binding and specific CRE-binding factors. A more likely explanation is that the CRE/AP1-binding factors alter the dimer form by changing each respective partner to bind CRE and/or AP1.


The adenovirus E3 region 14.7 kDa protein, heat and sodium arsenite inhibit the TNF-induced release of arachidonic acid. In this report we show that the adenovirus E3 region 14.7 kDa protein, heat and sodium arsenite, which have been defined previously as inhibitors of cytolysis, inhibit the tumor necrosis factor-alpha (TNF)-induced release of 3H-arachidonic acid from cycloheximide-sensitized C3HA fibroblasts. Since the A23187-induced release of 3H-a.a. was unaffected, our results suggest that these inhibitors provide resistance to lysis by selectively interfering with the lytic response pathway. Our results also show that heat and sodium arsenite can themselves induce the release of 3H-arachidonic acid. These results raise the possibility that stressor-induced resistance to TNF results from the selective desensitization of phospholipase A2.


High reactivity of aortic fibroblasts to vasoactive agents: endothelins, bradykinin and nucleotides. Cultured aortic fibroblasts express high affinity Et-1 binding sites that poorly discriminate between Et-1 and Et-3. Both endothelins activate phospholipase C hence indicating the presence of ETB receptors. Fibroblasts respond to bradykinin by large activations of phospholipase C and increases in [Ca2+]i in a manner that was abolished by D-Arg, [Hyp3,Thi5,8,D-Phe7]-bradykinin, thus indicating the presence of B2 kinin receptors. Finally, ATP, UTP and ADP increases [Ca2+]i in aortic fibroblasts via a nucleotide receptor that has a higher affinity for ATP and UTP (3 microM) than for ADP (50 microM) and that is distinct from P2x and P2y purinoceptors.


High pressure induced inactivation of ferrous cytochrome P-450 LM2 (IIB4) CO complex: evidence for the presence of two conformers in the oligomer. The effect of high pressure on the spectral properties of cytochrome P-450 LM2(Fe2+)-CO complex was studied. The application of high pressure was shown to induce the conversion of cytochrome P-450 to P-420. In the solution when P-450 was oligomeric only about 65% of the total converted to P-420. The remaining portion of cytochrome P-450 was stable at pressures up to 6 kbar. When P-450 was incorporated into membranes or when it was succinylated, the proportion of the pressure sensitive fraction was slightly higher (about 75%). Dissociation of P-450 oligomers into monomers was made by addition of 0.2% Triton N-101. Monomers were the most sensitive to pressure; they could be completely converted to P-420. These results have been interpreted as evidence for the existence of two different conformers of P-450 LM2, which differ in pressure stability. Splitting between these two states appears to be a result of the oligomeric organization of cytochrome P-450 in solution and in the membrane.


Tyr-179 and Lys-183 are essential for enzymatic activity of 11 beta-hydroxysteroid dehydrogenase. Tyr-179 and Lys-183 are likely to be functionally important residues in 11 beta-hydroxysteroid dehydrogenase, as these amino acids are absolutely conserved in all members of the "short chain dehydrogenase" family. We modified these residues by site-directed mutagenesis of rat cDNA and transfected these constructs into CHO cells. A highly but not absolutely conserved residue, Asp-110, was also studied. Mutation of Tyr-179 to Phe or Ser completely abolished enzymatic activity (interconversion of corticosterone and 11-dehydrocorticosterone), as did Lys-183-->Arg. Asp-110-->Asn affected activity only mildly. Tyr-179 and Lys-183 may be directly involved in the catalytic function of this class of enzymes.


Casein kinase-2 structure-function relationship: creation of a set of mutants of the beta subunit that variably surrogate the wildtype beta subunit function. Nine mutants of human casein kinase-2 beta subunit have been created and assayed for their ability to assemble with the catalytic alpha subunit to give, at a 1:1 molar ratio, a fully competent CK-2 holoenzyme as judged by the following criteria: 1) the generation of an active heterotetrameric form of CK-2 exhibiting the expected sedimentation coefficient and 2) the enhancement of catalytic activity of CK-2 alpha. Extended deletions of 71 and 44 residues from the C-terminal end, but not a 7 residue deletion (including the cdc2 phosphorylation site) prevent both reconstitution of the holoenzyme and, consequently, stimulation of activity. This indicates that residue(s) located in the 171-209 sequence is essential for reconstitution. Also a four residue's N-terminal deletion (removing the autophosphorylation site) and single to quintuple substitutions of alanine for the acidic residues clustered in the 55-70 sequence give rise to mutants that still assemble with the alpha subunit to give a tetrameric holoenzyme. However, in the case of the mutants A57,59, A63,64, A59-61,63,64 in vitro assembly with the CK-2 alpha subunit was not complete. There were also intermediate complexes, free alpha-subunit and beta-mutants found to sediment at various positions in the sucrose density gradient. In comparison to CK-2 beta +, mutants A57,59, A59-61 and A59-61,63,64 show an increased stimulation of the catalytic activity supporting the view that these residues play a crucial role in determining the basal activity of reconstituted CK-2 holoenzyme.


Iodothyronine 5-deiodinase in rat posterior pituitary. We first describe the presence of iodothyronine 5-deiodinase (5D) in the neural lobe of rat pituitary. 6-n-Propyl-2-thiouracil (PTU), a specific inhibitor of type-I deiodinase, had no effect, showing that 5D in neurohypophysis is of type-III isozyme, which is specific for 5-deiodination and has been found only in the brain, placenta and skin. The presence of 5D (type-III) together with our previous report of 5'-deiodinase (type-I in euthyroidism and type-II in hypothyroidism) shows that the isozymes of deiodinases in the neurohypophysis are quite similar to those in the brain. These data suggest a previously unrecognized role of thyroid hormone in posterior pituitary physiology.


Anchored anti-HLA class I monoclonal antibody fails to induce inhibition of PHA-activated lymphocytes proliferation. It is known that anti-HLA Class I antibodies inhibit the proliferative response of PHA-activated T-lymphocytes. We found that plastic- or sepharose-linked anti-HLA Class I monoclonal antibody 01.65 does not inhibit either [3H]Thymidine incorporation or recruitment in the cell cycle, nor does it reduce the expression of c-myc mRNA and the membrane expression of Interleukin-2 Receptor and Transferrin Receptor. Furthermore, particulate Protein Kinase C is not affected by anchored anti-HLA Class I monoclonal antibody 01.65. We suggest that anti-HLA Class I monoclonal antibody may act through crosslinking or internalization of HLA Class I antigens.


Endothelin-1 stimulates its own synthesis in human endothelial cells. We studied whether endothelin-1 (ET-1) would affect its own synthesis. Human umbilical cord vein endothelial cells in methionine-poor culture medium containing [35S] methionine were treated with synthetic ET-1 or ET-3. Immunoprecipitation of 35 S-labeled ET-1 was performed with rabbit ET-1 antiserum. ET-1 caused an 40 +/- 4% (mean +/- SEM) increase of immunoprecipitable 35 S-labeled ET-1 as confirmed by its elution point in reversed phase high power liquid chromatography (HPLC). ET-3 caused a 23 +/- 2% increase in ET-1 concentration. Amplification of cDNA by PCR showed both ET-1 and ETB receptor mRNAs in human cord vein endothelial cells. We conclude that ET-1 increases its own synthesis in endothelial cells. This suggests a positive autocrine feed-back action of ET-1 on its own synthesis, an effect which is probably mediated by non-specific ETB receptors.


Differential regulation of rat AT1a and AT1b receptor mRNA. Rat type 1 angiotensin II receptor has two subtypes, namely type 1a and type 1b. The regulation of the expressions of these two subtype receptor mRNAs was studied by using a competitive polymerase chain reaction method. The expression of the type 1a mRNA in the liver was negatively and that of the type 1b mRNA in the adrenal was positively modulated by bilateral nephrectomy. In the ventricle of 16 week old Spontaneously hypertensive rat, the expression level of the AT1b receptor mRNA was higher than that in the ventricle of the age-matched Wistar-Kyoto rat, while the expression levels of the AT1a mRNA in the ventricle were almost similar between the two strains at this age. Although type 1a and type 1b have almost similar functional properties, the expressions of their mRNAs were differentially regulated.


Tumor necrosis factor and interleukin-1 induce activin A gene expression in a human bone marrow stromal cell line. Activin A, a homodimer of the beta A chain, regulates hematopoiesis. In a human bone marrow-derived stromal cell line, KM-102, phorbol myristate acetate, tumor necrosis factor-alpha and interleukin-1 beta induced great increases in beta A chain mRNA levels and production of activin A activities. The phorbol ester-induced beta A chain gene expression was inhibited by cycloheximide and down regulation of protein kinase C, whereas the cytokine-induced expression was little affected by these treatments. These results indicate that the inflammatory cytokines directly stimulate beta A chain gene expression via protein kinase C-independent pathways.


A vasopressor factor partially purified from human parathyroid glands. Recently, a parathyroid hypertensive factor was postulated to play a role in the pathogenesis of hypertension in genetically hypertensive rats. Therefore it was examined, whether in human parathyroid glands a vasopressor substance can be detected. For this purpose, homogenates of hyperplastic parathyroid glands from 20 patients with tertiary hyperparathyroidism were deproteinized and fractionated by gel chromatography. The fractions obtained were tested for vasopressor activity in isolated perfused rat kidneys. A vasopressor fraction containing substances of 0.6-2.5 kDa was identified in the parathyroid glands. The responsible product was heat sensitive, peptidase-, trypsin- and carboxypeptidase y- sensitive and hydrophilic, as it did not bind to hydrophobic reversed-phase gel. These results suggest that parathyroid glands contain a hydrophilic peptide-like vasopressor substance different from the parathyroid hormone.


Insulin inhibits norepinephrine overflow from peripheral sympathetic nerve ending. The effects of insulin on peripheral nervous system are unknown. We therefore studied the effects of insulin on sympathetic nerve activity in isolated mesenteric arteries of Sprague-Dawley rats. The overflow of norepinephrine (NE) by electrical stimulation was used as the index of sympathetic nervous system activity. Insulin (0.5 to 1U/l) decreased the NE release in a dose-dependent fashion. This inhibitory effect was, however, reversed by either 5 x 10(-5)M cocaine or 5 x 10(-4)M ouabain treatment. Thus, we postulate that insulin attenuates NE overflow from peripheral sympathetic nerve endings, probably due to enhanced NE reuptake.


cDNA cloning of rat LRP, a receptor like protein tyrosine phosphatase, and evidence for its gene regulation in cultured rat mesangial cells. Protein tyrosine phosphatases (PTPases) are a family of enzymes that play a crucial role in the regulation of signal transduction mediated by reversible protein tyrosine phosphorylation. To understand the significance of PTPases in physiological and pathophysiological processes in the kidney, we isolated three cDNA segments encoding PTPases (LAR, LRP and a novel PTPase) from rat kidney by polymerase chain reaction (PCR). Using PCR product as a probe, we isolated a full-length cDNA of rat LRP. LRP cDNA encoded a single membrane spanning protein consisted of 796 amino acids, with two tandemly located intracellular PTPase domains. By Northern analysis, a ubiquitous pattern of LRP gene expression in rat tissues was demonstrated. In cultured rat mesangial cells, LRP mRNA was detected and the mRNA level was suppressed by either interleukin-1 or interleukin-6 treatment.


Effect of sphingoid bases on basal and insulin-stimulated 2-deoxyglucose transport in skeletal muscle. Incubation of rat soleus muscles with 50 microM sphingosine or 50 microM sphinganine augmented basal 2-deoxy-D-glucose (2DG) transport 32%, but reduced the response to 0.1 and 1.0 mU insulin/ml by 17 and 27%, respectively. When the muscles were incubated with 50 microM phytosphingosine, a 63-93% increase in basal 2DG transport was observed. However, this treatment had no effect on insulin-stimulated 2DG transport. The phytosphingosine-induced increase in basal 2-DG transport was inhibited 93 and 98% with 35 and 70 microM cytochalasin B, respectively, suggesting that it is mediated by glucose transporters. Cellular accumulation of L-glucose, which is not mediated by glucose transporters, was not affected by phytosphingosine. It is concluded that (a) both sphingosine and sphinganine increase basal 2DG transport in muscle but diminish insulin-stimulated transport, and (b) phytosphingosine stimulates basal 2DG transport in muscle by a mechanism involving glucose transporters.


Organotin-flavone complexes: a new class of fluorescent probes for F1F0ATPase. Fluorescent 5-coordinate organotin-flavone complexes of 3-hydroxy-flavone (Hof) and 3,5,7,2',4',-pentahydroxyflavone (morin) are good inhibitors of mitochondrial F1F0ATPase but do not inhibit F1-ATPase and they have been examined as possible fluorescent probes of F1F0ATPase. R2SnX (morin) complexes exhibit low fluorescence enhancement on binding to mitochondrial membranes with no displacement by equimolar tributyltin. In contrast R2SnX (of) complexes exhibit high fluorescence enhancement whose extent is variable and is displacable by equimolar tributyltin. Fluorescence enhancement by R2SnX (of) complexes correlates with the ATPase I50 values. Dialkyltin-3-hydroxy flavone, R2SnX(of), complexes act as a new class of fluorescent probes which titrate the F0 segment of F1F0ATPase.


Deficiency of 8-hydroxyguanine DNA endonuclease activity and accumulation of the 8-hydroxyguanine in mutator mutant (mutM) of Escherichia coli. In this report, we have characterized mutM mutant with respect to its ability to repair 8-hydroxyguanine (oh8Gua) in DNA. The oh8Gua DNA endonuclease activity in mutM strain was minimal as compared with that in the wild-type cells. The presence of oh8Gua in DNA of mutM was 6-fold that of the wild-type strain corresponding to a characteristically higher frequency of G.C-->T.A transversions in this mutant strain. These results suggest that mutator phenotype of mutM is at least partially due to a spontaneous accumulation of oh8Gua resulting from a greatly reduced oh8Gua DNA endonuclease activity.


Novel missense mutation in cardiac beta myosin heavy chain gene found in a Japanese patient with hypertrophic cardiomyopathy. We have analyzed the exon 9, 13, 14, 15, and 16 of cardiac beta myosin heavy chain gene in 96 Japanese patients with hypertrophic cardiomyopathy by using PCR-DNA conformation polymorphism analysis. The analysis revealed a sequence variation of the exon 16 in one patient. The sequence variation of a G to C transversion with replacement of Asn by Lys at the codon 615 was confirmed by sequencing and by dot-blot hybridization with an allele-specific oligonucleotide probe. Because the missense mutation was found at the residue conserved through birds to humans, this mutation was suggested to be a cause of hypertrophic cardiomyopathy in the patient. This is the first report of a mutant cardiac beta myosin heavy chain gene in the Japanese population.


The synthesis of cadystins, heavy metal chelating peptides, is induced in the fission yeast by wounds of the cell wall or by incubation with chitosan. It has been shown that heavy metal administration induced the synthesis of cadystins, the small metal chelating peptide with the general structure of (gamma-glu-cys)n-gly, in the fission yeast and in plants. Besides heavy metals, wounds to the cell surface or the incubation with chitosan induced the cadystin synthesis in the fission yeast. Under these induction conditions, the membrane permeability of the fission yeast significantly increased suggesting the structural alteration of the membrane. In these induction, the synthesized cadystins formed complexes with the cellular zinc ions together with or without glutathione.


Cloning of a major heparan sulfate proteoglycan from brain and identification as the rat form of glypican. We have obtained the complete coding sequence of a highly conserved heparan sulfate proteoglycan which we previously characterized biochemically after isolation from rat brain. An open reading frame of 558 amino acids encodes a protein with a molecular mass of 62 kDa containing three peptide sequences present in the isolated proteoglycan. The total sequence obtained is 3.5 kb long, including 1.6 kb of 3'-untranslated sequence and 0.2 kb of 5'-untranslated sequence. The deduced amino acid sequence and the 3'- and 5'-untranslated sequences have 89% and 66-80% identity, respectively, with those of a phosphatidylinositol-anchored human lung fibroblast heparan sulfate proteoglycan (glypican) for which mRNA is detectable in a large number of human cell lines. Our data therefore demonstrate that this major heparan sulfate proteoglycan of brain is the rat form of glypican.


Mutations at the putative junction sites of the yeast VMA1 protein, the catalytic subunit of the vacuolar membrane H(+)-ATPase, inhibit its processing by protein splicing. A single gene, VMA1, encodes the 69-kDa subunit of the vacuolar membrane H(+)-ATPase in the yeast Saccharomyces cerevisiae. We have proposed that the subunit is synthesized as a precursor of 120 kDa (1,071 amino acids) and then converted to the 69-kDa form by an unusual processing reaction, which removes the internal domain of 454 amino acids (residues 284-737) and joins the N- and C-terminal domains. Cysteine to serine mutations at residues 284 and 738, the residues that bracket the internal domain, were introduced into the VMA1 gene by site-directed mutagenesis, and the mutant genes were expressed in a null vma1 mutant. Cells harboring either of the mutant vma1 genes accumulate nonfunctional fragments of the subunit. The mutation of Cys-284 inhibited the cleavage of the N-terminal junction site. Cys-738-->Ser mutation appeared to block the processing at both junction sites although the mutant gene yielded a small fraction of the functional 69-kDa subunit.


Presence and turnover of adenosine diphosphate ribose in human erythrocytes. ADP-ribose was detected in human red blood cells (RBC) at 0.45 +/- 0.1 microM concentrations. These levels could be estimated after purification of ADP-ribose by means of three sequential HPLC fractionations of RBC extracts. Extraction was performed by sonication of RBC either in trichloroacetic acid, followed by centrifugation, or in carbonate-bicarbonate buffer, pH 10.0, followed by rapid ultrafiltration. Neither procedure of extraction caused artefactual formation of ADP-ribose. Prolonged incubation of intact RBC in isotonic buffer containing labeled orthophosphate resulted in the slow incorporation of radioactivity into ADP-ribose. Identification of the labeled ADP-ribose was confirmed upon incubation of the purified metabolite with nucleotide pyrophosphatase, yielding radioactive 5'-AMP and ribose 5-phosphate, while its exposure to a nonspecific deaminase resulted in the quantitative formation of labeled inosine diphosphate ribose.


Preliminary characterization of a nuclear factor interacting with the silencer element at the 3'-side of the chicken alpha-globin gene domain. A silencer element has been identified previously on the 3'-side of the chicken alpha-globin genes placed next to the major enhancer in this domain (Recillas Targa et al., unpublished). Deletion fragments of this negative element show the requirement of the entire DNA segment for maximum silencing activity. Additionally, the sub-fragments including the previously defined SF1 (Silencer Factor 1) sequence seem to play an important role on the negative action of this silencer. Preliminary characterization of the nuclear factor interacting with the SF1 sequence was carried out. Methylation interference experiments show the contact points and, by electrophoretic retardation analysis with SF1 and SF1-mutated oligonucleotides, the critical role of these contacts points on the formation of the DNA-protein complex could be demonstrated. These results provide a basis for further experiments on the characterization and understanding of the mechanisms of action of the coupled positive and negative regulatory elements.


Characterization of cysteine residues of glutathione S-transferase P: evidence for steric hindrance of substrate binding by a bulky adduct to cysteine 47. Glutathione S-transferase P (GST-P) lost the enzymatic activity by 7-fluoro-4-sulfamoyl-2, 1, 3-benzodiazole (ABD-F), a thiol-group chemical modifier, but did not by methylmethanethiol-sulfonate. Both ABD-F and methylmethanethiolsulfonate reacted with Cys47 and Cys101. These two cysteine residues were site-directedly mutated with serine residues. Only the Cys101Ser lost the enzymatic activity by the treatment of ABD-F. On carbon 13 NMR experiments, a NMR signal of S-[13C]CH3 adduct to Cys47 did not show any change by the addition of S-hexylglutathione. These facts revealed that Cys47 did not locate at the active site, and a bulky adduct to Cys47 hindered the binding of substrates to the active site.


An S1 nuclease-sensitive homopurine/homopyrimidine domain in the PDGF A-chain promoter contains a novel binding site for the growth factor-inducible protein EGR-1. Transcription of the platelet-derived growth factor (PDGF) A-chain gene is activated in cells exposed to growth factors. We now have identified a homopurine/homopyrimidine domain in the promoter region of the PDGF A-chain gene that exhibits S1 nuclease sensitivity in vitro and that contains a novel binding site (5'-TCCTCCTCCTCCTC-3') for the growth factor inducible transcription factor EGR-1 as demonstrated in gel mobility shift assays. Sequences similar to this novel EGR-1 binding site were observed also in five growth-related genes and shown to bind to EGR-1 in competition assays, suggesting that EGR-1 may influence the transcriptional regulation of these growth-related genes.


Localization of an adipocyte-specific retinoic acid response domain controlling S14 gene transcription. S14 gene transcription is induced by retinoic acid (RA) in cultured adipocytes, but not preadipocytes. Transfection of 3T3-L1 cells with S14-CAT fusion genes showed that adipocyte-specific RA responsive cis-acting elements are located between -1588 and -1381 bp upstream from the 5' end of the S14 gene. This region has enhancer-like properties. In contrast, an artificial RA response element conferred RA control to a CAT gene in both preadipocyte and adipocyte phenotypes indicating that the RA regulatory network is functional in both phenotypes. These studies show that RA receptors interact with adipocyte-specific transcription factors to control S14 gene expression.


The interplay between basicity, conformation, and enzymatic reduction in biliverdins. Biliverdins with extended conformations are reduced by biliverdin reductase (BvR) at higher rates than biliverdins with helical conformations. To find out the molecular basis for this important feature of BvR mechanism, helical and extended biliverdins were titrated for their acid-base equilibria in a protic solvent (methanol). It was found that the basicity of biliverdins increases with the stretching of the conformation. Biliverdin IX gamma (all-syn) has a pKa = 3.6; 5,10,15-syn,syn,anti-biliverdin has a pKa = 3.7; 5,10,15-syn,anti,syn-biliverdin has a pKa = 6.1; 5,10,15-syn,anti,anti-biliverdin has a pKa = 6.4; and 5,10,15-all-anti-biliverdin has a pKa = 7.9. The increase in basicity with progressive stretching of conformations closely parallels the increase in the reduction rates by BvR. A biliverdin constrained by a four carbon chain to a helical conformation and which is a very weak base (pKa = 0.4) is not reduced by BvR. Nucleophilic additions of 2-mercaptoethanol at the C10 in biliverdins closely parallel their basicities, as can be expected if the formation of a positive mesomeric species at C10 is linked to the basicity (i.e., the ease of protonation) of the N23 on the pyrrolenine ring.


Structure and chromosomal location of the human granulin gene. Granulins are a family of cysteine rich polypeptides some of which have growth modulatory activity. We showed previously that the granulins are encoded within the same precursor consisting of seven granulin domains arranged in tandem. Here we report the chromosomal location and structural organization of the protein coding region of the granulin gene. The granulin gene was assigned to chromosome 17 using DNA from human-hamster somatic cell hybrids. The protein-coding region of the granulin gene was shown to comprise 12 exons covering about 3700 bp. Each tandem granulin repeat is encoded by two non-equivalent exons, a configuration unique to the granulins that would permit the formation of hybrid granulin-like proteins by alternate splicing.


Further evidence of the SA gene as a candidate gene contributing to the hypertension in spontaneously hypertensive rat. We have recently reported that the allele of the SA gene of the Spontaneously hypertensive rat (SHR) has a capacity to influence blood pressure in a F2 rat population prepared from SHR and Wistar-Kyoto rat. In the present study, we have undertaken a similar genetic co-segregation analysis of the F2 rat population prepared from SHR and Lewis rat. The result indicated that, although overall effects of the SA gene genotypes on blood pressure were not significant, a correlation of the genotypes of the SA gene with blood pressure was significantly observed in the female rats of this population. The present results further strengthen our hypothesis that the SA gene, or a gene closely linked to this gene, has a capacity to influence blood pressure.


Effect of a mechanical tension on the hydration of DNA in fibres. Fiber X-ray diffraction and measurement of fibre dimensions yield information about the effects of a mechanical tension on hydration of DNA in fibres. At a given relative humidity, the mechanical tension changes the DNA conformation but does not modify the number of water molecules associated to a nucleotide. The number of water molecules per nucleotide necessary to maintain B form decreases for increasing tensions applied to the DNA fibre. Form transitions can be opposed by mechanical tensions; an energy of 1 Kcal per mole of nucleotide pairs is sufficient to prevent the B to A transition.


Expression and possible function of glucose transporter protein GLUT1 during preimplantation mouse development from oocytes to blastocysts. A micro-Western analysis method both sensitive and quantitative enough to analyze oocytes and embryos is developed. GLUT1 protein is present in mouse oocytes and preimplantation embryos and levels are increased by fertilization and with time in ensuing embryonic development; the levels were 20-fold greater in blastocysts than in unfertilized oocytes. Similar increases were observed in glucose uptake by oocytes and embryos, suggesting that they may depend on GLUT1 expression. These results suggest that GLUT1 expression may explain a switch in substrate preference of the embryo from pyruvate to glucose during preimplantation development.


Binding of myosin subfragment-1 to F-actin. During a part of the hydrolytic cycle, myosin head (S1) carries no nucleotide and binds strongly to an actin filament forming a rigor bond. At saturating concentration of S1 in rigor, S1 is well known to form 1:1 complex with actin. However, we have provided evidence that under certain conditions S1 could also form a complex with 2 actin monomers in a filament (Andreev, O.A. & Borejdo, J. (1991) Biochem. Biophys. Res. Comm. 177, 350-356). This view was recently challenged by Carlier & Didry (Carlier, M-F. & Didry, D. (1992) Biochem. Biophys. Res. Comm. 183, 970-974) who interpreted our data by suggesting that F-actin underwent a simple depolymerization and implied that, when only actin in the F-form was scored, the real stoichiometry in our experiments was 1:1. We show here that under conditions of our experiments less than 8% of actin was depolymerized. Moreover, we have repeated the experiments in the presence of phalloidin and show that under these conditions too, when S1 was added slowly to a fixed concentration of F-actin, it formed a different complex with F-actin than when it was added quickly. This confirms our original conclusion that S1 can bind actin in two different ways and shows that depolymerization of F-actin is not responsible for this finding.


Fibronectin stimulates protein synthesis in cultured fibroblastic cells. Protein synthesis in mouse embryo fibroblastic (NIH-L1) cells was stimulated by addition of fibronectin (FN) to the culture medium as a soluble factor. This stimulatory activity of FN appeared only when the cells were cultured over their saturated density. A hexapeptide (GRGDSP) containing the RGDS cell attachment site of FN could not prevent the stimulation of protein synthesis. Moreover, activity of the stimulatory FN was not affected by inclusion of cytochalasin B, which is known to release FN from cell surface by disorganizing actin cytoarchitecture. These results suggest that the signaling of change in protein synthesis rate by FN may be distinct from signaling involving cell attachment and spreading.


Monomer-dimer equilibrium of nitroso-aromatic spin trap 3,5-dibromo-4-nitrosobenzenesulfonic acid, sodium salt (DBNBS), in aqueous solution. 3,5-Dibromo-4-nitrosobenzenesulfonic acid, sodium salt (DBNBS), which is widely used as a spin trap in biological systems, was allowed to form the monomer-dimer equilibrium in aqueous solution, and its equilibrium constant (Kc) was determined by NMR measurement and visible spectrum analysis. The Kc values obtained from the two different methods were virtually identical and evaluated as 1.3 x 10(-3) mol dm-3 at 25 degrees C. Further, the molar extinction coefficient of DBNBS monomer (epsilon 760) was determined to be 34 dm3mol-1cm-1. The present results indicate that only a minor portion (ca. 20%) of dissolved DBNBS (10 mmol dm-3) is practically capable of spin trapping under the conditions most widely used in biological studies.


Fatty acid binding protein inhibits glycolithocholate sulfation. Sulfation of hepatotoxic monohydroxy bile salts is viewed as an important detoxification mechanism. Bile salts are bound by fatty acid binding protein (FABP) with decreasing affinity as the extent of their hydroxylation increases. This binding has the potential to interfere with sulfation of monohydroxy bile salts and to augment their toxicity. FABP inhibits monohydroxy bile salt sulfation via bile salt sulfotransferases BST 1 and 2. With BST 1, the main BST, we obtained a maximal reduction of sulfation by 42.8 +/- 8.1%, using 10 microM glycolithocholate as substrate. FABP had no effect on sulfation of either 10 microM glycodeoxycholate or glycochenodeoxycholate. FABP may therefore specifically alter hepatotoxicity of lithocholate and its metabolites.


Isolation and characterization of riboflavin carrier protein from human amniotic fluid. A specific protein exhibiting immunological cross-reactivity with chicken riboflavin carrier protein has been purified to homogeneity from human amniotic fluid by use of ion-exchange and affinity chromatography. The protein is similar to its avian counterpart in terms of molecular size, distribution of 125I-labelled tryptic peptides during finger printing, and preferential binding to riboflavin. Immunologically, they are homologous since most of the monoclonal antibodies raised against the avian protein cross-react with the purified human vitamin carrier.


Superoxide dismutase and glutathione peroxidase activities in rat pineal gland. Activities of the antioxidant enzymes superoxide dismutase (E.C.1.15.1.1) and glutathione peroxidase (E.C.1.11.1.9) were measured in the pineal gland of adult rats. For comparative purpose superoxide dismutase was also measured in the anterior hypothalamic area (suprachiasmatic region) and the liver and glutathione peroxidase in the liver. The mean activities obtained for superoxide dismutase were: pineal (103.13 +/- 4.70 N.U.), anterior hypothalamus (132.88 +/- 4.73 N.U.), liver (253.91 +/- 0.10 N.U.) and, for glutathione peroxidase were: pineal (0.027 +/- 0.005 I.U.) and liver (0.103 +/- 0.031 I.U.). It was shown for the first time that superoxide dismutase and glutathione peroxidase are present in pineal gland being part of its antioxidant defense system.


Fusion of phospholipid vesicles induced by phospholipase D in the presence of calcium ion. Fusion of phospholipid vesicles composed of various combination of phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid was induced by Ca2+ in the presence or absence of phospholipase D from Streptomyces chromofuscus. For the vesicles composed of 20:50:30 molar ratio of phosphatidylcholine/phosphatidylethanolamine/phosphatidic acid, the initial fusion rate was much faster than the expected value when only the conversion of phosphatidylcholine and phosphatidylethanolamine into phosphatidic acid by phospholipase D is taken into account. These observations are discussed in terms of the involvement of outer monolayer of the vesicles and the enzyme activity itself in the fusion process.


Interaction of Artocarpus lectins with human IgA does not involve asparagine-linked oligosaccharide of the immunoglobulin. In view of the controversy with respect to the interaction of jacalin with human IgA2, a study was undertaken to assess the reactivity of the Artocarpus heterophyllus lectin, as well as the lectin from Artocarpus integer (lectin C), with subclasses of human immunoglobulin A by ELISA. Our data is consistent with the view that Artocarpus lectins have no affinity for the IgA2 immunoglobulins. In further support of the findings, we have established that N-linked oligosaccharide moieties of IgA have no significant bearing in the lectin-immunoglobulin binding. Interaction was also not affected in the presence of 1% (w/v) BSA.


Oxalate production from glyoxylate by lactate dehydrogenase in vitro: inhibition by reduced glutathione, cysteine, cysteamine. Lactate dehydrogenase is known to act as a dismutase converting glyoxylate to oxalate and glycolate. LDH (sources: human erythrocytes, human plasma; rabbit muscle; rat liver) activity was assayed at 340 nm using glyoxylate (5.0 mmol/l) and NADH. The LDH activity (approx. % of control) in all the cases decreased respectively in the presence of 5.0 and 10.0 mmol/l of cysteine (45 and 20), cysteamine (45 and 20), and GSH (55 and 30). This decrease in LDH activity resulted in decreased oxalate production from glyoxylate (0.5 mmol/l). A 50% inhibition in oxalate production was observed in presence of 0.3 mmol/l cysteine, 0.35 mmol/l cysteamine, and 2.0 mmol/l GSH. The results suggest that the net LDH activity towards oxalate production may be regulated by the free SH-groups in the cell. This possibility needs evaluation as a tool to lower endogenous oxalate production and the associated risk of stone formation.


Chemical modification of arginine residues of protein kinase C. Modification of basic residues of protein kinase C by phenylglyoxal results in a reversible, dose-dependent inhibition of autophosphorylation and substrate phosphorylation. The inhibition is not due to specific modification of the Ca2+ or ATP binding sites. Modified PKC bound more [3H] phorbol ester than unmodified providing further evidence that the binding of lipids and the catalytic phosphotransferase activity have different regulatory sequences. Additionally, the effects of these modifying reagents were different on enzyme stimulated by phorbol esters or by the endogenous activator, 1,2-diacylglycerol. These studies suggest that there is at least 1 arginine residue that is unique in the binding site of these different activators.


Destruction of vitamin K1 of cultured carrot cells by ultraviolet radiation and its effect on plasma membrane electron transport reactions. The effect of ultraviolet radiation on plasma membrane electron transport reactions was studied in cultured carrot cells. It was found that a 90 min treatment inhibited transmembrane hexacyanoferrate reduction greater than 50%. Extraction of lipophilic quinones from irradiated cells showed that vitamin K1 and coenzyme Q were totally destroyed, while control unirradiated cells showed the presence of 0.4 mumole vitamin K1 g dry wt.-1. The addition of exogenous vitamin K1 in concentrations of 1-10 microM partially restored plasma membrane electron transport with impermeable hexacyanoferrate as the electron acceptor. Total restoration of activity was given by growing irradiated cells in vitamin K1 supplemented growth media for 6 days. This shows that vitamin K1 may function as a member of the transplasma membrane electron transport chain in cultured carrot cells.


Detection of human satellite II DNA-binding polypeptides in HeLa cellular and nuclear extracts. Highly-repetitive satellite DNA's are generally located in centromeric heterochromatin, yet little is known about how they are maintained and condensed. In an effort to address this question, we have searched for specific satellite DNA-binding proteins in cell-free extracts (both total cellular and nuclear), prepared from HeLa cells, using southwestern blotting. Four polypeptides were detected that specifically bound to a human 1797 bp EcoRI satellite II DNA, which consists in large part of tandemly repeated pentamer (5' TTCCA 3') units, but not to a control probe from plasmid pBR322. These proteins have apparent molecular weights of 100, 93, 77 and 34 kDa and were termed satellite binding protein (Sbp) -1, -2, -3, and -4, respectively.


Putrescine uptake by rabbit intestinal brush-border membrane vesicles. The transport of putrescine in rabbit intestinal brush-border membrane vesicles is a time-dependent, Na+ and H+ gradient-independent slow process, reaching a high equilibrium level from 1 hr onwards. An approximately 12-fold concentration gradient of 5.4 microM putrescine was generated in the vesicles after 3 h of incubation at 25 degrees C, suggesting a substantial binding of the polyamine to membrane components. Putrescine uptake did not appear to occur via a polyamine-H+ exchange pathway, but the activity of transport was strongly reduced when the medium pH was lowered from 7.5 to 6.0. Although it was inhibited in the presence of SCN-, putrescine uptake was found neither to involve a preferred anion nor to be sensitive to changes in the membrane potential. Putrescine accumulation was substantially stimulated in the presence of a 10 mM concentration of the divalent cations Ca2+, Mg2+ or Ba2+, unlike the monovalent cations K+, Na+ or Li+. Despite the presence of a Ca(2+)-dependent transglutaminase activity, vesicular accumulation does not appear to result from covalent binding of putrescine to membrane peptides. Inhibition of the uptake by polyamines, unlike choline and dibasic amino acids, shows that putrescine may bind noncovalently to other specific anionic sites.


Evaluation of protein assay methods for pollen and fungal spore extracts. The usual procedures available for protein estimation of biological extracts often give variable results due to presence of many peptides and coloured materials. To identify a suitable method for allergenic extracts, protein was estimated from common pollen and fungal antigens by modified Lowry's (ML), Bradford (B), micro-Kjeldahl (MK), Bicinchoninic acid (BCA) and modified BCA (MBCA) assays. Bradford assay resulted in low protein values, whereas BCA method gave very high values in general. Statistical analysis of the results revealed similarity between protein values quantitated by MK, ML and MBCA methods for most of the extracts. Graded volumes of the extracts on subjecting to protein estimation by these three methods showed linear response, while recovery of a protein (bovine serum albumin) added to the extracts was greater than 90%.


Validation of the MTT dye assay for enumeration of cells in proliferative and antiproliferative assays. Increasing use is being made of colorimetric assays to quantitate viable cells, e.g., the cellular reduction of the tetrazolium salt, MTT, to formazan by mitochondrial succinate dehydrogenase. We validated this assay for cell proliferation in mixed lymphocyte cultures as compared with 3H-thymidine uptake, and for inhibition of cell proliferation induced by interferon with results compared by direct cell counting. We also found that cells do not, as previously assumed, require functional mitochondria: there were no differences in formazan production by normal cells or respiratory defective cells in which mitochondria had been poisoned by the nucleic acid toxin, ethidium bromide. The high reproducibility of MTT metabolism by lymphoblasts and various cultured cell lines establishes the reliability and versatility of this method for quantitating cell numbers.


Stimulation of the protein synthesis by fibronectin is specific for untransformed fibroblastic cells. In the preceding paper, we showed that fibronectin (FN) in solution has the ability to stimulate protein synthesis in NIH-L1 fibroblastic cells. We show here that FN stimulated protein synthesis also in other normal fibroblast cell lines, WI-38 and BALB/3T3, but did so only slightly in their transformed cell lines, L1-4NQOH10, WI-38 VA-13 and KN(7)8; and in rat hepatocytes in primary culture and rat liver epidermoid cells (GR-1) it hardly affected the synthesis. Augmentation of the protein synthesis rate was not attributable to stimulation of collagen synthesis. This stimulatory activity of FN was enhanced remarkably by proteolytic digestion of FN with trypsin.


Active site mapping studies of malate dehydrogenase : identification of essential amino acid residues by o-phthalaldehyde. Mitochondrial malate dehydrogenase (MDH) was found to be rapidly inactivated by o-phthalaldehyde. MDH-o-phthalaldehyde adduct gives a characteristic absorption maximum at 337 nm. Moreover, this derivative shows fluorescence emission maxima at 405 nm when excited at 337 nm and 280 nm. These results were consistent with isoindole ring formation in which the -SH group of cysteine and epsilon-NH2 group of lysine participate in the reaction. The enzyme was found to be protected against o-phthalaldehyde inactivation by NADH, indicating that the essential residues are present at or near the coenzyme binding site. Stoichiometric results indicate that 4 isoindole derivatives are formed per enzyme molecule upon complete inactivation. However, 90% of the activity loss was accompanied by the formation of 2 moles of isoindole per mole of the enzyme. These approaches give consistent evidence that two cysteines along with two lysines in close proximity are essential for the enzymatic activity.


Synthesis and characterization of some collagen sequence analogs. Some analogs of natural collagen sequences (773-779) were synthesized. The peptides were hydrolyzed at the Gly-Ile bond not only by crude collagenase isolated from normal rat liver, but also by the bacterial Clostridium histolyticum collagenase. The reason for this unusual cleavage site in the latter case may lie in the unordered secondary structure of the substrates measured by CD spectroscopy.


Comparative study of the effect of ethanol on the fluidity of subcellular hepatic membranes. The aim of this study has been to compare the effects of chronic ethanol administration on the lipid composition and fluidity of chick-liver microsomes and mitochondria. Cholesterol levels were increased only in microsomal membranes whilst the fatty-acid profiles were considerably altered in the major phospholipids in both membranes. By using fluorescence polarization we found that ethanol produces a disorganisation of acyl chains in the core of microsomes and mitochondria and consequently causes higher fluidity. In addition, opposite changes were induced by ethanol in the polar region of the microsomes, inducing a more rigid area in the membrane.


A novel fluorescent pH indicator for the acidic range. Reaction of a commercial preparation of lissamine-rhodamine B sulfonyl chloride with primary amines gives rise to two sulfonamide derivatives, of which one behaves as a pH indicator with maximal fluorescence emission in the acidic range and a pKa of about 4.6. The structure of this derivative and the mechanism of fluorescence dependence on pH, elucidated by means of NMR, is presented.