bluuebunny/crossref_metadata_2025 huggingface dataset:
A competitive inhibitor of phospholipase A2 decreases surfactant phosphatidylcholine degradation by the rat lung
Authors: Fisher A B , Dodia C , Chander A , Jain M
Published: August 2015

We have shown previously that radiolabelled phosphatidylcholine (PC) in liposomes or natural surfactant is removed from the alveolar space and metabolically recycled in a process that is stimulated by cyclic AMP (cAMP). In this study, we evaluated the effect of a transition-state phospholipid analogue (MJ33; 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol) that competitively inhibited acidic phospholipase A2 (PLA2) activity (pH 4.0) of lung homogenate by more than 97%, but had no effect on PLA2 activity at pH 8.5. MJ33 incorporated into unilamellar liposomes (dipalmitoyl PC/egg PC/cholesterol/phosphatidylglycerol, molar proportions 10:5:3:2) or co-sonicated with biosynthesized natural surfactant was instilled into the trachea of the anaesthetized rat; lungs were then removed for 2 h perfusion in the absence or presence of 0.1 mM-8-bromo cAMP. Total uptake for phospholipid was unchanged in the presence of the inhibitor MJ33. Degradation of labelled PC during 2 h perfusion in the absence of MJ33 was approx. 26% of that instilled for choline-labelled liposomal PC, 16% for liposomal PC labelled in the second fatty-acyl position, and 33% for choline-labelled natural surfactant. Degradation of PC was decreased by approx. 25-40% for each substrate in the presence of MJ33. Inhibition of lipid degradation depended on the mole fraction of MJ33 in the liposomes and was maximal at 1 mol%. These studies demonstrate a significant role for acidic Ca(2+)-independent PLA2 in the degradation of internalized alveolar PC, but further indicate that this enzyme accounts for a minor fraction of total lung PC metabolism.
Inhibition of proliferation, but not of Ca2+ mobilization, by cyclic AMP and GMP in rabbit aortic smooth-muscle cells
Authors: Assender J W , Southgate K M , Hallett M B , Newby A C
Published: August 2015

The effects on cellular proliferation and Ca2+ mobilization of analogues of cyclic AMP (cAMP) and cyclic GMP (cGMP) and of agents that elevate the intracellular concentrations of cyclic nucleotides were compared in closely similar preparations of first-passage rabbit aortic vascular smooth-muscle cells. Proliferation induced by foetal-bovine serum was inhibited by 78% by 1 mM-8-bromo cAMP and by 42% by 1 mM-8-bromo cGMP. In the presence of 100 microM-isobutylmethylxanthine, 100 microM-forskolin increased intracellular cAMP concentration 5-fold and inhibited proliferation by 87%, but did not affect cGMP concentration or cell viability (ATP concentration). Similarly in the presence of 100 microM-isobutylmethylxanthine, 1 mM-SIN-1 (3-morpholinosydnonimine) elevated cGMP concentration 4-fold and inhibited proliferation by 48%, but did not affect cAMP or ATP concentration. Isobutylmethylxanthine (1 mM) elevated cAMP concentration by 3-fold and cGMP concentration by 20-fold and inhibited proliferation by 81%. Concentrations of 8-bromo cAMP, 8-bromo cGMP, forskolin or SIN-1 that inhibited proliferation did not affect the elevation of intracellular free Ca2+ concentration caused by 2% (v/v) foetal-bovine serum, 100 nM-5-hydroxytryptamine or 10 nM-angiotensin II. The results demonstrate that elevation of intracellular cAMP and cGMP concentrations both independently inhibit vascular smooth-muscle cell proliferation, but these effects on proliferation are not mediated by inhibition of Ca2+ mobilization.
Cell-mediated degradation of type IV collagen and gelatin films is dependent on the activation of matrix metalloproteinases
Authors: Atkinson S J , Ward R V , Reynolds J J , Murphy G
Published: August 2015

The ability of normal rabbit dermal fibroblasts to degrade films of type IV collagen and gelatin when stimulated by phorbol ester was shown to be dependent on the induction, secretion and activation of 95 kDa gelatinase B and the secretion and activation of 72 kDa gelatinase A and stromelysin. Degradation was inhibited by exogenous human recombinant tissue inhibitor of metalloproteinases-1, specific antibodies to gelatinase and stromelysin and by the reactive-oxygen-metabolite inhibitor catalase. We discuss the various pathways for activation of matrix metalloproteinases in this model system and conclude that, although plasmin may play a key role in the activation of gelatinase B and stromelysin, gelatinase A is activated by a mechanism which has yet to be elucidated. The involvement of oxygen radicals in the direct activation of matrix metalloproteinases in this model is thought to be unlikely.
Intracellular Ca2+ rise in human platelets induced by polymorphonuclear-leucocyte-derived cathepsin G
Authors: Molino M , Di Lallo M , de Gaetano G , Cerletti C
Published: August 2015

Cathepsin G, a serine protease released by polymorphonuclear-leucocyte azurophilic granules upon stimulation, activates human platelets, inducing an increase in intra-platelet Ca2+ concentration ([Ca2+]i) in a concentration-dependent manner (50-200 nM). The [Ca2+]i rises elicited by low (50-80 nM) cathepsin G concentrations in fura-2-loaded platelets showed a biphasic mode, with a first small peak followed by a greater and more prolonged Ca2+ transient. Higher (100-200 nM) cathepsin G concentrations induced a monophasic increase in intracellular Ca2+. Acetylsalicylic acid, nordihydroguaiaretic acid and ketanserin did not affect platelet activation by cathepsin G, whereas the ADP-scavenger system phosphocreatine/creatine kinase significantly decreased Ca2+ mobilization, platelet aggregation and 5-hydroxytryptamine secretion by cathepsin G. Preventing cathepsin G-induced platelet aggregation with the synthetic peptide RGDSP (Arg-Gly-Asp-Ser-Pro) did not significantly affect cathepsin G-induced Ca2+ transients. Ni2+ (4 mM), a bivalent-cation-channel inhibitor, decreased the cathepsin G-induced fluorescence rise by more than 90%. This effect was reversed by either decreasing Ni2+ or increasing cathepsin G concentration. Preventing Ca2+ influx across the plasma membrane with 4 mM-EGTA totally abolished Ca2+ transients. However, EGTA also strongly decreased catalytic activity of cathepsin G, which is essential for platelet activation. Evidence of a rapid and sustained bivalent-cation channel opening in the platelet membrane was obtained by adding Mn2+ to the platelet suspension 30 s or 3 min after cathepsin G. No accumulation of InsP3 could be detected when platelets were stimulated with cathepsin G. All these data indicate that cathepsin G induces a [Ca2+]i increase mainly through an influx across the plasma membrane. This massive Ca2+ entry is probably due to opening of receptor-operated channels and is amplified by endogenous ADP release.
Partial purification of Golgi-bound arabinosyltransferase and two isoforms of xylosyltransferase from French bean (Phaseolus vulgaris L.)
Authors: Rodgers M W , Bolwell G P
Published: August 2015

The purification of glycosyltransferases involved in wall matrix polysaccharide synthesis has been attempted. A number of activities readily demonstrated in isolated Golgi membranes are lost following detergent solubilization. However, solubilization releases pyrophosphorylases and phosphatases that hydrolyse the substrate in enzyme assays, whether UDP-glucose, -arabinose or -xylose is used. This hydrolysis, which cannot be completely inhibited, appears to be the major factor in the apparent loss of activity. Separation of this hydrolytic activity during further purification by ion-exchange and gel exclusion leads to recovery of glycosyltransferase activity. Thus two xylosyltransferases and one arabinosyltransferase could be partially purified. These appeared to be differentially expressed. The arabinosyltransferase of apparent M(r) 70,000 on size-exclusion chromatography was isolated from cells undergoing rapid growth and division. A xylosyltransferase of apparent M(r) 38,000 on size-exclusion chromatography was associated with cell expansion and primary wall synthesis. A second xylosyltransferase, which was purified to near homogeneity with M(r) 40,000, showed a peak of activity during the period of maximum secondary wall synthesis.
The effect of mevalonic acid deprivation on enzymes of DNA replication in cells emerging from quiescence
Authors: Silber J R , Galick H , Wu J M , Siperstein M D
Published: August 2015

We have investigated the biochemical basis of the mevalonate dependence of DNA replication. Stimulating quiescent rat hepatoma cells to proliferate in the presence of compactin, an inhibitor of mevalonate synthesis, prevented DNA replication in as many as 80% of these cells. The percentage of cells that failed to replicate DNA increased with the increased duration of quiescence. Aphidicolin-sensitive DNA polymerase and ornithine decarboxylase activities were selectively decreased in compactin-treated cells, whereas RNA and protein synthesis, the level of dihydrofolate reductase and aphidicolin-resistant DNA polymerase activity were unaffected. Adding putrescine, the product of ornithine decarboxylase and the precursor of other polyamines, did not restore DNA replication. Our results demonstrate that the decreased activities of at least two DNA-replication enzymes are among the proximal causes of the failure of mevalonate-deprived cells to synthesize DNA. More importantly, our data indicate that a mevalonate-dependent factor(s) is progressively depleted during quiescence, and that inability to resynthesize this factor(s) may be the ultimate cause of the failure of resting cells to replicate DNA when stimulated to proliferate in the absence of mevalonate.
Effect of tumour-promoting phorbol ester, thrombin and vasopressin on translocation of three distinct protein kinase C isoforms in human platelets and regulation by calcium
Authors: Crabos M , Fabbro D , Stabel S , Erne P
Published: August 2015

Protein kinase C (PKC) acts in synergy with Ca2+ mobilization for the activation of platelets. Three different PKC subtypes that specifically react with antibodies to alpha- beta- and zeta-PKC have been detected in human platelets. We have compared the subcellular redistribution of these isoforms in platelets after exposure to the tumour-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) and to two physiological agonists, thrombin and vasopressin. In the presence of PMA, beta-PKC is most rapidly translocated to membranes, followed by zeta-PKC and alpha-PKC [membrane contents of 39 +/- 6, 31 +/- 4 and 24 +/- 4% (means +/- S.E.M.) respectively after 2 min incubation]. In contrast, both thrombin and vasopressin induced a biphasic translocation of PKC isoforms. For both agonists, the first phase of translocation occurred within 1 min and was identical for the three isoforms. However, during the second phase, the translocation of zeta-PKC by thrombin and vasopressin differed [membrane contents (mean +/- S.E.M.) of 24 +/- 3 and 46 +/- 4% respectively after 10 min]. These results suggest a differential activation of zeta-PKC by vasopressin and thrombin. PMA-induced translocation of alpha-PKC was decreased from 278 +/- 27 to 198 +/- 24 (mean +/- S.E.M., P = 0.02; percentage increase over control value) in the presence of 1 mM-EDTA, whereas chelation of intracellular Ca2+ by Quin2-AM does not influence this response. These results suggest that the PMA-induced translocation of alpha-PKC depends on the presence of 1 mM concentration of extracellular Ca2+. In addition, the chelation of either extracellular or intracellular Ca2+ inhibited both vasopressin- and thrombin-induced translocation of all three isoforms, suggesting that Ca2+ is an important requirement for the translocation of alpha-, beta- and zeta-PKC by physiological agonists. In conclusion, the translocation of PKC varies between different isoforms and between different agonists.
Cytosolic calcium as a second messenger for collagen-induced platelet responses
Authors: Smith J B , Selak M A , Dangelmaier C , Daniel J L
Published: August 2015

We showed previously that direct platelet activation by collagen involves an increase in the platelet cytosolic free Ca2+ concentration ([Ca2+]i) but that this increase is not required for the adhesion of platelets to collagen. We now report that collagen-induced arachidonic acid liberation, myosin phosphorylation and 5-hydroxytryptamine secretion are dependent on increases in [Ca2+]i, as they were markedly inhibited in platelets loaded with the acetoxymethyl ester of the Ca2+ chelator BAPTA but not in cells loaded with the acetoxymethyl ester of the non-chelating diazo-3. BAPTA also partially inhibited the rate of collagen-induced phosphatidic acid (PtdA) formation but had little effect on increases in phosphorylation of pleckstrin (47 kDa protein; P47). From these results we infer that collagen-induced increases in [Ca2+]i are required for dense granule secretion and arachidonic acid liberation, but are not necessary for stimulation of the protein kinase C pathway.
Heparin increases chromatin accessibility by binding the trypsin-sensitive basic residues in histones
Authors: Villeponteau B
Published: August 2015

Recent evidence indicates that chromatin accessibility to transcription factors is of regulatory significance. The polyanion heparin is known to increase chromatin accessibility to DNAase I and to stimulate both RNA and DNA synthesis. In the present study, chromatin structure and its modification by polyanions were examined by using trypsin and micrococcal nuclease as probes. Both heparin and poly(glutamic acid) were found to be equivalent to trypsin digestion of histones in their ability to increase nuclease accessibility in chromatin. However, no increase in nuclease accessibility was observed when trypsin-digested chromatin was further treated with heparin, indicating that polyanions and trypsin are not additive in their effects on chromatin accessibility. Moreover, sucrose-gradient analysis demonstrated that heparin binds tightly to intact nucleosomes but not to trypsin-digested nucleosomes. These data suggest that polyanions interact predominantly with the trypsin-sensitive lysine and arginine residues in histone H1 and the N-terminal segments of the core histones. The possible relevance of these results to the chromatin structure of actively transcribed regions is discussed.
Evidence for the regulation of guinea-pig heart microsomal phosphatidylcholine-hydrolysing phospholipase A1 by guanosine 5′-[γ-thio]triphosphate
Authors: Badiani K , Lu X , Arthur G
Published: August 2015

We have recently characterized lysophospholipase A2 activities in guinea-pig heart microsomes and postulated that these enzymes act sequentially with phospholipases A1 to release fatty acids selectively from phosphatidylcholine (PC) and phosphatidylethanolamine, thus providing an alternative route to the phospholipase A2 mode of release. In a further investigation of the postulated pathway, we have characterized the PC-hydrolysing phospholipase A1 in guinea-pig heart microsomes. Our results show that the enzyme may have a preference for substrates with C16:0 over C18:0 at the sn-1 position. In addition, although the enzyme cleaves the sn-1 fatty acid, the rate of hydrolysis of PC substrates with C16:0 at the sn-1 position was influenced by the nature of the fatty acid at the sn-2 position. The order of decreasing preference was C18:2 > C20:4 = C18:1 > C16:0. The hydrolyses of the molecular species were differentially affected by heating at 60 degrees C. An investigation into the effect of nucleotides on the activity of the enzyme showed that guanosine 5′-[gamma-thio]triphosphate (GTP[S]) inhibited the hydrolysis of PC by phospholipase A1 activity, whereas GTP, guanosine 5′-[beta-thio]diphosphate (GDP[S]), GDP, ATP and adenosine 5′-[gamma-thio]triphosphate (ATP[S]) did not affect the activity. The inhibitory effect of GTP[S] on phospholipase A1 activity was blocked by preincubation with GDP[S]. A differential effect of GTP[S] on the hydrolysis of different molecular species was also observed. Taken together, the results of this study suggest the presence of more than one phospholipase A1 in the microsomes with different substrate specificities, which act sequentially with lysophospholipase A2 to release linoleic or arachidonic acid selectively from PC under resting conditions. Upon stimulation and activation of the G-protein, the release of fatty acids would be inhibited.
Glutathione S-transferase π in an arsenic-resistant Chinese hamster ovary cell line
Authors: Lo J F , Wang H F , Tam M F , Lee T C
Published: August 2015

A glutathione S-transferase (GST) was purified from an arsenic-resistant Chinese hamster ovary cell line, SA7. The SA7 GST was shown to catalyse the conjugation of glutathione and ethacrynic acid, a specific substrate for Pi class GST. Its N-terminal amino-acid sequence has 80% identical residues to that of rat GST P and human GST pi. Thus, the GST purified from SA7 cells belongs to the Pi family. Treatment with Cibacron Blue or ethacrynic acid, which are GST inhibitors, significantly decreased the resistance of SA7 cells to sodium arsenite. On the other hand, pretreatment of SA7N cells, a partial revertant of SA7 cells, with sublethal doses of sodium arsenite, cadmium acetate or zinc sulphate resulted in re-elevation of GST activities and the cells regained the arsenic resistance. The regained arsenic resistance was well correlated with the levels of GST pi which were induced dose-dependently by zinc sulphate. Heat-shock treatment (45 degrees C for 10 min) did not increase GST pi expression or arsenic resistance of SA7N cells. The results indicate that GST pi is possibly involved in the mechanism of arsenic detoxification.
Expression of high levels of nitrobenzylthioinosine-sensitive nucleoside transport in cultured human choriocarcinoma (BeWo) cells
Authors: Boumah C E , Hogue D L , Cass C E
Published: August 2015

We have examined binding of [3H]nitrobenzylthioinosine (NBMPR) and influx of [3H]thymidine in adherent cultures of human choriocarcinoma (BeWo) cells and, for comparison, cervical-carcinoma (HeLa) cells. Specific association of NBMPR with BeWo cells at 22 degrees C required 1.5 h to reach an equilibrium between free and bound ligand, whereas association with HeLa cells required 20-30 min. Scatchard analysis of NBMPR binding to low-density cultures of BeWo cells revealed a total of 27 x 10(6) sites per cell, consisting of two distinct populations that differed in their affinities for NBMPR. One population bound NBMPR with ‘high’ affinity (Bmax.1 15.0 pmol/10(6) cells; Kd1 0.6 nM) and the other, larger, population bound NBMPR with ‘low’ affinity (Bmax.2 29.0 pmol/10(6) cells; Kd2 14.5 nM). By contrast, HeLa cells possessed only 4.1 x 10(5) sites per cell, and these sites all bound NBMPR with the same affinity (Bmax. 0.7 pmol/10(6) cells; Kd 0.5 nM). Interaction of NBMPR with both populations of sites in BeWo cells could be blocked by nitrobenzylthioguanosine (NBTGR), dilazep or dipyridamole. Concentration-effect relationships for dilazep inhibition of binding of 1 nM- and 25 nM-NBMPR to BeWo cells were monophasic, with virtually complete inhibition achieved at 0.1 microM and 1 microM respectively. Plasma-membrane preparations from BeWo cells also had high numbers of NBMPR-binding sites, and u.v. irradiation of site-bound [3H]NBMPR in such preparations labelled polypeptides that migrated in electrophoretograms as a broad band with a peak M(r) of 60,000. The concentration-effect relationship for NBMPR inhibition of thymidine transport by BeWo cells was biphasic, with an IC50 for inhibition of the ‘NBMPR-sensitive’ component of 1.6 nM and a substantial (15-20%) component of flux that was not inhibited by 10 microM-NBMPR and was thus ‘NBMPR-insensitive’. Vmax. values for thymidine transport by BeWo cells were 20-30-fold larger than the corresponding values for transport by HeLa cells. Elimination of the Na+ gradient had no effect on initial rates of thymidine fluxes measured in either the presence or the absence of NBMPR. Our results demonstrate that BeWo cells have an unusually large capacity for NBMPR-sensitive nucleoside transport, apparently resulting from high levels of expression of ‘erythrocyte-like’ transport elements, identified by their high-affinity interaction with NBMPR. The relationship of the low-affinity binding sites to NBMPR-sensitive transporter elements is uncertain.
Leupeptin-binding site(s) in the mammalian multicatalytic proteinase complex
Authors: Savory P J , Rivett A J
Published: August 2015

The multicatalytic proteinase (MCP) complex is a major nonlysosomal proteinase which plays an important role in non-lysosomal pathways of protein degradation and which has recently been implicated in antigen processing. The mammalian MCP complex is composed of more than 20 different types of polypeptide, but it is not yet clear which of these components are responsible for its proteolytic activities. The complex has at least three distinct types of proteolytic activity. One of these, the so-called ‘trypsin-like’ activity, which involves cleavage on the carboxy side of basic amino acid residues, can be selectively and completely inhibited by peptidyl arginine aldehydes (such as leupeptin and antipain), and is also the most sensitive to inhibition by thiol-reactive reagents. In the present study N-[ethyl-1-14C]ethylmaleimide has been used to specifically label thiol groups protected by leupeptin binding. The results suggest that one or two polypeptide components within the complex can be protected against modification by N-ethylmaleimide. These components may be responsible for the ‘trypsin-like’ activity of the complex or may be adjacent to the catalytic component(s) and play an important role in substrate binding.
Monitoring of changes in hepatic fatty acid and glycerolipid metabolism during the starved-to-fed transition in vivo. Studies on awake, unrestrained rats
Authors: Moir A M B , Zammit V A
Published: August 2015

1. The technique of selective labelling of hepatic fatty acids in vivo [Moir and Zammit (1992) Biochem. J. 283, 145-149] has been used to monitor non-invasively the metabolism of fatty acids in the livers of awake unrestrained rats during the starved-to-refed transition. Values for the incorporation of labelled fatty acid into liver and plasma glycerolipids and into exhaled carbon dioxide after injection of labelled lipoprotein and Triton WR 1339 into rats with chronically cannulated jugular veins were obtained for successive 1 h periods from the start of refeeding of 24 h-starved rats. 2. Starvation for 24 h resulted in marked and reciprocal changes in the incorporation of label into glycerolipids and exhaled 14CO2, such that a 4-fold higher value was obtained for the oxidation/esterification ratio in livers of starved rats compared with fed animals. 3. Refeeding of starved rats did not return this ratio to the value observed for fed animals for at least 7 h; during the first 3 h of refeeding the ratio was at least as high as that for starved rats. Between 4 h and 6 h of refeeding the ratio was still approx. 70% of that in starved animals, and 2.5-fold higher than in fed rats. 4. These data support the hypothesis that the capacity of the liver to oxidize fatty acids is maintained at a high level during the initial stages of refeeding [Grantham and Zammit (1986) Biochem. J. 239, 485-488] and that control of the flux of hepatic fatty acids into the oxidative pathway is largely lost from the reaction catalysed by mitochondrial overt carnitine palmitoyltransferase (CPT I) during this phase of recovery from the starved state. 5. Refeeding also resulted in a rapid (< 1 h) increase in hepatic malonyl-CoA concentrations to values intermediate between those in livers of fed and starved animals. The sensitivity of CPT I to malonyl-CoA inhibition in isolated liver mitochondria was only partially reversed even after 5 h of refeeding. 6. Refeeding resulted in an acute 35% inhibition of the fraction of synthesized triacylglycerol that was secreted into the plasma; the maximal effect occurred 2-3 h after the start of refeeding. The inhibition of the fractional secretion rate was fully reversed after 5 h of refeeding. 7. The amount of 14C label that was incorporated into phospholipids as a fraction of total glycerolipid synthesis was doubled within 2 h of the start of refeeding.(ABSTRACT TRUNCATED AT 400 WORDS)
Limited proteolysis of rat liver nucleolin by endogenous proteases: effects of polyamines and histones
Authors: Suzuki T , Suzuki N , Hosoya T
Published: August 2015

Nucleolin is a major nucleolar phosphoprotein and is presumably involved in rDNA transcription and ribosome biosynthesis. This protein is known to be very labile and to be cleaved by endogenous proteases into many small peptides. We found that, when rat liver nucleolar suspension (Nu-1) or nucleolin-rich extract (Nu-2) was incubated under conventional conditions, polyamines and histones interacted with the nucleolin to lead to its preferential degradation to 60 kDa phosphopeptide (p60). The peptide p60 was identified as a peptide containing the N-terminal half of the nucleolin molecule, as judged from peptide-map analysis. Whereas spermine binding to the purified nucleolin was decreased by KCl concentrations above 50 mM, histones (H1, H2B and H3) were able to bind to the nucleolin in the presence of up to 300 mM KCl. A distinct difference between H1 and other histones was found in that H1 could produce p60 from nucleolin in both Nu-1 and Nu-2, whereas H2B and H3 stimulated the degradation of nucleolin to p60 only when Nu-2 was used for the source of nucleolin. A possible relationship between p60 formation and rRNA synthesis is discussed, but its exact role remains to be studied.
Synthesis of antimony complexes of yeast mannan and mannan derivatives and their effect on Leishmania-infected macrophages
Authors: Cantos G , Barbieri C L , Iacomini M , Gorin P A G , Travassos L R
Published: August 2015

Antimony(Sb)-yeast mannan complexes were synthesized as a strategy to introduce Sb into macrophages infected with Leishmania amastigotes. The complexes were taken up by endocytosis after specific recognition by alpha-D-mannosyl receptors on the macrophage membrane. About 90% of the intracellular parasites were destroyed by Sb-mannan in vitro, whereas the corresponding Sb concentration used as the pentavalent antimonial drug glucantime destroyed about 60% of the amastigotes. None of the Sb complexes prepared with mannan acid or basic derivatives was as effective as the simple Sb-mannan complex in clearing macrophage infection by Leishmania (L) amazonensis. The leishmanicidal effect of Sb-mannan was also demonstrated in vivo with infected hamsters. The alternative use of Sb-mannan complex in the treatment of human leishmaniasis is envisaged on the basis of parasite-killing efficiency and the use of a low antimony dose.
Fluxionate Lewis acidity of the Zn2+ ion in carboxypeptidase A
Authors: Mock W L , Freeman D J , Aksamawati M
Published: August 2015

Competitive inhibition constants Ki for a series of phenol-ring-substituted derivatives of alpha-(2-hydroxyphenyl)benzenepropanoic acid have been ascertained by observing their influence on the catalytic hydrolysis of a peptide substrate by the zinc enzyme carboxypeptidase A. The pH-dependence of Ki shows that binding is maximal between two pKa values: one is that of the phenol group of the inhibitor, and the other uniformly has a value of 6, the pKa of a Zn(2+)-bound water molecule on the enzyme in the absence of substrate or inhibitor. This is the dependence expected if phenolate binds to the Zn2+ displacing its bound H2O/HO-. A log-log plot of the dissociation constants for the productive forms of inhibitor plus enzyme versus the acid dissociation constants of the phenolic residues in the inhibitors yields a straight line with a slope of +0.76. This number indicates that the active-site metal ion has special capacity for dispersing negative charge, such as builds up on the oxygen atom of a carboxamide group undergoing nucleophilic addition.
Bovine kidney β-mannosidase: purification and characterization
Authors: Sopher B L , Traviss C E , Cavanagh K T , Jones M Z , Friderici K H
Published: August 2015

Lysosomal beta-mannosidase was purified 160,000-fold in 24% yield from bovine kidney by a four-step purification procedure, which included concanavalin A-Sepharose, immunoaffinity, TSK-butyl and h.p.l.c. cation-exchange chromatography. When analysed by SDS/PAGE and detected by Coomassie Blue or silver staining, the purified enzyme preparation consists of two prominent peptides (100 and 110 kDa) and a third minor peptide (84 kDa). These three peptides are immunologically related and are consistently associated with beta-mannosidase activity in all chromatographic steps. Removal of N-linked carbohydrate from the 84, 100 and 110 kDa peptides decreases their molecular sizes to 75, 86 and 91 kDa respectively. Bovine kidneys lacking beta-mannosidase, activity, acquired from calves affected with beta-mannosidosis, do not contain detectable quantities of the three beta-mannosidase peptides, as judged by monoclonal- and polyclonal-antibody reactivity.
Sequence-dependent reactivity of linear DNA to chemical cleavage by Cu(II):thiol combinations including cysteine or glutathione
Authors: John D C A , Douglas K T
Published: August 2015

The chemical cleavage of linear DNA by Cu(II):thiol combinations was investigated using end-labelled double-stranded restriction fragments. Single-strand cleavage of the target DNA occurred with apparent sequence preference, but there appeared to be no apparent consensus sequence for such preferentially cleaved sites based on studies of three different restriction fragments. For any given restriction fragment, the observed Cu(II):thiol DNA-cleavage sequence preference was found to be independent of the nature of the thiol used. Cleavage of 3′-end-labelled DNA generated fragments bearing 5′-phosphoryl termini, and cleavage of 5′-end-labelled DNA gave rise to fragments bearing 3′-phosphoryl and 3′-‘phosphatase-inert’ termini in equal proportions. No appreciable amounts of fragments bearing 3′- or 5′-hydroxy termini were detected. This pattern of cleavage products is similar to that observed using the hydroxyl-radical-generating systems, 60Co gamma-irradiation or methidium propyl EDTA: Fe(II), but is dissimilar to that found with the Cu(II):phenanthroline or bleomycin systems. Cu(II):thiol cleavage of DNA fragments containing inserts of repeated dinucleotide sequence showed alternating patterns of relative cleavage intensities within the insert-sequence regions which were not seen in the sequences flanking the insert sequence. The assembled experimental data indicate that a reaction product of the Cu(II):thiol interaction, probably the hydroxyl radical, causes DNA cleavage. The Cu(II):thiol interaction may occur free in solution or via a copper species which is bound non-specifically to DNA. The observed low sequence-dependence of the cleavage reaction probably reflects the nature of the DNA structure itself, the Cu(II):thiol system serving as a structural probe of sequence-dependent structural variations in the DNA structure.
Differential cholera-toxin- and pertussis-toxin-catalysed ADP-ribosylation of G-proteins coupled to formyl-peptide and leukotriene B4 receptors
Authors: Schepers T M , McLeish K R
Published: August 2015

N-Formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe) and leukotriene B4 (LTB4) induce disparate second-messenger generation and functional responses in neutrophils and HL-60 granulocytes. Receptors for these chemoattractants couple to a common pool of G-proteins which are substrates for both pertussis-toxin- and cholera-toxin-catalysed ADP-ribosylation. The hypothesis that formyl-peptide and LTB4 receptors induce different receptor-specific conformations of activated G-proteins was tested. The ability of pertussis toxin and cholera toxin to ADP-ribosylate G(i) proteins coupled to formyl-peptide or LTB4 receptors in membranes isolated from HL-60 granulocytes was used to assess the conformational state of the alpha subunits. Cholera-toxin-catalysed ADP-ribosylation of alpha 40 (40 kDa alpha subunit) was inhibited by guanosine 5′-[beta gamma-imido]triphosphate and GDP in a concentration-dependent manner. Addition of fMet-Leu-Phe, but not LTB4, re-established cholera-toxin labelling of alpha 40 in the presence of either guanine nucleotide. In the absence of guanine nucleotides, fMet-Leu-Phe and C5a enhanced cholera-toxin-catalysed labelling of alpha 40, whereas LTB4 and platelet-activating factor had no effect. Preincubation with fMet-Leu-Phe, but not LTB4, inhibited pertussis-toxin labelling of alpha 40 in the presence of guanosine 5′-[gamma-thio]triphosphate and in the absence of guanine nucleotides. Preincubation with fMet-Leu-Phe or LTB4 enhanced pertussis-toxin labelling of alpha 40 in the presence of GDP. These data suggest that activated G(i) proteins coupled to formyl-peptide and LTB4 receptors exist in different conformations determined by the receptor with which they interact.
Effects of CoA and acyl-CoAs on GTP-dependent Ca2+ release and vesicle fusion in rat liver microsomal vesicles
Authors: Comerford J G , Dawson A P
Published: August 2015

(1) CoA (IC50 23 microM) and acyl-CoAs (IC50 values 15-18 microM) inhibit GTP-dependent vesicle fusion in rat liver microsomal vesicles. Acyl-CoAs of carbon chain length C8 and C20 are much less effective than acyl-CoAs of carbon chain length C14-C18. The effect of CoA is mimicked by dephospho-CoA, but not by desulpho-CoA. High acyl-CoA concentrations (50 microM) appear to favour formation of small vesicles (budding), while 50 microM CoA does not. (2) Low concentrations of CoA (EC50 2 microM) and palmitoyl-CoA (10 microM) cause re-accumulation of Ca2+ released in response to GTP. This re-accumulation is into an Ins(1,4,5)P3-sensitive compartment. By investigation of the effects of CoA and palmitoyl-CoA on the thapsigargin-induced passive leak rate of Ca2+, and on the latency of the mannose-6-phosphatase of the vesicles, we conclude that CoA and palmitoyl-CoA cause decreased vesicle permeability rather than stimulation of Ca2+ pumping activity. (3) It is suggested that GTP-induced membrane fusion in rat liver microsomes involves an as yet uncharacterized acylation-deacylation reaction which is required to produce complete vesicle sealing.
Human fetoacinar pancreatic protein: an oncofetal glycoform of the normally secreted pancreatic bile-salt-dependent lipase
Authors: Mas E , Abouakil N , Roudani S , Miralles F , Guy-Crotte O , Figarella C , Escribano M J , Lombardo D
Published: August 2015

A fetoacinar pancreatic protein (FAP) associated with the ontogenesis, differentiation and oncogenic transformation of the human exocrine pancreas has been purified from pancreatic juices of patients suffering from pancreatitis or duodenal cancers invading the pancreas [Escribano and Imperial (1989) J. Biol. Chem. 264, 21865-21871]. This protein has striking similarities, i.e. M(r), amino acid composition and N-terminal sequence, to the bile-salt-dependent lipase (BSDL) of normal human pancreatic secretion. The aim of this study was to gain further insight into the nature of the two proteins. Reactivity with the mouse monoclonal antibody J28 (mAb J28), which characterizes FAP, and enzyme activity could not be dissociated during biochemical purification of BSDL. Furthermore, a polyclonal antiserum raised against purified human BSDL reacted completely with FAP in Western-blot analysis giving additional support to the idea of similar molecular structures for BSDL and FAP. However, by the same technique, mAb J28 reacted with a relatively restricted population of BSDL molecules. The classical BSDL preparation could be separated into molecules bearing the J28 epitope and those devoid of it by immunoaffinity on immobilized mAb J28. The two subpopulations had identical N-terminal sequences and some differences in their amino acid compositions. However, they had different carbohydrate compositions. J28-epitope-bearing molecules were active on BSDL substrates, although their specific activity was decreased. These results are consistent with the existence of two closely related polypeptide chains with different glycan counterparts. Therefore, if the name FAP is reserved for molecules bearing the J28 epitope, which is linked to a carbohydrate-dependent structure. FAP could represent an oncofetal-related variant of BSDL. Our result is the first demonstration of the existence of an oncofetal-type subpopulation of an otherwise normally secreted human pancreatic enzyme.
Cloning and characterization of the rat bone sialoprotein gene promoter
Authors: Li J J , Sodek J
Published: August 2015

To study the transcriptional regulation of the rat bone sialoprotein (BSP) gene, the nucleotide sequence of a approximately 1 kb HindIII/KpnI subfragment from a genomic clone containing the 5′ flanking sequence, exon 1 and part of intron 1 was determined and the transcription start site defined. This region includes an inverted TATA element (nt -24 to -19), an inverted CCAAT box, a homeobox-binding site, a putative 1,25-dihydroxyvitamin D3 response element (VDRE) sequence overlapping the inverted TATA sequence, and a novel 18 nt palindrome that may control the tissue-specific transcription of the BSP gene. The shortest promoter sequence capable of directing bacterial chloramphenicol acetyltransferase reporter gene expression included the inverted TATA element and the inverted CCAAT box. However, the promoter activity was down-regulated by 1,25-dihydroxyvitamin D3, indicating that the unique VDRE-like sequence overlapping the TATA element is functional. Thus the rat BSP gene promoter is characterized by novel cis-acting elements that may be involved in hormone- and tissue-specific regulation of transcription.
A protein kinase inhibitor, staurosporine, enhances the expression of phorbol dibutyrate binding sites in human polymorphonuclear leucocytes
Authors: Combadière C , Pedruzzi E , Hakim J , Périanin A
Published: August 2015

Staurosporine, a potent protein kinase C (PKC) inhibitor, was studied for its effects on the binding of phorbol 12,13-dibutyrate (PDBu) to human polymorphonuclear leucocytes (PMNs). Treatment of PMNs with staurosporine concentrations in the range 50 nM-2 microM at 37 degrees C (but not at 4 degrees C) and with 1 nM [3H]PDBu at both temperatures enhanced specific PDBu binding to PMNs by approx. 10-600% relative to control values. The potentiation was rapid (detectable within 1 min) and reached a plateau after 10 min of cell treatment. Scatchard analysis of the binding showed that staurosporine increased the total number of PDBu-binding sites (Bmax) from (178 +/- 9) x 10(3) (control) to (324 +/- 15) x 10(3) sites per PMN and lowered the apparent dissociation constant (Kd) from 9.6 +/- 0.8 (control) to 3.3 +/- 0.3 nM. In Ca(2+)-depleted cells, staurosporine induced similar changes in Kd values, whereas the Bmax. increased by 60%. Treatment of PMNs with 500 nM staurosporine enhanced PDBu binding in the particulate fraction by 120 +/- 7% and decreased PDBu binding in the soluble fraction by 69 +/- 4%, whereas PKC histone-phosphorylating activity of both fractions was almost completely inhibited. Incubation of staurosporine-pretreated particulate fractions with soluble fractions enriched the particulate fraction in PDBu-binding sites at the expense of the soluble fraction, without altering the binding affinity of PDBu for either fraction. Stimulation of PMNs with chemotactic N-formyl peptides induced a transient increase in PDBu binding. This effect was potentiated by approx. 52% by staurosporine. These results show that, in addition to its interference with PKC protein-phosphorylating activity, staurosporine enhances both PDBu-binding capacity and affinity to PMNs, through a mechanism involving Ca(2+)-dependent and -independent processes. Alterations of PDBu binding to soluble and particulate fractions suggest that the enhanced binding capacity in intact PMNs may be due to translocation of PDBu receptors, presumably PKC units. This phenomenon may affect PKC-dependent cellular responses mediated by physiological stimulation.
Direct desaturation of intact galactolipids by a desaturase solubilized from spinach (Spinacia oleracea) chloroplast envelopes
Authors: Schmidt H , Heinz E
Published: August 2015

In plants, polyenoic fatty acids are synthesized by desaturase enzymes which use acyl groups of membrane lipids as substrates. To provide direct ‘in vitro’ evidence for this reaction, we solubilized envelope membranes from spinach (Spinacia oleracea) chloroplasts with Triton X-100 to release a membrane-bound n-6 desaturase. In the presence of oxygen and reduced ferredoxin, the solubilized enzyme desaturated a variety of substrates, such as free oleic acid, free erucic acid, 1-oleoyl-sn-glycerol 3-phosphate and the three galactolipids 1-oleoyl-2-(7′-cis-hexadecenoyl)-3-beta-D-galactopyranosyl-sn-glycerol, 1,2-dioleoyl-3-beta-D-galactopyranosyl-sn-glycerol and the ether analogue 1,2-di-(9′-cis-octadecenyl)-3-beta-D-galactopyranosyl-sn- glycerol. The in vitro desaturation of these exogenously added complex lipids with ester- and ether-linked substrate chains is unambiguous evidence for lipid-linked desaturation. The enzyme measures the insertion of the new double bond from the methyl end and the existing (n-9)-cis-double bond of an appropriate acyl or alkyl chain. The distal part of the substrate group, normally the carboxy end of a fatty acyl residue, is of less importance and, in particular, its activation in thioester form is not required.
Diacylglycerol kinase is phosphorylated in vivo upon stimulation of the epidermal growth factor receptor and serine/threonine kinases, including protein kinase C-ε
Authors: Schaap D , van der Wal J , van Blitterswijk W J , van der Bend R L , Ploegh H L
Published: August 2015

In signal transduction, diacylglycerol (DG) kinase attenuates levels of the second messenger DG by converting it to phosphatidic acid. A previously cloned full-length human 86 kDa DG kinase cDNA was expressed as fusion protein in Escherichia coli, to aid in the generation of DG-kinase-specific monoclonal antibodies suitable for immunoprecipitation experiments. To investigate whether phosphorylation of DG kinase is a possible mechanism for its regulation, COS-7 cells were transiently transfected with the DG kinase cDNA and phosphorylation of the expressed DG kinase was induced by various stimuli. Activation of both cyclic AMP-dependent protein kinase and protein kinase C (PKC) resulted in phosphorylation of DG kinase on serine residues in vivo, and both kinases induced this phosphorylation within the same tryptic phosphopeptide, suggesting that they may exert similar control over DG kinase. No phosphorylation was observed upon ionomycin treatment, intended to activate Ca2+/calmodulin-dependent kinases. Co-transfections of DG kinase with either PKC-alpha or PKC-epsilon cDNA revealed that both protein kinases, when stimulated, are able to phosphorylate DG kinase. For PKC-epsilon, DG kinase is the first in vivo substrate identified. Stimulation with epidermal growth factor (EGF) of COS-7 cells transfected with both DG kinase and EGF-receptor cDNA results mainly in phosphorylation of DG kinase on tyrosine. Since the EGF receptor has an intrinsic tyrosine kinase activity, this finding implies that DG kinase may be a direct substrate for the activated EGF receptor.
Mechanisms of hepatic phosphatidylcholine synthesis in the developing guinea pig: contributions of acyl remodelling and of N-methylation of phosphatidylethanolamine
Authors: Burdge G C , Kelly F J , Postle A D
Published: August 2015

Hepatic phosphatidylcholine (PC) from the immature fetal guinea pig at day 55 of gestation comprised mainly unsaturated molecular species containing C18:2(n-6) and C22:6(n-3) at the sn-2 position, reflecting placental permeability to essential fatty acids. At both day 55 and term (day 68), [Me-14C]choline was incorporated in utero over 3 h largely into sn-1-C16:0 PC species, with incorporation into sn-1-C18:0 PC species increasing by 18 h of incubation. Comparison of specific radioactivities after 3 h and 18 h suggests PC acyl remodelling by phospholipase A1. No incorporation into C20:4(n-6)-containing PC species could be detected of either [Me-14C]choline in vivo or CDP-[Me-14C]choline in isolated microsomes. The major phosphatidylethanolamine (PE) species were 16:0/22:6 and 18:0/22:6. Although [14C]ethanolamine was initially incorporated mainly into sn-1-C16:0 species, specific-radioactivity analysis suggested differential turnover rather than acyl remodelling. [1,2-14C]Ethanolamine and [Me-14C]methionine incorporation into PC molecular species indicated that both newly synthesized and total PE pools were available for N-methylation. Since the PC pool synthesized from PE included C20:4- and C22:6-containing species, N-methylation may provide a mechanism for supplying essential long-chain fatty acids to developing tissues that can be regulated independently from bulk PC synthesis.
Folate transport in intestinal brush border membrane: involvement of essential histidine residue(s)
Authors: Said H M , Mohammadkhani R
Published: August 2015

We examined the possible existence of histidine residue(s) in the folate transporter of rabbit intestine. This was done with use of the histidine-specific reagent diethyl pyrocarbonate (DEPC) and purified intestinal brush-border-membrane vesicles. DEPC caused significant concentration- and time-dependent inhibition of folic acid transport. The inhibition was only seen when transport was examined in vesicles incubated in buffer at pH 5.2 and not in those incubated in buffer at pH 7.4. The addition of unlabelled folic acid to vesicle suspension before treatment with DEPC (2.5 mM) led to a significant (P < 0.01) protection (84%) against the inhibition of folic acid transport. Treating vesicles pretreated with DEPC (2.5 mM) with reducing reagents (dithiothreitol, 2-mercaptoethanol and 2,3-dimercaptopropanol, all at a final concentration of 10 mM) did not reverse the inhibitory effect of DEPC on folic acid transport. On the other hand, treating the DEPC-pretreated vesicles with hydroxylamine (140 mM) led to a significant reversal (P < 0.01) (54%) of the inhibition of folic acid transport. The inhibitory effect of DEPC on carrier-mediated folic acid transport was found to be mediated through a decrease in the Vmax. (i.e. a decrease in the number and/or activity) of the carriers and an increase in the apparent Km (i.e. a decrease in their affinity), classifying the effect as a mixed-type inhibition. These results demonstrate the existence of critical histidine residue(s) in the intestinal brush-border-membrane folate transporter which is essential for its interaction with, and transport of, the vitamin. These findings also suggest that the histidine residue(s) is located at (or near) the substrate-binding site.
Purification and characterization of a DNA-binding heterodimer of 52 and 100 kDa from HeLa cells
Authors: Zhang W W , Zhang L X , Busch R K , Farrés J , Busch H
Published: August 2015

In studies of protein binding to the upstream region of the human proliferation-associated antigen p120 gene, a heterodimer of 52 and 100 kDa proteins was purified from HeLa cells. A 1:1 ratio of p52 and p100 was constant throughout the purification. The heterodimer was localized to cell nuclei, as shown by immunofluorescence. The pI values of the p52 and p100 were 7.8 and 8.6 respectively. The peptide sequences obtained for p52 (QSNKTFNLEKQNHTPRKKHQ and PLRGKQLRVRFAAHSASLTVR) and for p100 (PGGPKPGGGPGLSTPGGHPKPPHRGGGEPPRGRQ and GPGPGQSGPKPPIPPPPPHQQ) were not found in the computer databanks. One p52 peptide sequence, PLRGKQLRVRFA, shows considerable sequence similarity to a conserved motif in topoisomerase II of multiple species. The p52/100 heterodimer bound to different DNA probes. The binding was competed by poly(dI-dC), sonicated salmon sperm DNA, and circular or linearized plasmid DNA. The optimal DNA binding for the heterodimer was at pH 7-9 with low salt. The DNA-binding subunit of the heterodimer was the p100 polypeptide, as shown by u.v.-cross-linking assays and Southwestern blots.