bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of poly(ADP-ribose) polymerase during neutrophilic and monocytic differentiation of promyelocytic (NB4) and myelocytic (HL-60) leukaemia cells
Authors: Bhatia M , Kirkland J B , Meckling-Gill K A
Published: August 2015

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme which has been shown to play a role in the differentiation of haematopoietic cells. We report here that neutrophils are the first nucleated mammalian cell type demonstrated to be devoid of immunoreactive PARP. Both NB4 acute promyelocytic leukaemia and HL-60 (acute myelocytic leukaemia) cells were differentiated into non-malignant neutrophils with all-trans-retinoic acid (ATRA). Western blot analysis demonstrated that ATRA had no effect on PARP expression in HL-60 cells. However, PARP was completely down-regulated in NB4 cells within 36 h of treatment initiation. This decrease in PARP polypeptide coincided with growth arrest and preceded the appearance of neutrophilic differentiation features. NB4 cells require a combination of 1,25-dihydroxyvitamin D3 (1,25-D3) and phorbol 12-myristate 13-acetate (PMA) to differentiate completely into monocyte/macrophages, whereas HL-60 cells can be made to differentiate by combined or single agents. PARP expression was up-regulated 90-fold when NB4 cells were treated with PMA and 1,25-D3 together, and this increase accompanied expression of the monocyte/macrophage phenotype. Only modest changes in PARP expression were observed when each agent was used alone in NB4 cells or when HL-60 cells were differentiated along the monocyte/macrophage pathway. In addition, PARP activity was modulated in a pattern similar to protein levels when NB4 cells were induced to differentiate along the neutrophilic and monocyte/macrophage pathways. This suggests that the activity of PARP may be controlled through regulation of protein levels during NB4 cell differentiation. We conclude that PARP levels are dramatically modulated during monocyte/macrophage and neutrophilic differentiation. On the basis of the tremendous changes in PARP polypeptide and total activity during myeloid differentiation, we propose that modulation of PARP gene expression is required for cellular maturation in both lineages.
Glutathione analogues as novel inhibitors of rat and human glutathione S-transferase isoenzymes, as well as of glutathione conjugation in isolated rat hepatocytes and in the rat in vivo
Authors: Ouwerkerk-Mahadevan S , van Boom J H , Dreef-Tromp M C , Ploemen J H T M , Meyer D J , Mulder G J
Published: August 2015

Inhibitors of rat and human Alpha- and Mu-class glutathione S-transferases that effectively inhibit the glutathione (GSH) conjugation of bromosulphophthalein in the rat liver cytosolic fraction, isolated rat hepatocytes and in the rat liver in vivo have been developed. The GSH analogue (R)-5-carboxy-2-gamma-(S)-glutamylamino-N-hexylpentamide [Adang, Brussee, van der Gen and Mulder (1991) J. Biol. Chem. 266, 830-836] was used as the lead compound. To obtain more potent inhibitors, it was modified by replacement of the N-hexyl moiety by N-2-heptyl and by esterification of the 5-carboxy group with ethyl and dodecyl groups. In isolated hepatocytes, the branched N-2-heptyl derivatives were stronger inhibitors of GSH conjugation of bromosulphophthalein than the N-hexyl derivatives. The ethyl ester compounds were more efficient than the corresponding unesterified derivatives. The dodecyl ester of the N-2-heptyl analogue was the most effective inhibitor in isolated hepatocytes, but was relatively toxic in vivo. However, the corresponding ethyl ester was a potent in vivo inhibitor: GSH conjugation of bromosulphophthalein (as assessed by biliary excretion of the conjugate) was decreased by 70% after administration of a dose of 200 mumol/kg. The isoenzyme specificity of the inhibitors towards purified rat and human glutathione S-transferases was also examined. The unesterified compounds were more potent than the esterified analogues, and inhibited Alpha- and Mu-class isoenzymes of both rat and human glutathione S-transferase (Ki range 1-40 microM). Other GSH-dependent enzymes, i.e. GSH peroxidase, GSH reductase and gamma-glutamyltranspeptide, were not inhibited. Thus (R)-5-ethyloxycarbonyl-2-gamma-(S)-glutamylamino-N-2-hept ylpentamide, the in vivo inhibitor of GSH conjugation, may be useful in helping to assess the role of the Alpha and Mu classes of glutathione S-transferases in cellular biochemistry, physiology and pathology.
Effects of lactation on the signal transduction systems regulating lipolysis in sheep subcutaneous and omental adipose tissue
Authors: Vernon R G , Doris R , Finley E , Houslay M D , Kilgour E , Lindsay-Watt S
Published: August 2015

The effect of lactation on the regulation of lipolysis by beta- and alpha 2-adrenergic agents and by adenosine has been investigated. When changes in adipocyte mean cell volume (which decreases with lactation) are allowed for, lactation increased the maximum response both to beta-adrenergic agents and to the adenosine analogue N6-phenylisopropyladenosine, but had no apparent effect on the responsiveness of the alpha 2-adrenergic system in both subcutaneous and omental adipocytes. For subcutaneous adipocytes, there was no significant change in the number of beta-adrenergic or alpha 2-adrenergic receptors, but the amount of Gs and the maximum (forskolin-stimulated) adenylate cyclase activity were increased by lactation. In contrast, in omental adipocytes, the number of beta- (but not alpha 2-) adrenergic receptors and the amount of Gs were increased, whereas forskolin-stimulated adenylate cyclase activity was unchanged by lactation. In both types of adipocyte, cyclic AMP phosphodiesterase and total protein kinase A activities were unchanged. Lactation had no effect on the number of adenosine receptors but increased the amounts of the Gi isoforms expressed in both types of adipocyte. These various adaptations differ markedly in a number of respects from those described previously in the rat. Lactation, then, while having a similar overall effect on the response to beta-adrenergic agonists of adipocytes, achieves this by depot-specific mechanisms. In contrast, changes in response to adenosine appear to involve the same mechanism in the two depots investigated.
Human acetyl-coenzyme A:α-glucosaminide N-acetyltransferase. Kinetic characterization and mechanistic interpretation
Authors: Meikle P J , Whittle A M , Hopwood J J
Published: August 2015

Acetyl-CoA: alpha-glucosaminide N-acetyltransferase (N-acetyltransferase) is an integral lysosomal membrane protein which catalyses the transfer of acetyl groups from acetyl-CoA on to the terminal glucosamine in heparin and heparan sulphate chains within the lysosome. In vitro, the enzyme is capable of acetylating a number of mono- and oligo-saccharides derived from heparin, provided that a non-reducing terminal glucosamine is present. We have prepared highly enriched lysosomal membrane fractions from human placenta by a combination of differential centrifugation and density-gradient centrifugation in Percoll. This preparation was used to investigate the kinetics of the enzyme with three acetyl-acceptor substrates, i.e. glucosamine and a disaccharide and a tetrasaccharide derived from heparin, each containing a terminal glucosamine residue. The enzyme showed a pH optimum at 6.5, extending to 8.0 for the mono- and di-saccharide substrates but falling off sharply above pH 6.5 for the tetrasaccharide substrate. We identified two distinct Km values for the glucosamine substrate at both pH 7.0 and pH 5.0, whereas the tetrasaccharide substrate displayed only a single Km value at each pH. The Km values were found to be highly pH-dependent, and at pH 5.0 the values for the acetyl-acceptor substrates showed a decreasing trend as the size of the substrate increased, suggesting that the enzyme recognizes an extended region of the non-reducing terminus of the heparin or heparan sulphate polysaccharides. Double-reciprocal analysis, isotope exchange between N-acetylglucosamine and glucosamine, and inhibition studies with desulpho-CoA indicate that the enzyme operates by a random-order ternary-complex mechanism. Product inhibition studies display a complex pattern of dead-end inhibition. Taken in context with what is known about lysosomal utilization and physiological levels of acetyl-CoA, these results suggest that in vivo the enzyme operates via a random-order ternary-complex mechanism which involves the utilization of cytosolic acetyl-CoA to transfer acetyl groups on to the terminal glucosamine residues of heparin within the lysosome.
Characterization of the manganese superoxide dismutase cDNA and gene from the human parasite Onchocerca volvulus
Authors: Henkle-Dührsen K , Tawe W , Warnecke C , Walter R D
Published: August 2015

The manganese-containing superoxide dismutase (MnSOD) is a major component of the cellular defence mechanisms against the toxic effects of the superoxide radical. Within the framework of studies on anti-oxidant enzymes and their protective role in the human parasitic nematode Onchocerca volvulus, sequences encoding the MnSOD were isolated and examined in this study. Degenerate primers were designed based upon conserved regions of MnSOD sequences from other organisms, and were used in PCR on reverse-transcribed O. volvulus total RNA and genomic DNA to identify partial cDNA and genomic DNA fragments encoding the O. volvulus MnSOD (OvMnSOD). The genomic DNA PCR product was used to screen an O. volvulus adult worm lambda unizap II cDNA library and the nucleotide sequence of the longest clone determined. The complete 5′-end of the OvMnSOD cDNA was obtained using the rapid amplification of cDNA ends (RACE) procedure with O. volvulus total RNA and was found to possess a spliced leader sequence at the 5′-terminus. The deduced primary sequence encodes a 25 kDa protein, which has the conserved residues required for enzyme activity and metal binding. The 24 N-terminal amino acids encoded by the OvMnSOD cDNA comprise a putative mitochondrial transit peptide. The OvMnSOD gene was also isolated from an O. volvulus adult worm lambda fix II genomic library, a restriction map was constructed and the nucleotide sequence determined. The OvMnSOD gene was found to possess five exons and four introns with consensus splice-site junctions. Potential regulatory elements were identified in the 5′ genomic flanking sequence. Southern-blot analysis with total worm genomic DNA indicates a single-copy gene, with a restriction pattern consistent with that of the isolated gene.
Probing the details of the HIV-1 Rev-Rev-responsive element interaction: effects of modified nucleotides on protein affinity and conformational changes during complex formation
Authors: Renwick S B , Critchley A D , Adams C J , Kelly S M , Price N C , Stockley P G
Published: August 2015

The solution structure of the human immunodeficiency virus type 1 (HIV-1) Rev-responsive element (RRE) has been investigated by enzymic and chemical structural probing of a 71 nt RRE transcript. The minimum sequence information required to maintain recognition by the Rev protein has previously been mapped to a 29 nt stem-loop structure, known as minSLIIB. The key recognition target is a single-stranded RNA bubble at the base of the RNA stem. The fine details of RNA recognition have been probed using chemically synthesized minSLIIBs containing variant base or sugar residues at sites within the bubble. These have been analysed by gel retardation assays and their relative affinities for Rev protein determined. Complex formation between the wild-type minSLIIB RRE and Rev protein was also monitored using CD spectroscopy, which suggests a change in RNA conformation upon Rev binding. The spectral change is consistent with localized melting of RNA, leading to a decrease in the level of base stacking and/or a change in base tilting, during formation of the complex. Deoxynucleotide substitutions on just one side, the 5′ side, of the bubble inhibit the conformational change detected by CD. The data are consistent with a dynamic interaction between Rev and its target site. The contact points between Rev and the RRE were probed directly using photo-cross-linking with either ribo-5-bromouridine- or ribo-4-thiouridine-substituted minSLIIBs. The data are consistent with protein-RNA contacts at the bottom of the bubble.
Effects of magnesium on cyclic GMP hydrolysis by the bovine retinal rod cyclic GMP phosphodiesterase
Authors: Srivastava D , Fox D A , Hurwitz R L
Published: August 2015

Knowledge of the kinetics of the rod cyclic GMP phosphodiesterase is essential for understanding the kinetics and gain of the light response. Therefore, the interactions between Mg2+, cyclic GMP, and purified, trypsin-activated bovine rod cyclic GMP phosphodiesterase (EC 3.1.4.17) were examined. The effects of Mg2+ and of cyclic GMP on the rod phosphodiesterase activity were mutually concentration-dependent. Formation of a free Mg-cyclic GMP complex is unlikely due to its high dissociation constant (Kd = 19 mM). Plots of 1/velocity versus 1/[cyclic GMP] as a function of [Mg2+] and 1/velocity versus 1/[Mg2+] as a function of [cyclic GMP] intersected to the left of the 1/velocity axis. This is consistent with the formation of a ternary complex between the phosphodiesterase, Mg2+, and cyclic GMP. A competitive inhibitor of the phosphodiesterase relative to cyclic GMP, 3-isobutyl-1-methylxanthine, non-competitively inhibited the enzyme relative to Mg2+, Pb2+, a competitive inhibitor of the phosphodiesterase relative to Mg2+ [D. Srivastava, R.L. Hurwitz and D. A. Fox (1995) Toxicol. Appl. Pharmacol, in the press] non-competitively inhibited the enzyme relative to cyclic GMP. Collectively these results are suggestive of a rapid equilibrium random binding order of Mg2+ and cyclic GMP to the rod phosphodiesterase.
Mitochondrial gene expression in small intestinal epithelial cells
Authors: Mayall T P , Bjarnason I , Khoo U Y , Peters T J , Macpherson A J S
Published: August 2015

Most mitochondrial genes are transcribed as a single large transcript from the heavy strand of mitochondrial DNA, and are subsequently processed into the proximal mitochondrial (mt) 12 S and 16 S rRNAs, and the more distal tRNAs and mRNAs. We have shown that in intestinal epithelial biopsies the steady-state levels of mt 12 S and 16 S rRNA are an order of magnitude greater than those of mt mRNAs. Fractionation of rat small intestinal epithelial cells on the basis of their maturity has shown that the greatest ratios of 12 S mt rRNA/cytochrome b mt mRNA or 12 S mt rRNA/cytochrome oxidase I mt mRNA are found in the surface mature enterocytes, with a progressive decrease towards the crypt immature enteroblasts. Cytochrome b and cytochrome oxidase I mt mRNA levels are relatively uniform along the crypt-villus axis, but fractionation experiments showed increased levels in the crypt base. The levels of human mitochondrial transcription factor A are also greater in immature crypt enteroblasts compared with mature villus enterocytes. These results show that the relative levels of mt rRNA and mRNA are distinctly regulated in intestinal epithelial cells according to the crypt-villus position and differentiation status of the cells, and that there are higher mt mRNA and mt TFA levels in the crypts, consistent with increased transcriptional activity during mitochondrial biogenesis in the immature enteroblasts.
Molecular cloning of a novel splice variant of human type IVA (PDE-IVA) cyclic AMP phosphodiesterase and localization of the gene to the p13.2-q12 region of human chromosome 19
Authors: Horton Y M , Sullivan M , Houslay M D
Published: August 2015

We have isolated from a human T-cell Jurkat cDNA library a novel human cDNA (2EL) that is closely related to the human type-IV PDE splice variant family ‘A’ (PDE-IVA) cDNA characterized previously by us [Sullivan, Egerton, Shakur, Marquardsen and Houslay (1994) Cell. Signalling 6, 793-812]; (h6.1, PDE-IVA/h6.1; HSPDE4A7). (PDE stands for cyclic nucleotide phosphodiesterase). The novel cDNA 2EL (PDE-IVA/2EL; HSPDE4A8) contains two regions of unique sequence not found in PDE-IVA/h6.1. These are a distinct 5′-end and a 34 bp insert which occurs within a domain thought to encode the type-IV PDE catalytic site and which can be expected to result in premature truncation of any expressed protein. HSPDE4A8 appeared to be catalytically inactive. Isolation and characterization of a human genomic cosmid clone revealed that 2EL and h6.1 represent alternative splice variants of the human PDE-IVA gene. Using a unique sequence found at the 5′-end of the 2EL cDNA, a probe was generated which was used to screen the DNA of human-hamster hybrids. This located the human gene for PDE-IVA to human chromosome 19. Through both the analysis of genomic DNAs from a human-hamster somatic cell hybrid panel and also using fluorescent in situ hybridization, it was shown that the human PDE-IVA gene is located on human chromosome 19, between p13.2 [corrected] and q12. This region on chromosome 19 has been shown to be related to genetic diseases such as the autosomal dominant cerebrovascular disease CADASIL, susceptibility to late-onset Alzheimer's disease and changes seen in benign pituitary and thyroid adenomas.
Inhibition of interleukin-2-mediated DNA synthesis in activated human T-lymphoblasts by okadaic acid is accompanied by hyperphosphorylation of lck
Authors: Churcher Y , Moss S E
Published: August 2015

We have previously shown that, during interleukin-2-driven G1/S transition in activated human T-lymphoblasts, a restricted group of cellular proteins become tyrosine phosphorylated de novo, and that p56lck is the major active tyrosine kinase at this stage of the cell cycle. We now report that okadaic acid, a potent and specific inhibitor of protein phosphatases type 1 and type 2A, inhibits S-phase entry, and that this occurs with the simultaneous disappearance of a 56 kDa tyrosine phosphoprotein. We show that this protein is the lck tyrosine kinase and that okadaic acid stimulates a mobility shift to 59 and 64 kDa forms. These two forms of lck were found to have decreased autocatalytic activity, as judged by immune-complex kinase assays. Two-dimensional phosphopeptide mapping and phosphoamino-acid analyses revealed that, in the presence of okadaic acid, lck becomes phosphorylated mainly on serine and to a lesser extent threonine, and that phosphorylation occurs at novel sites. These results show that the kinase activity of lck is at least partly regulated by protein phosphatases, and suggest a role for lck in directing growth-factor-mediated DNA synthesis during T-cell proliferation.
Cloning and analysis of human gastric mucin cDNA reveals two types of conserved cysteine-rich domains
Authors: Klomp L W J , Van Rens L , Strous G J
Published: August 2015

Human gastric mucin was isolated by successive CsCl-gradient ultracentrifugation in the presence of guanidinium hydrochloride to prevent degradation of the polypeptide moieties of the molecules. The amino acid sequence of a tryptic fragment of this molecule was identical to that of a tryptic fragment of tracheobronchial mucin. An oligonucleotide based on this sequence hybridized specifically to human stomach mRNA and was subsequently used to screen a human stomach lambda ZAPII cDNA library. The largest of 10 positive clones encoded 850 amino acid residues, including the tryptic fragment, with high amounts of threonine, serine and proline residues. Interestingly, cysteine accounted for almost 8% of the amino acid residues. The 3′ part of the sequence was very similar but not identical to the 3′ region of human tracheobronchial cDNA. No tandem repeated sequences were present and the deduced polypeptide sequence contained two potential N-linked glycosylation sites. Four cysteine-rich clusters were detected, one of which was apparently homologous to the D-domains present in other mucins and in von Willebrand factor. The arrangement of the cysteines in three other cysteine-rich clusters was conserved in the human gastric mucin cDNA in a similar fashion as in two domains in the MUC2 gene product. The cysteine-rich domains were separated by short stretches of non-repetitive amino acid residues with a very high content of threonine and serine residues. These data suggest that the encoded polypeptide of this clone may be involved in disulphide-bond-mediated oligomerization of the mucin, and provide new insights into the molecular organization of mammalian apomucins.
Identification of a lysosomal protein causing lipid transfer, using a fluorescence assay designed to monitor membrane fusion between rat liver endosomes and lysosomes
Authors: Kuwana T , Mullock B M , Luzio J P
Published: August 2015

In the present and previous studies [Mullock, Perez, Kuwana, Gray and Luzio (1994) J. Cell Biol. 126, 1173-1182], we have attempted to investigate endosome-lysosome fusion using an assay based on the dilution of the self-quenching fluorescent lipid probe octadecylrhodamine. Although some characteristics of fluorescence dequenching were consistent with those observed in other cell-free assays, we have now demonstrated that increased fluorescence was due to leakage of an intralysosomal lipid-transfer protein. This protein was purified and found to be a 22 kDa molecule with sequence, immunological and functional characteristics strongly suggesting that it is the rat homologue of human GM2-activator protein. Both the 22 kDa protein and recombinant human GM2-activator protein caused fluorescence dequenching either when mixed with octadecylrhodamine-loaded endosomes and lysosomal membranes or in a liposome system. The data were consistent with GM2-activator protein acting as an octadecylrhodamine-transfer protein. Antibodies to the 22 kDa protein added to cell-free endosome-lysosome content-mixing assays had no effect, although they could inhibit fluorescence dequenching caused by the protein. Thus this protein is not required in any fusion event involved in delivery of ligands from endosomes to lysosomes. The existence within an intracellular organelle of a protein capable of acting as an octadecylrhodamine-transfer protein suggests the need for caution in the interpretation of fluorescence-dequenching assays using mammalian subcellular fractions.
Mechanisms of NADPH oxidase activation in human neutrophils: p67phox is required for the translocation of rac 1 but not of rac 2 from cytosol to the membranes
Authors: Dusi S , Donini M , Rossi F
Published: August 2015

NADPH oxidase is the enzyme complex responsible for the production of oxygen radicals in phagocytes. On neutrophil stimulation, the cytosolic components of NADPH oxidase, p67phox and p47phox, as well as the Ras-related G-protein rac 2, are translocated from the cytosol to cell membranes where they associate with a flavocytochrome b to form a functional complex. Besides rac 2, rac 1 G-protein is also involved in the activation of the NADPH oxidase, but, to date, it has not been documented whether it is also translocated in activated neutrophils. In this paper we show that: (a) in neutrophils stimulated with formylmethionyl-leucylphenylalanine, concanavalin A or phorbol 12-myristate 13-acetate, both rac 1 and rac 2 are translocated from cytosol to the membranes; (b) in neutrophils from a patient with a form of chronic granulomatous disease in which p67phox is absent, rac 2 and p47phox were translocated as in normal neutrophils on stimulation with the above agonists, but rac 1 failed to be translocated from the cytosol to the membranes. This is the first demonstration that, in activated neutrophils, rac 1 is translocated from the cytosol to the membranes and this translocation requires p67phox. These results, coupled with those showing that rac 2 is not translocated in activated neutrophils lacking p47phox [El Benna, Ruedi and Babior (1994) J. Biol. Chem. 269, 6729-6734], may suggest that the assembly of the cytosolic components of NADPH oxidase on the plasma membrane takes place through selective coupling of activated rac 1 and rac 2 with p67phox and p47phox respectively.
Phosphatidylinositol 3-kinase and the actin network are not required for the stimulation of glucose transport caused by mitochondrial uncoupling: comparison with insulin action
Authors: Tsakiridis T , Vranic M , Klip A
Published: August 2015

In L6 myotubes insulin stimulates glucose transport through the translocation of glucose transporters GLUT1, GLUT3 and GLUT4 from intracellular stores to the plasma membrane. An intact actin network and phosphatidylinositol 3-kinase activity are required for this process. Glucose transport is also stimulated by the mitochondrial ATP-production uncoupler dinitrophenol. We show here that, in serum-depleted myotubes, dinitrophenol induced translocation of GLUT1 and GLUT4, but not GLUT3. This response was not affected by inhibiting phosphatidylinositol 3-kinase or disassembling the actin network. Insulin, but not dinitrophenol, caused tyrosine phosphorylation of several polypeptides, including the insulin-receptor substrate-1 and mitogen-activated protein kinase. Similarly, insulin, but not dinitrophenol, caused actin reorganization, which was inhibited by wortmannin. We conclude that insulin and dinitrophenol stimulate glucose transport by different mechanisms.
Characterization of an exocellular serine-thiol proteinase activity in Paracoccidioides brasiliensis
Authors: Carmona A K , Puccia R , Oliveira M C F , Rodrigues E G , Juliano L , Travassos L R
Published: August 2015

An exocellular proteinase activity has been characterized in Paracoccidioides brasiliensis culture filtrates. Chromatographic analysis showed that the activity was eluted from an anion-exchange Resource Q column at 0.08-0.1 M NaCl, and by gel filtration near ovalbumin elution, in a single peak. Purification of the proteinase, however, was hampered by the low protein yield, in contrast to the high peptidase activity. Numerous chromogenic peptidyl p-nitroanilide derivatives and internally quenched fluorescent peptides, flanked by Abz (O-aminobenzoyl) and EDDnp (ethylenediaminedinitrophenyl), were tested as substrates. Cleavage was observed with Abz-MKRLTL-EDDnp, Abz-FRLVR-EDDnp, and Abz-PLGLLGR-EDDnp at Leu-Thr, Leu-Val and Leu-Leu/Leu-Gly bonds respectively as determined by isolation of the corresponding fragments by HPLC. Leucine at P1 seemed to be restrictive for the activity of the exocellular enzyme, but threonine (P'1) and leucine (P'2) in Abz-MKRLTL-EDDnp apparently were not essential. Also, a pair of alanines could substitute for lysine (P3) and arginine (P2) in this substrate, with a decrease in the Km values. The exocellular peptidase activity of P. brasiliensis had an optimum pH of > 9.0 and was irreversibly inhibited by PMSF, mercuric acetate and p-hydroxymercuribenzoate. Inhibition of the mercuriate compounds could be partially reversed by Cys/EDTA. E-64 [trans-epoxysuccinyl-L-leucylamido-(4-guanido)butene] was a weak and reversible inhibitor, whereas EDTA and pepstatin were not inhibitory. These results suggest that P. brasiliensis exocellular enzyme belongs to the subfamily of SH-containing serine proteinases.
Biosynthesis of rat MUC2 in colon and its analogy with human MUC2
Authors: Tytgat K M A J , Bovelander F J , Opdam F J M , Einerhand A W C , Büller H A , Dekker J
Published: August 2015

In order to identify the mucins synthesized and secreted in the rat colon, we studied their biochemical characteristics and biosynthesis and evaluated their analogy to human colonic mucins. Purified mucin from both species appeared similar with respect to composition, buoyant density and mobility on SDS/PAGE. Isolated rat colonic mucin (RCM) was used to elicit a polyclonal antiserum, which was used in metabolic labelling studies to identify mucins and mucin precursors. RCM is synthesized as a 600 kDa precursor protein, which oligomerizes before O-glycosylation. The mature, high-molecular mass mucin is secreted and displays an anomalous molecular mass on SDS/PAGE of approximately 650 kDa. Polymorphism in precursor size was found among different rats, suggesting genetic heterogeneity. Molecular mass, biosynthesis and secretion of RCM appeared similar to human MUC2. Moreover, RCM precursor could be immunoprecipitated using specific anti-(human MUC2) antisera, indicating that the RCM can be designated rat MUC2. This study describes the biosynthesis of two homologous mucins in two different species. The high degree of similarity suggests functional analogy.
Characterization of three types of human αs1-casein mRNA transcripts
Authors: Johnsen L B , Rasmussen L K , Petersen T E , Berglund L
Published: August 2015

Here we report the molecular cloning and sequencing of three types of human alpha s1-casein transcripts and present evidence indicating that exon skipping is responsible for deleted mRNA transcripts. The largest transcript comprised 981 bp encoding a signal peptide of 15 amino acids followed by the mature alpha s1-casein sequence of 170 amino acids. Human alpha s1-casein has been reported to exist naturally as a multimer in complex with kappa-casein in mature human milk, thereby being unique among alpha s1-caseins [Rasmussen, Due and Petersen (1995) Comp. Biochem. Physiol., in the press]. The present demonstration of three cysteines in the mature protein provides a molecular explanation of the interactions in this complex. Tissue-specific expression of human alpha s1-casein was indicated by Northern-blot analysis. In addition, two cryptic exons were localized in the bovine alpha s1-casein gene.
Binding of gelatinases A and B to type-I collagen and other matrix components
Authors: Allan J A , Docherty A J P , Barker P J , Huskisson N S , Reynolds J J , Murphy G
Published: August 2015

Matrix sequestration of matrix metalloproteinases may be important for the facilitation of remodelling events and the migration of cells through the extracellular matrix. Using an ELISA technique we studied the ability of pro and active forms of gelatinases A and B (GLA and GLB) to bind to matrix components and the contribution made by the different enzyme domains. Pro and active forms of GLA and GLB bound to type-I and type-IV collagens, gelatin and laminin films. Binding to collagens occurred exclusively via the N-terminal portion of the molecule in both of the gelatinases; deletion of the fibronectin-like domain in GLA abolished binding. Fibronectin was shown to compete with GLA, confirming that binding occurs through this domain. GLA and GLB competed for binding to collagen type I, whereas collagenase and stromelysin bound to different sites and could be co-localized with the gelatinases. We conclude that gelatinases have different binding specificities from those previously documented for stromelysin and collagenase, which bind through their C-terminal domains to collagen fibrils.
Expression, purification and characterization of the ubiquitous protein kinase C-related kinase 1
Authors: Palmer R H , Parker P J
Published: August 2015

The recently described protein kinase C-related kinase (PRK) family is comprised of at least three members: PRK1, PRK2 and PRK3. Here the expression, purification and characterization of the ubiquitously expressed isoform, PRK1, is described. The enzyme was expressed in COS 7 cells and subsequently purified to apparent homogeneity by sequential column chromatography. The purified PRK1 protein migrates as a single 120 kDa polypeptide on SDS/PAGE. It displays a substrate specificity that in part resembles that of protein kinase C (PKC); however, unlike PKC, it is not activated by any combination of phorbol esters, diacylglycerol and Ca2+. Nevertheless, it can be activated by limited proteolysis, indicating a negative regulatory role for the N-terminal domain(s). PRK1 is also activated by phospholipids. The physiological relevance of this activation is discussed.
Differential calcium dependence in the activation of c-Jun kinase and mitogen-activated protein kinase by muscarinic acetylcholine receptors in rat 1a cells
Authors: Mitchell F M , Russell M , Johnson G L
Published: August 2015

Carbachol stimulation of the muscarinic acetylcholine m1 receptor (m1R), stably expressed in Rat 1a fibroblasts, resulted in a calcium-dependent activation of c-Jun kinase (JNK). Stimulation of the muscarinic acetylcholine m2 receptor (m2R), stably expressed in Rat 1a fibroblasts, resulted in a G1-mediated activation of JNK that was weak relative to that observed with the m1R. Chelation of calcium inhibited the m2R-mediated activation of JNK but not the robust m2R stimulation of mitogen-activated protein kinase (MAPK) activity. These findings demonstrate a role for the second messenger, calcium, in the differential regulation of the activity of JNK and MAPK in Rat 1a cells.
Serum amyloid A protein enhances the activity of secretory non-pancreatic phospholipase A2
Authors: Pruzanski W , de Beer F C , de Beer M C , Stefanski E , Vadas P
Published: August 2015

The acute-phase proteins serum amyloid A protein (SAA) and secretory phospholipase A2 (sPLA2) are simultaneously expressed during inflammatory conditions. SAA associates with high-density lipoprotein (HDL) altering its physicochemical composition. We found that purified acute-phase SAA, but not the constitutive form, markedly enhances the lipolytic activity of sPLA2 in a dose-related manner with phosphatidylcholine/lysophosphatidylcholine or phosphatidylethanolamine/lysophosphatidylethanolamine liposomal substrates. Normal HDL was found to reduce activity of sPLA2 in a dose-dependent manner, but when acute-phase HDL containing 27% SAA was tested, it enhanced sPLA2 activity. Immunopurified monospecific antibodies against SAA completely abolished the enhancing activity of SAA and acute-phase HDL. Given the central role of HDL in lipoprotein metabolism, the interaction between HDL, SAA and sPLA2 may account for changes detected in lipoprotein metabolism during the acute phase.
Squid spermiogenesis: molecular characterization of testis-specific pro-protamines
Authors: Wouters-Tyrou D , Martin-Ponthieu A , Ledoux-Andula N , Kouach M , Jaquinod M , Subirana J A , Sautière P
Published: August 2015

Cuttlefish spermiogenesis is characterized by a two-step nuclear protein transition: histones-->spermatid-specific protein (protein T)-->sperm protamine (protein Sp). A similar situation can be observed in another Cephalopod species, the squid Loligo pealeii. The protein T from Loligo consists of two structural variants, T1 and T2 (molecular masses: 10788 and 10791 Da respectively), phosphorylated to different degrees (2-6 phosphate groups). The primary structures of these two variants and of the protamine variant Sp2 were established from sequence analysis and mass spectrometric data of the proteins and their fragments. T1 and T2 are closely related proteins of 79 residues. The complete structural identity of the C-terminal domain (residues 22-79) of protein T2 with the sperm protamine Sp2 (molecular mass 8562 Da, 58 residues) strongly suggests that the testis-specific protein T2 is indeed the precursor of the protamine. The transition between the precursor protein T and protein Sp results from a hydrolytic cleavage similar to that found in many proteins that are synthesized as precursors. The processing mechanism involves the specific cleavage of a Gly-Arg bond in the sequence Met/Leu18-Lys-Gly-Gly-Arg-Arg23. Furthermore, the study provides molecular evidence on the taxonomic relationship between Loligo and Sepia.
Isolation and characterization of the flavin-binding domain of flavocytochrome b2 expressed independently in Escherichia coli
Authors: Balme A , Brunt C E , Pallister R L , Chapman S K , Reid G A
Published: August 2015

Flavocytochrome b2 consists of two distinct domains. The N-terminal domain contains protohaem IX and the larger, C-terminal domain contains flavin mononucleotide (FMN). We describe here the isolation of the flavin-binding domain expressed in Escherichia coli independent of the cytochrome domain. The isolated domain is an efficient lactate dehydrogenase with ferricyanide as electron acceptor but reduces cytochrome c, the physiological oxidant for flavocytochrome b2, extremely poorly; electron transfer from the flavin-binding domain to the separately expressed cytochrome domain is undetectable. FMN reduction by lactate occurs as a single exponential process in the isolated flavin-binding domain, in contrast to the biphasic kinetics observed with native flavocytochrome b2.
Functional expression of the taste specific G-protein, α-gustducin
Authors: Hoon M A , Northup J K , Margolskee R F , Ryba N J P
Published: August 2015

The taste-specific G-protein alpha-subunit, alpha-gustducin, was expressed using a baculovirus based system. alpha-Gustducin was demonstrated to be myristoylated and was also palmitoylated in insect larval cells. Recombinant alpha-gustducin was purified to homogeneity. Neither receptors nor effectors that interact with gustducin in taste are known. However, alpha-gustducin has a close structural similarity to the visual G-protein, alpha-transducin. Therefore alpha-gustducin was reconstituted with components of the visual system to determine the degree of its functional similarity with alpha-transducin. Despite the fact that the sequences of alpha-gustducin and alpha-transducin share only 80% identity with each other, the interactions and functions of these two proteins were quantitatively identical. These included the interaction with receptor, bovine rhodopsin, with effector, bovine retinal cyclic GMP-phosphodiesterase, and with bovine brain and retinal G-protein beta gamma-heterodimers; receptor-catalysed GDP-GTP exchange and the intrinsic GTPase activity of alpha-gustducin and alpha-transducin were also identical. Gi alpha which is 70% identical with alpha-transducin interacts with different receptor and effector proteins and has very different guanine-nucleotide binding properties. Therefore, the functional equivalence of alpha-gustducin and alpha-transducin suggest that taste buds are likely to contain receptor and effector proteins that share many properties with their retinal equivalents.
The src-family protein-tyrosine kinase p59hck is located on the secretory granules in human neutrophils and translocates towards the phagosome during cell activation
Authors: Möhn H , Le Cabec V , Fischer S , Maridonneau-Parini I
Published: August 2015

The src-family protein-tyrosine kinase p59hck is mainly expressed in neutrophils; however, its functional role in these cells is unknown. Several other src-family members are localized on secretory vesicles and have been proposed to regulate intracellular traffic. We have established here the subcellular localization of p59hck in human neutrophils. Immunoblotting of subcellular fractions showed that approx. 60% of the p59hck per cell is localized on the secretory granules; the other 40% is distributed equally between non-granular membranes and the cytosol. Immunofluorescence of neutrophils and HL60 cells suggests that the p59hck-positive granules are azurophil granules. Granular p59hck is highly susceptible to degradation by an azurophil-granule proteinase. Different forms of p59hck occur in the three subcellular compartments: a 61 kDa form is mainly found in the granules, a 59 kDa form is predominant in the non-granular membranes, whereas cytosolic p59hck migrates as a doublet at 63 kDa. During the process of phagocytosis-linked degranulation, induced by serum-opsonized zymosan in neutrophils or HL60 cells, granular p59hck translocates towards the phagosome. The subcellular localization of p59hck suggests that the enzyme could be involved in the regulation of the degranulation process.
Sequence variation of a novel heptahelical leucocyte receptor through alternative transcript formation
Authors: Barella L , Loetscher M , Tobler A , Baggiolini M , Moser B
Published: August 2015

Chemoattractants, including chemokines such as interleukin 8 (IL-8) and related proteins, activate leucocytes via seven-transmembrane-domain G-protein-coupled receptors. A cDNA for a novel receptor of this kind consisting of 327 amino acids was isolated from a human blood monocyte cDNA library. The polypeptide, termed monocyte-derived receptor 15 (MDR15), is an alternative form of the Burkitt's lymphoma receptor 1 (BLR1) encoded by a human Burkitt's lymphoma cDNA [Dobner, Wolf, Emrich and Lipp (1992) Eur. J. Immunol. 22, 2795-2799]. MDR15 and BLR1 cDNAs differ in the 5′ region, where the open reading frame of MDR15 is shorter by 45 codons. Southern-blot analysis indicates that the two transcripts for MDR15 and BLR1 are encoded by the same gene. Northern-blot analysis using a probe that hybridizes with both mRNAs demonstrated high-level expression in chronic B-lymphoid leukaemia and non-Hodgkin's lymphoma cells and, to a lesser extent, peripheral blood monocytes and lymphocytes. Reverse transcription-PCR studies with MDR15- and BLR1-specific primers showed similar levels of transcripts for both receptors in RNA that was positive in Northern-blot analysis. MDR15 and BLR1 have high structural similarity to receptors for human IL-8 (about 40% amino acid identity) and other chemokines. However, none of a series of radiolabelled chemokines (IL-8, NAP-2, GRO alpha, PF4, IP10, MCP-1, MCP-2, MCP-3, I-309, RANTES and MIP-1 alpha) and other ligands (C3a and leukotriene B4) bound to Jurkat transfectants that stably expressed either MDR15 or BLR1 mRNA. The fact that MDR15 and BLR1 are expressed on leucocytes and show marked sequence similarity to chemokine receptors suggests the existence of as yet unidentified chemokines. Alternative transcript formation affecting the 5′-terminal part of the coding region may be a way to modify ligand-binding selectivity.
The phosphorylation state of the microtubule-associated protein tau as affected by glutamate, colchicine and β-amyloid in primary rat cortical neuronal cultures
Authors: Davis D R , Brion J P , Couck A M , Gallo J M , Hanger D P , Ladhani K , Lewis C , Miller C C J , Rupniak T , Smith C , Anderton B H
Published: August 2015

The effects of the excitatory amino acid glutamate, the microtubule destabilizing agent colchicine, and beta 25-35-amyloid peptide on the phosphorylation state of tau were studied in rat cortical neurons in primary culture. Using immunocytochemistry and Western-blot analysis, we demonstrated that a proportion of tau in these cultures is normally highly phosphorylated, but most of this tau fraction is dephosphorylated after treatment of the cultures with glutamate or colchicine, but not with beta-amyloid; the glutamate- and colchicine-induced changes in tau phosphorylation commenced before cell death, as assessed by release of lactate dehydrogenase. Dephosphorylation of tau was readily revealed by using the monoclonal antibodies Tau.1 and AT8, which have phosphate-sensitive epitopes that both centre around serine-199 and -202 (numbering of the largest tau isoform). On Western blots and by immunocytochemistry, AT8 labelling strongly decreased after glutamate and colchicine treatments, whereas Tau.1 staining was more intense. Neurofilament monoclonal antibodies, including RT97, 8D8, SMI31 and SMI310, all additionally known to recognize tau in a phosphorylation-dependent manner, also demonstrated that glutamate and colchicine treatments of the cultures induced a dephosphorylation of tau. We also showed immunocytochemically that there is an increase in tau immunoreactivity in neuronal perikarya in response to glutamate and colchicine treatment, and this occurs concomitantly with the dephosphorylation of tau. Treatment of the primary rat cortical neuronal cultures with beta 25-35-amyloid peptide, under conditions which induce neuronal degeneration, did not induce a change in tau phosphorylation, and failed to act synergistically with glutamate to produce an increase in dephosphorylation of tau over that produced by glutamate treatment alone. These findings demonstrate that glutamate and colchicine induce tau dephosphorylation, as opposed to increased tau phosphorylation, which would be more indicative of Alzheimer-type neurodegeneration.
The napEDABC gene cluster encoding the periplasmic nitrate reductase system of Thiosphaera pantotropha
Authors: Berks B C , Richardson D J , Reilly A , Willis A C , Ferguson S J
Published: August 2015

The napEDABC locus coding for the periplasmic nitrate reductase of Thiosphaera pantotropha has been cloned and sequenced. The large and small subunits of the enzyme are coded by napA and napB. The sequence of NapA indicates that this protein binds the GMP-conjugated form of the molybdopterin cofactor. Cysteine-181 is proposed to ligate the molybdenum atom. It is inferred that the active site of the periplasmic nitrate reductase is structurally related to those of the molybdenum-dependent formate dehydrogenases and bacterial assimilatory nitrate reductases, but is distinct from that of the membrane-bound respiratory nitrate reductases. A four-cysteine motif at the N-terminus of NapA binds a [4Fe-4S] cluster. The DNA- and protein-derived primary sequence of NapB confirm that this protein is a dihaem c-type cytochrome and, together with spectroscopic data, indicate that both NapB haems have bis-histidine ligation. napC is predicted to code for a membrane-anchored tetrahaem c-type cytochrome that shows sequence similarity to the NirT cytochrome c family. NapC may be the direct electron donor to the NapAB complex. napD is predicted to encode a soluble cytoplasmic protein and napE a monotopic integral membrane protein, napDABC genes can be discerned at the aeg-46.5 locus of Escherichia coli K-12, suggesting that this operon encodes a periplasmic nitrate reductase system, while napD and napC are identified adjacent to the napAB genes of Alcaligenes eutrophus H16.
Proliferative and cytotoxic effects of mildly oxidized low-density lipoproteins on vascular smooth-muscle cells
Authors: Augé N , Pieraggi M T , Thiers J C , Nègre-Salvayre A , Salvayre R
Published: August 2015

We have investigated the role of low-density lipoprotein (LDL) oxidation in the proliferative effect of LDLs on cultured bovine aortic smooth-muscle cells and compared it with their effect on bovine aortic endothelial cells. The following conclusions were reached. (1) Non-toxic doses of mildly oxidized LDLs elicit a proliferative effect on smooth-muscle cells significantly higher than that of native LDLs or lipoprotein-depleted serum. The proliferative effect is dependent on time (relatively slow), dose (high doses are cytotoxic) and the level of LDL oxidation. (2) The proliferative effect on smooth-muscle cells is counterbalanced at high concentrations of mildly oxidized LDLs (or at high oxidation levels) by their cytotoxic effect. (3) The same dose of mildly oxidized LDLs exhibits no proliferative effect on endothelial cells but rather a cytotoxic one. Endothelial cells may therefore be intrinsically more susceptible to the cytotoxic effect of mildly oxidized LDLs than are smooth-muscle cells. (4) The proliferative effect of native LDLs on smooth-muscle cells results (at least in part) from cell-induced LDL oxidation during cell culture as suggested by (i) the progressive LDL oxidation over the 3 days of contact between LDLs and smooth-muscle cells and (ii) the concomitant inhibition of LDL oxidation and proliferative effect by butylated hydroxytoluene. The hypothetical mechanisms and potential involvement in atherogenesis are discussed.