bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel primary Ca2+‐transport system from Saccharomyces cerevisiae
Authors: OKOROKOV Lev A. , TANNER Widmar , LEHLE Ludwig
Published: March 2005

A novel primary Ca2+‐transport system in membrances from Saccharomyces cerevisiae is described. Ca2+ transport is strictly dependent on the presence of ATP; other nucleotides like GTP, UTP and CTP do not efficiently (<10% of the rate of ATP) drive uptake. Transport is inhibited by sodium vanadate with an IC50 of 130 μM, but is insensitive to carbonylcyanide p‐trifluoromethoxy‐phenylhydrazone, valinomycin, gramicidin or calmodulin. Ca2+ accumulates in a free form and can be readily released by the Ca2+ ionophore A‐23187 or by osmotic shock. The apparent Km values of transport activity for free Ca2+ was determined to be 0.11 μM and 5 μM for Mg. ATP, respectively. Taken together the results indicate that the Ca2+ transport described here does not belong to the plasma‐membrane‐type Ca2+‐ATPase family but rather to the family of endomembrane‐type ATPases. Cell‐fractionation studies of crude membranes on sucrose gradient centrifugation have shown that the Ca2+‐transport activity separates from marker enzymes for endoplasmic reticulum, vacuole, or plasma membrane and migrates with GDPase activity, a marker for the yeast Golgi complex.
Structure and metabolic control of the Yarrowia lipolytica peroxisomal 3‐oxoacyl‐CoA‐thiolase gene
Authors: BERNINGER Gabriele , SCHMIDTCHEN Robert , CASEL Gaby , KNORR Annegret , RAUTENSTRAUSS Karin , KUNAU Wolf‐Hubert , SCHWEIZER Eckhart
Published: March 2005

Using a Yarrowia lipolytica genomic library, several overlapping clones of the peroxisomal 3‐oxoacyl‐CoA‐thiolase gene, POT1, were isolated. The library was prepared in the bacterial expression vector λgt11, thus allowing an immunological screening of recombinant bacteriophages with specific antibodies raised against purified peroxisomal thiolase. The isolated POT1 clones hybridized to a 1.4 kb RNA species, which was induced approximately 30‐fold when oleate was the carbon source. A 3634‐bp segment of the cloned DNA was sequenced. This segment contained, on both strands, three major overlapping open‐reading frames of 678, 1122 and 1242 bp. Northern‐hybridization analysis showed that only the largest of these reading frames was transcribed. It encodes a protein of 414 amino acids and molecular mass 43.059 kDa. Its deduced amino acid sequence has 30–60% identity and 50–70% sequence similarity when compared to other known thiolases. According to both the amount (68–71%) and location of conserved amino acids, the encoded protein belongs to the peroxisomal rather than the mitochondrial or cytoplasmic class of thiolases. Compared to bacterial and yeast cytosolic thiolases, the POT1 gene product contains a N‐terminal extension of 25 amino acids which clearly differs from typical mitochondrial import signals. One of the isolated clones contained, in addition to the POT1 coding sequence, 784 bp of the corresponding 5′ flanking region. Nevertheless, it was efficiently expressed in Escherichia coli suggesting the correct recognition of this fungal promoter by the prokaryotic transcriptional and translational machinery. The Y. lipolytica genomic POT1 gene was disrupted by replacing 120 bp of its coding sequence with 2.7 kbp of DNA including the Y. lipolytica LEU2 gene. The resulting pot1: :LEU2 cells were free of immunologically cross‐reacting thiolase. Western‐blot analysis showed that the product of the non‐disrupted gene had a molecular mass of approximately 42 kDa. This corresponds well to the molecular mass of purified Y. lipolytica peroxisomal thiolase. Disruption of POT1 abolished the ability of Y. lipolytica cells to grow on solid media with oleate as a carbon source. This inability to grow in the presence of oleate suggests both the catabolic function of POT1 and the absence of additional catabolic thiolases in Y. lipolytica. However, the pot1: :LEU2 cells were unaffected in their ability to elongate externally added tridecanoic acid to its higher‐chain‐length homologues. Hence, another, POT1‐independent and biosynthetic 3‐oxoacyl‐CoA thiolase must be responsible for this reaction in Y. lipolytica.
Isolation of three antibacterial peptides from pig intestine: gastric inhibitory polypeptide(7–42), diazepam‐binding inhibitor(32–86) and a novel factor, peptide 3910
Authors: AGERBERTH Birgitta , BOMAN Anita , ANDERSSON Mats , JÖRNVALL Hans , MUTT Viktor , BOMAN Hans G.
Published: March 2005

Two antibacterial peptides, cecropin P1 and PR‐39 (39‐residue proline/arginine‐rich peptide), from the upper part of pig small intestine have previously been isolated and characterized. We have now continued our search for antibacterial peptides in different side fractions generated during the isolation of intestinal hormones. Starting from one such fraction and monitoring activity againstBacillus megaterium, we isolated three homogeneous peptides by three consecutive chromatographic steps. Amino acid sequence analysis in combination with mass spectrometry identified two of the peptides as gastric inhibitory polypeptide(7–42) [GIP(7–42)] and diazepam‐binding inhibitor(32–86) [DBI(32–86)], derived from factors already known. However, intact GIP and DBI have hardly any antibacterial activity by themselves. The third peptide constitutes a previously unknown structure, designated as peptide 3910 from its molecular mass. All three peptides showed good activity againstB. megaterium.In addition. GIP(7–42) showed some activity againstStreptococcus pyogenesand anEscherichia colimutant with a defect in its outer membrane.
Bovine lens acylpeptide hydrolase
Authors: SHARMA K. Krishna , ORTWERTH Beryl J.
Published: March 2005

An acylpeptide hydrolase has been purified from bovine lens tissue by anion‐exchange and hydrophobic‐interaction chromatography. The enzyme, purified over 27000‐fold with 44% recovery, has a molecular mass of 300 kDa under native conditions. Under denaturing conditions it shows a subunit molecular mass of 75 kDa. The enzyme is inhibited by diisopropylfluorophosphate (iPr2P‐F), phenylmethylsulfonyl fluoride and N‐ethylmaleimide, indicating the presence of an essential serine residue and ‐SH group. Each subunit of the enzyme has one active serine residue which can be labelled with [3H]iPr2P‐F. Nα‐blocked amino acids in L form act as competitive inhibitors of the enzyme. The antibiotics penicillin‐G and ampicillin partially inhibit the enzyme. Exposure of the purified enzyme to the proteases trypsin, chymotrypsin or elastase do not result in any loss of activity. Digestion of the native enzyme with bovine trypsin generates a 55‐kDa protein containing the active‐site serine and a 22‐kDa polypeptide, indicating the presence of a unique trypsin site. N‐terminal amino acid sequencing of the 55‐kDa polypeptide shows that the bovine lens enzyme has a sequence at the trypsin cleavage site identical to the porcine liver acylpeptide hydrolase sequence 196–215. The data show that the split enzyme is as active as the native enzyme towards the synthetic substrate Ac‐Ala‐p‐nitroanilide. The enzyme activity decreases with increasing urea, but 15% of the activity remains even in the presence of 6.0 M urea. On removal of urea, complete recovery of the enzyme activity is observed. However, treatment with 1 M guanidine/HCl completely inactivates the enzyme.
Tyrosine‐kinase activity in rabbit platelets stimulated with platelet‐activating factor
Authors: MURPHY Christine T. , KELLIE Stuart , WESTWICK John
Published: March 2005

The temporal relationship of tyrosine phosphorylation of proteins in platelet‐activating‐factor‐(PAF)‐stimulated rabbit platelets was characterised by Western blotting using a monoclonal anti‐phosphotyrosine antibody, demonstrated to be specific for detecting only tyrosine phosphorylated proteins. In addition, the protein tyrosine kinase (PTKase) inhibitor genistein, was used to investigate the role of endogenously activated PTKase(s) in the regulation of receptor‐stimulated changes in both signal molecule production and in platelet functional responses. Several tyrosine phosphorylated protein bands (52–62 kDa) were observed in unstimulated platelets, however, within 5 s of PAF stimulation, two further groups of tyrosine phosphorylated protein bands were observed (35–45 kDa and 66–90 kDa) and within 30 s of PAF stimulation a further group was detected (90–150 kDa). Under conditions where intracellular Ca2+ was chelated with acetoxymethyl 1,2‐bis(O‐aminophenoxy)ethane‐N,N,N′N′‐tetraacetate (BAPTA‐AM) and extracellular Ca2+ was chelated with EGTA, the number of tyrosine‐phosphorylated bands was greatly reduced. Tyrosine phosphorylation of the proteins induced by PAF stimulation were differentially inhibited by treatment with genistein. Genistein inhibited PAF‐induced elevation of the signal molecule inositol 1,4,5‐trisphos‐phate and also inhibited both mobilization of Ca2+ and the influx of Ca2+ through the plasma membrane. These results suggest a role for endogenously activated PTKase(s) in the early stages of signal transduction in PAF‐stimulated platelets. Moreover, inhibition of genistein‐sensitive PTKase(s) also caused an inhibition of PAF‐induced thromboxane B2 generation, dense‐granule release and platelet aggregation, indicating a role for PTKase(s) in the regulation of platelet functional responses. Platelets stimulated with α‐thrombin, ionomycin and 12‐O‐tetradecanylphorbol 13‐acetate gave a similar pattern of phosphorylated proieins to PAF‐stimulated platelets, however, whereas genistein inhibited protein phosphorylation, it had no significant effect on functional responses in platelets stimulated with these agents, suggesting that an alternative signalling pathway exists.
Complete sequence of human fast‐type and slow‐type muscle myosin‐binding‐protein C (MyBP‐C)
Authors: WEBER Franz E. , VAUGHAN Kevin T. , REINACH Fernando C. , FISCHMAN Donald A.
Published: March 2005

Myosin‐binding‐protein C (MyBP‐C) is a myosin‐associated protein of unknown function found in the cross‐bridge‐bearing zone (C region) of A bands in striated muscle. Using a cDNA clone encoding the fast‐type isoform of chicken MyBP‐C, we screened a human fetal muscle cDNA library and isolated clones encoding the full‐length human fast‐type isoform of MyBP‐C. cDNA clones encoding the slow‐type isoform of human MyBP‐C, were also isolated and fully sequenced. Northern‐blot analysis demonstrated skeletal muscle‐specific expression of these gene products. Using human/hamster somatic‐cell hybrids, we were able to map the slow‐type MyBP‐C to human chromosome 12, and the fast‐type MyBP‐C to chromosome 19.

The cDNA for human fast‐type MyBP‐C encodes a polypeptide of 1142 amino acids with an expected molecular mass of 128.1 kDa. Comparison of this cDNA with other members of the MyBP family reveals extensive primary‐sequence conservation. Each MyBP‐C contains seven immunoglobulin C2 motifs and three fibronectin type‐III repeats in the arrangement C2‐C2‐C2‐C2‐C2‐III‐III‐C2‐III‐C2. Regions of high identity shared by the chicken and the two human proteins are not restricted to the immunoglobulin and fibronectin motifs. Sequence comparison of all three proteins has allowed us to map a highly conserved region between the first and second C2 motifs, the only large spacer sequence present between motifs in these proteins.
Post‐translational modifications of theDictyostelium discoideumglycoprotein PsA
Authors: HAYNES Paul A. , GOOLEY Andrew A. , FERGUSON Michael A. J. , REDMOND John W. , WILLIAMS Keith L.
Published: March 2005

Prespore‐specific antigen (PsA) is a cell‐surface glycoprotein isolated fromDictyostelium discoideum, which is post‐translationally modified by addition of carbohydrate to threonine residues of the carboxy‐terminal peptide domain, and a glycosylphosphatidylinositol (GPI) anchor which attaches the glycoprotein to the cell membrane. The GPI anchor was isolated by proteolytic cleavage of the protein, and the structure of the lipid and glycan portions of the anchor were determined. The lipid moiety of the anchor is an inositolphosphoceramide which contains C18:0 phytosphingosine as a long chain base, and a mixture of fatty acids with a C18:1 mono‐unsaturated fatty acid as the major component. The purified GPI anchor was susceptible to digestion by a bacterial phosphatidylinositol‐specific phospholipase‐C enzyme. The glycan of the GPI anchor consisted of two molecular species present in the ratio 55:45, the structures of which were determined by exoglycosidase sequencing and found to be Manα1‐2Manα1‐6Manα1‐4GlcNH2and Manα1‐2Manα1‐2Manα1‐6Manα1‐4GlcNH2. The glucosamine in both structures is glycosidically linked to the inositol ring of the inositolphosphoceramide. The GPI glycan structures are consistent with the conserved core structure of all characterised GPI anchors, and the structure of theD. discoideumGPI moiety has features in common with structures from yeast, protozoa and higher eukaryotes.

Compositional analysis of the carbohydrate attached to threonine residues in the carboxy‐terminal peptide domain is also presented. The oligosaccharides bind to wheat germ agglutinin, and contain glucosamine and fucose as the major constituents.
Does oligosaccharide‐phosphatidylinositol (glycosyl‐phosphatidylinositol) hydrolysis mediate prolactin signal transduction in granulosa cells?
Authors: FANJUL Luisa F. , MARRERO Isabel , GONZÁLEZ Juan , QUINTANA José , SANTANA Pino , ESTÉVEZ Francisco , MATO Jose María , RUIZ DE GALARRETA Carlos M.
Published: March 2005

Initial biosynthetic radiolabelling experiments with cultured granulosa cells revealed the presence of an oligosaccharide‐phosphatidylinositol (glycoyl‐phosphatidylinositol; (Ose)nPtdIns) structurally related to (Ose)nPtdIns‐lipids isolated from other cell types. Prolactin (PRL) stimulated [3H]glucosamine‐(Ose)nPtdIns turnover and the rapid generation of [3H]myristoyl‐diacylglycerol in cultured follicle‐stimulating hormone‐(FSH)‐primed granulosa cells endowed with PRL receptors. In parallel experiments performed with [3H]myo‐inositol‐labelled granulosa cells, treatment with PRL stimulated (Ose)nPtdIns hydrolysis in a similar manner, whereas no effect on phosphoinositide (PtdIns, PtdInsP and PtdInsP2) turnover could be observed. These results strongly suggest that the cleavage of (Ose)nPtdIns by phosphodiesterase followed by the subsequent generation of diacylglycerol and a soluble phosphoinositol‐oligosaccharide (inositol‐phosphoglycan; (Ose)nInsP) moiety could be part of the signal‐transduction mechanism linking PRL receptors to their biological effects in granulosa cells. To test this hypothesis, we examined the effect of PRL and purified (Ose)nInsP moiety (from rat liver membranes) on granulos cell 3β‐hydroxysteroid dehydrogenase/Δ5–4 isomerase (3β‐HSD) enzyme activity. Results presented show that, in FSH‐primed granulosa cells, PRL (40 nM) and (Ose)nInsP (5 μM) prevented gonadotropin‐stimulated 3β‐HSD activity. Further‐more, in undifterentiated granulosa cells where PRL receptors are absent, no effect of the hormone on 3β‐HSD activity could be observed, whereas, (Ose)nInsP (1–10 μM) inhibited enzyme activity in a dose‐dependent manner.
A study of colchicine tubulin complex by donor quenching of fluorescence energy transfer
Authors: BHATTACHARYYA Anusree , BHATTACHARYYA Bhabatarak , ROY Siddhartha
Published: March 2005

The utility of collisional quenching of energy donors in fluorescence energy transfer is described. In multi‐donor single acceptor systems, which contain different classes of donors (as distinguished by their accessibility towards a collisional quencher), donor quenching may be used to assess the fraction of energy transfer from each class of donor. The tubulin‐colchicine complex was used as a donor‐acceptor system to show that two inaccessible tryptophans are at or near the colchicine binding site.
The structure of N‐linked oligosaccharides of human pancreatic bile‐salt‐dependent lipase
Authors: SUGO Tsukasa , MAS Eric , ABOUAKIL Nezha , ENDO Tamao , ESCRIBANO Maria‐Juana , KOBATA Akira , LOMBARDO Dominique
Published: March 2005

This study describes the structure of the N‐linked oligosaccharide chains of bile‐salt‐dependent lipase isolated from the pancreatic juice of a normal donor. After hydrazinolysis, neurtral sugar chains were separated from acidic chains by paper electrophoresis and were fractionated using serial column chromatography with immobilized lectins and Bio‐Gel P‐4 filtration. Structural analysis was performed by means of sequential glycosidase digestion and revealed that the neutral sugar chains are mainly of the biantennary complex type. Fucose residues were identified for some trimannosyl core structures and were α(i‐6) or α(1–2) linked to the innermost GlcNAc residue and a terminal Gal residue, respectively. Sialyl residues were also involved in the oligosaccharide structures. Most of these structures have no linear N‐acetyllactosamine repeats. Evidence from several approaches suggests that the sugar chains of the human pancreatic bile‐salt‐dependent lipase possess a blood‐group‐related antigenic determinant.
Erythropoietin induces the association of phosphatidylinositol 3′‐kinase with a tyrosine‐phosphorylated protein complex containing the erythropoietin receptor
Authors: MAYEUX Patrick , DUSANTER‐FOURT Isabelle , MULLER Odile , MAUDUIT Philippe , SABBAH Michèle , DRUKER Brian , VAINCHENKER William , FISCHER Sigmund , LACOMBE Catherine , GISSELBRECHT Sylvie
Published: March 2005

Stimulation of sensitive cells with erythropoietin results in rapid induction of protein tyrosine phosphorylation. Other than tyrosine phosphorylation of one chain of the erythropoietin receptor, the identities of the remaining tyrosine‐phosphorylated proteins are undefined. In this report, we demonstrate that the stimulation of the erythropoietin‐sensitive human UT7 cells by erythropoietin rapidly resulted in the appearance of phosphatidylinositol 3‐kinase activity in anti‐phosphotyrosine immunoprecipitates. Erythropoietin action was rapid, detectable after as early as 1 min stimulation, transient, returning to control level after 30 min stimulation and was observed using the erythropoietin concentrations able to stimulate the cell proliferation. Anti‐(phosphatidylinositol 3‐kinase) antibodies specifically immunoprecipitated 125I‐erythropoietin bound to its receptor, strongly suggesting that phosphatidylinositol 3‐kinase associated with a protein complex containing the activated erythropoietin receptor. To confirm this result, phosphatidylinositol 3‐kinase was immunoprecipitated from erythropoietin‐stimulated cells using mild conditions followed by Western analysis using anti‐phosphotyrosine antibodies. Five tyrosine phosphorylated proteins were revealed: the cloned chain of the erythropoietin receptor, the regulatory subunit of phosphatidylinositol 3‐kinase and three unidentified proteins of 111, 97 and 64 kDa. None of these tyrosine phosphorylated proteins was detected in anti‐(phosphatidylinositol 3‐kinase) immunoprecipitates from unstimulated cells. Thus, our results show that phosphatidylinositol 3‐kinase associates with a tyrosine‐phosphorylated protein complex containing the activated erythropoietin receptor.
Biosynthesis of platelet‐activating factor in cultured mast cells.
Authors: COLARD Odile , BIDAULT Jocelyne , BRETON Michelyne , NINIO Ewa
Published: March 2005

We have recently demonstrated that arachidonate [20:4(5,8,11,14)] was primarily linked to the hexadecyl (16:0) and octadecenyl (18:1) species of alkylacyl derivatives of glycerolphosphocholine (GroPCho). Consistent with the involvement of arachidonate‐specific CoA‐independent transacylase in the synthesis of platelet‐activating factor (PAF: 1‐O‐alkyl‐2‐acetyl‐GroPCho), 16:0 and 18:1 PAF species were formed upon antigen stimulation [Joly, F., Breton, M., Wolf, C., Ninio, E. & Colard, O. (1992) Biochim. Biophys. Acta 1125, 305–312]. In the present work, addition of lyso‐PAF to mast cells resulted in PAF production. We analyzed the PAF species formed in the presence of a defined lyso‐PAF molecular species in order to differentiate between either direct acetylation or involvement of the membrane precursor. The 18:1 lyso‐PAF was more effective than the 16:0 in producing PAF which was composed of 95% 18:1 PAF, the balance being 16:0, indicating that part of the acetylated lyso‐PAF originated from the cellular pool of alkyl‐arachidonyl‐GroPCho in resting cells. Consistent with alkyl‐arachidonyl‐GroPCho species content and acetyltransferase specificity, similar amounts of 16:0 and 18:1 PAF species were formed when mast cells were stimulated with antigen. Supplemented with 16:0 or 18:1 lyso‐PAF, antigen‐stimulated mast cells responded by 230% and 125% increase in PAF synthesis, respectively. As expected, the amount of the PAF species corresponding to the added lyso‐PAF was increased. More interestingly, addition of 16:0 lyso‐PAF almost doubled the amount of 18:1 PAF content as compared to antigen alone, thus indicating that the lyso‐PAF formed via the CoA‐independent transacylase was significantly used for PAF synthesis. despite a large excess of exogenous lyso‐PAF. The CoA‐independent transacylase, measured using [3H]lyso‐PAF as a substrate in sonicates from antigen‐stimulated cells, was decreased concurrently with PAF formation. In conclusion, we show that when lyso‐PAF is added to mast cells, a direct acetylation may occur. However, PAF is preferentially synthesized through a mechanism involving the CoA‐independent transacylase reaction.
Cloning of two related genes encoding the 56‐kDa and 123‐kDa subunits of trehalose synthase from the yeast Saccharomyces cerevisiae
Authors: VUORIO Outi E. , KALKKINEN Nisse , LONDESBOROUGH John
Published: March 2005

Preparations of intact trehalose synthase contain three polypeptides with molecular masses of 56, 102 and 123 kDa. We have cloned the genes TSSI and TSLI coding for the 56‐ and 123‐kDa subunits, respectively. These genes are located on chromosomes II (TSS1) and XIII (TSLI). The TSS1 gene was found to be identical with CIF1, a gene required for normal growth on glucose. The product of the entire TSS1 gene exibits 37% identity with a 502‐amino‐acid stretch from the middle of the TSL1 product. Disruption of the TSS1 gene in yeast eliminates both trehalose 6‐phosphate synthase (Tre6P synthase) and trehalose 6‐phosphate phosphatase (Tre6Pase) activities, and reintroduction of this gene restores these activities. Transformation of Escherichia coli with TSS1 increases its Tre6P synthase activity. Specific proteolytic degradation of the 123‐kDa polypeptide from the N‐terminus greatly influences the Tre6P synthase activity, decreasing its inhibition by phosphate and activatability by fructose 6‐phosphate but has little effect on the Tre6Pase activity. These results suggest that this N‐terminal part confers regulatory properties upon the Tre6P synthase activity.
Gene sequence, cDNA construction, expression in Escherichia coli and genetically approached purification of porcine interleukin‐1β
Authors: VANDENBROECK Koen , FITEN Pierre , BEUKEN Erik , MARTENS Erik , JANSSEN Ann , VAN DAMME Jo , OPDENAKKER Ghislain , BILLIAU Alfons
Published: March 2005

A genomic clone (PIL3) covering the 8.8‐kb prointerleukin‐1β (‘catabolin’) gene of the domesticated swine (Sus scrofa domestica) was isolated from a genomic library and characterized by nucleotide sequencing. Typical features of the gene include a seven‐exon structure, with the highest degree of nucleotide and amino acid conservation among human and porcine genes being found in the receptor‐binding portion encoded by exons six and seven. Three 250‐bp repetitive elements with a >75% similarity to the pig repetitive element‐1 family sequence are located in untranslated gene segments. Southern‐hybridization experiments disclosed extensive genomic heterogeneity of the porcine interleukin‐1β (IL‐1β) gene region, suggesting a duplication of at least the 3′ half of the gene in the porcine genome. Since similar hybridization patterns were observed for wild boar (Sus scrofa) genomic DNA, it was concluded that this gene rearrangement had preceded domestication of the wild swine. In addition, the cDNA for processed porcine IL‐1β was constructed through polymerase‐chain‐reaction‐mediated exon fusion by overlap extension starting from the genomic template. Recombinant IL‐1β was expressed in Escherichia coli as a fusion protein containing an N‐terminal hexahistidine tag followed by a factor‐Xa‐cleavage site. The protein was efficiently purified through adoption of a scheme that consisted of four alternating cycles of immobilized metal‐ion‐affinity chromatography and size‐exclusion chromatography. 13.8 mg highly purified recombinant porcine IL‐1β was obtained starting from a 900‐ml thermo‐induced E. coli culture (final endotoxin concentration < 0.22 ng/ml). The protein behaved homogeneously as a monomeric species, which was reactive in Western‐blot experiments with an anti‐(human‐IL‐1β) serum and which appeared to induce gelatinase B in MDBK cells in a dose‐dependent fashion.
A role for protein kinase C‐α in zymosan‐stimulated eicosanoid synthesis in mouse peritoneal macrophages
Authors: HUWILER Andrea , PFEILSCHIFTER Josef
Published: March 2005

A possible regulatory function of protein kinase C (PKC) isoenzymes in zymosan‐stimulated eicosanoid synthesis was studied in mouse peritoneal macrophages in culture. The addition of zymosan to intact cells labelled with [3H]arachidonic acid stimulated a time‐dependent and concentration‐dependent release of the fatty acid. There was a simultaneous marked increase in the synthesis of prostaglandin E2 and leukotriene C4. The protein‐kinase inhibitor K‐252a and the selective PKC inhibitor CGP41251 completely blocked zymosan‐triggered arachidonic acid release as well as prostaglandin E2 and leukotriene C4 synthesis. In contrast, an inactive staurosporine derivative, CGP42700, failed to inhibit any of the zymosan‐induced responses. The down‐regulation of PKC by long‐term treatment with phorbol 12‐myristate 13‐acetate eliminated zymosan‐stimulated arachidonic acid release and eicosanoid synthesis (after 4–6 h treatment). By using specific antibodies it was observed that mouse macrophages express five PKC isoenzymes, PKC‐α, ‐β, ‐δ, ‐ζ and ‐ζ. No PKC‐γ isoenzyme was detected. After exposure to phorbol 12‐myristate 13‐acetate, a complete depletion of PKC‐β was observed within 1 h and the complete depletion of PKC‐α and PKC‐δ isotypes was observed within 4 h. In contrast, PKC‐ε was only partially down‐regulated after a 24‐h treatment with phorbol 12‐myristate 13‐acetate and PKC‐ζ was not affected at all. These data indicate that PKC‐α and PKC‐δ isoenzymes are candidates for regulating prostaglandin and leukotriene production. From the potent inhibitory activities of K‐252a and CGP41251, two compounds that reportedly display a higher selectivity for PKC‐α compared to PKC‐δ, it is suggested that PKC‐α triggers arachidonic acid mobilization and eicosanoid synthesis in peritoneal macrophages.
Correlation between the inhibition of cell growth by accumulated polyamines and the decrease of magnesium and ATP
Authors: HE Yong , KASHIWAGI Keiko , FUKUCHI Junichi , TERAO Kiyoshi , SHIRAHATA Akira , IGARASHI Kazuei
Published: March 2005

The mechanism of the antiproliferation effect of spermidine and spermine was studied using a cell culture system of mouse FM3A cells. The addition of either 10 mM spermidine or 2 mM spermine to the growth medium containing 0.9 mM Mg2+ greatly inhibited cell growth (more than 90%). A decrease in the Mg2+ concentration to 50 μM in the growth medium, but without the polyamine addition, did not influence cell growth. However, the concentrations of spermidine and spermine necessary for the inhibition of cell growth when cells were cultured in the presence of 50 μM Mg2+ were much smaller (2 mM spermidine and 0.15 mM spermine). Nevertheless, the amount of polyamines accumulating in cells which could cause the inhibition of cell growth was almost the same, regardless of the large difference in the added polyamine concentrations. At the early stage of polyamine accumulation, the inhibition of cell growth correlated with the decrease of Mg2+ content, but not with a decrease of the ATP content. The decrease in Mg2+ content correlated well with the inhibition of macromolecular synthesis, especially protein synthesis. Thus, the inhibition of cell growth at the early stage of polyamine accumulation was thought to be due to the inactivation of ribosomes through the replacement of Mg2+ on magnesium‐binding sites by polyamines. The decrease in Mg2+ content was mainly caused by the inhibition of Mg2+ transport by polyamines. At the later stage of polyamine accumulation, a decrease in ATP content was also observed. This was followed by swelling of the mitochondria, which may be a symptom of the subsequent cell death.
The mannose‐specific lectins from ramsons (Allium ursinum L.) are encoded by three sets of genes
Authors: VAN DAMME Els J. M. , SMEETS Koen , TORREKENS Sophie , VAN LEUVEN Fred , PEUMANS Willy J.
Published: March 2005

Lectin cDNA clones encoding the two mannose‐binding lectins from ramsons (Allium ursinum L.) bulbs, AUAI and AUAII (AUA, Allium ursinum agglutinin), were isolated and characterized. Sequence comparison of the different cDNA clones isolated revealed three types of lectin clones called LECAUAG0, LECAUAG1 and LECAUAG2, which besides the obvious differences in their sequences also differ from each other in the number of potential glycosylation sites within the C‐terminal peptide of the lectin precursor. In vivo biosynthesis studies of the ramson lectins have shown that glycosylated lectin precursors occur in the organelle fraction of radioactively labeled ramson bulbs.

Despite the similarities between the A. ursinum and the A. sativum (garlic) lectins at the protein level, molecular cloning of the two ramson lectins has shown that the lectin genes in A. ursinum are organized differently. Whereas in A. sativum the lectin polypeptides of the heterodimeric ASAI are encoded by one large precursor, those of the heterodimeric AUAI lectin are derived from two different precursors. These results are confirmed by Northern blot hybridization of A. ursinum RNA which, after hybridization with a labeled lectin cDNA, reveals only one band of 800 nucleotides in contrast to A. sativum RNA which yields two bands of 1400 and 800 nucleotides. Furthermore it is shown that the two mannose‐binding lectins are differentially expressed.
Heterogeneity of glycosylphosphatidylinositol‐anchored alkaline phosphatase of calf intestine
Authors: BUBLITZ Renate , ARMESTO Julio , HOFFMANN‐BLUME Eva , SCHULZE Margarete , RHODE Heidrun , HORN Anton , AULWURM Steffen , HANNAPPEL Ewald , FISCHER Werner
Published: March 2005

A method is described for large‐scale purification of glycosylphosphatidylinositol‐anchored alkaline phosphatase from intestinal mucosa and chyme to homogeneity. Both enzyme preparations contain approximately 2 mol fatty acid/mol subunit and exhibit a very similar fatty acid composition with octadecanoate and hexadecanoate as prevalent components.

No significant differences between native glycosylPtdIns‐anchored and hydrophilic alkaline phosphatases from both sources were found regarding Km, Vmax, the type of inhibition and inhibition constants of the amino acids l‐leucine, l‐phenylalanine, and l‐tryptophan. The purified enzymes of both sources yield diacylglycerol and phosphatidic acid, after treatment with phosphatidylinositol‐specific phospholipase C (PtdIns‐PLC) and glycosylphosphatidylinositol phospholipase D (PLD), respectively.

Enzyme preparations of both sources appear as heterogeneous mixtures of five fractions separable by octyl‐Sepharose chromatography. Fractions I corresponds to the anchorless enzyme, fractions II–V differ in their susceptibility to phospholipases. Fractions II and IV are completely split by PtdIns‐PLC or PLD action, almost 50% of fraction III is split by PtdIns‐PLC, while fraction V is resistant. The susceptibility of these two fractions toward the action of PLD is considerably higher. Fatty acid analysis yields molar ratios of fatty acids/alkaline phosphatase subunit of 1.78, 2.58, 2.24, and 3.37 for fractions II, III, IV, and V, respectively.

Aggregates of glycosylPtdIns‐anchored alkaline phosphatase of all fractions are seen in native PAGE in the presence of Triton X‐100. By gel chromatography in the presence of Brij 35, fractions II–V form stable multiple aggregates of dimers and may bind different amounts of the detergent.

These data, together with fatty acid analysis, can be interpreted by the following model. Fractions II and IV are tetramers and octamers with two molecules fatty acid/subunit. Fraction III is a tetramer, bearing one additional fatty acid molecule, localized on the dimer. Fraction V is an octamer, containing glycosylPtdIns‐anchor molecules with three molecules fatty acids/anchor molecule. The additional fatty acid residue is possibly located on inositol and responsible for the reduced susceptibility to PtdIns‐PLC.

The similarity of all measured parameters of both enzymes suggests that the glycosylPtdIns‐anchored alkaline phosphatase of the mucosa is released into the chyme without changing the anchor molecule constituents.
Effect of hydrostatic pressure and cholesterol depletion on the expression of a tumor‐associated antigen
Authors: GESMUNDO Nicla , CALONGHI Natalia , SARTOR Giorgio , FARRUGGIA Giovanna , MIOTTI Silvia , CANEVARI Silvana , COLNAGHI M. Ines , MASOTTI Lanfranco
Published: March 2005

The molecular events related to the expression of three tumor‐associated epitopes, Ca‐MOv17, Ca‐MOv18 and Ca‐MOv19 have been addressed. The epitopes are carried by a 38‐kDa glycoprotein (gp38), recently cloned and identified as a human folate‐binding protein. They were found to be coexpressed on the surface of the ovarian carcinoma cell line OVCA432, while they are not coordinately expressed on other adenocarcinoma cell lines (IGROV1, HT‐29). This lack of coexpression was investigated from a molecular point of view. We studied three carcinoma cell lines, characterized by a different reactivity with the three relevant monoclonal antibodies MOv17, MOv18 and MOv19. The epitope expression was examined after modifying the membrane properties by using hydrostatic pressure and/or the variation of cholesterol content. Measurement of the expression after cell labelling by mAbs was performed by indirect immunofluorescence, using both fluorescence microscopy and flow cytometry. At variance with HT‐29 cells, treatment of ovarian carcinoma IGROV1 cells with hydrostatic pressure failed to exert any effect. On IGROV1, instead, cholesterol depletion affected the expression Ca‐MOv17, increasing, in the indirect immunofluorescence tests, the proportion of positive cells from 0 to 66 ± 9%. Moreover, restoring the cholesterol content of the plasma membrane did not reverse the induced epitope expression. In parallel, ìmmunoprecipitation experiments confirmed that, on IGROV1 surface, gp38 was recognized by all three mAbs. The data presented suggest that in IGROV1 cells the selective lacking of the epitope expression is related to the physical state of the plasma membrane. An explanation is provided by the model of membrane microdomains in which epitope expression may be influenced by the cholesterol level of different plasma membrane regions.
Differential regulation of the human H1°‐histone‐gene transcription in human tumor‐cell lines
Authors: BOUTERFA Hakim L. , TRIEBE Suzane M. , DOENECKE Detlef R.
Published: March 2005

Cloning and sequence analysis of about 2 kb of the 5′ flanking region of the human H1° histone gene reveals several potential regulatory elements upstream of the transcribed portion of this gene. Transfection studies using the chloramphenicol acetyl tranferase (CAT) gene as a reporter gene with a series of promoter deletions revealed that the expression of the H1° gene may depend on a complex interplay of several transcription factors, including members of the retinoic acid and/or thyroid‐hormone‐receptor superfamily, at the 5′ flanking region of the H1° gene. CAT assays demonstrate varied patterns of expression and regulation in different human tumor‐cell lines. The leukemia cell line HL60 does not express H1° mRNA and shows no CAT activity. HeLa cells strongly express the CAT gene under the control of the H1° promoter. Under the same conditions, HepG2 cells also transcribe the CAT gene, although at a lower rate than HeLa cells. Using different promoter‐deletion clones, the CAT activity differs in HepG2 and HeLa cells in the very distal promoter region. In both cell lines, the CAT activity decreases several fold when the region between nucleotides –450 and –600 upstream of the mRNA start site is deleted. It also decreases when just the proximal portion but not the distal promoter region is deleted. In summary, the regulatory patterns of these three cell lines differ, indicating a cell‐type‐specific regulation of the human H1°‐histone‐gene expression.
Interactions of chloroaluminium‐tetramethyl‐tetrapyridino‐porphyrazine with DNA
Authors: GANTCHEV Tsoetan G. , ALI Hasrat , van LIER Johan E.
Published: March 2005

The interaction of chloroaluminium‐tetramethyl‐tetrapyridinoporphyrazine (ClAlTMPyPa) with DNA was investigated by absorption/emission and circular‐dichroic spectroscopy. Formation of a DNA‐porphyrazine complex was evidenced by quenching of the ClAlTMPyPa fluorescence, a hypochromic red shift of the absorption band in the visible part of the spectrum and induction of a characteristic CD band with high positive ellipticity. Scatchard analysis of the spectral changes in low ionic strength buffer suggested an association constant of 9.7 ± 0.6 × 107 M−1. Quenching of the fluorescence of a DNA‐ethidium complex by added ClAlTMPyPa was shown to result from direct DNA‐dye interaction rather than the displacement of ethidium from DNA. Our data suggest that porphyrazine binds to the outside of the DNA helix, involving interactions which are not limited to electrostatic forces only. The computer assisted modelling of ClAlTMPyPa‐DNA interactions showed that the energy‐minimized intermolecular orientation involves face‐on binding of the ligand, preferably on the minor DNA groove.
A mutant T7 phage promoter is specifically transcribed by T7‐RNA polymerase in mammalian cells
Authors: LIEBER Andre , SANDIG Volker , STRAUSS Michael
Published: March 2005

The phage T7 promoter/polymerase system is highly specific in bacteria in contrast to that observed in mammalian cells. A number of cell lines exhibit a considerable level of expression from the T7 promoter, even in the absence of T7‐RNA polymerase. Here, we demonstrate that nuclear‐factor‐including components of the TFIID fraction, bind to the T7 promoter and inhibit transcription by T7‐RNA polymerase. In order to increase the specificity of the promoter for T7‐RNA polymerase and to abolish binding of nuclear factors, a novel strategy for the selection of randomly mutated promoters was established. The strategy involves adsorption of mutant promoters to HeLa extracts and binding of the free oligonucleotides to T7‐RNA polymerase, cloning, and functional testing of the recombinants. After selection, the resulting mutant promoters showed an increase in specificity for transcription by T7‐RNA polymerase.
Identification and characterization of a soluble form of the plasma cell membrane glycoprotein PC‐1 (5′‐nucleotide phosphodiesterase)
Authors: BELLI Sabina I. , van DRIEL Ian R. , GODING James W.
Published: March 2005

PC‐1 is a membrane glycoprotein, found on the surface of plasma cells and a few types of non‐lymphoid cells, which has recently been found to have 5′‐nucleotide phosphodiesterase activity. In this paper, we demonstrate the existence of enzymically active water‐soluble forms of PC‐1 in ascites from plasmacytoma‐bearing mice, normal mouse serum, and in supernatants of cultured mouse plasmacytoma cells and mouse L cells transfected with a cDNA encoding the membrane form of PC‐1. The water‐soluble enzyme activity can be specifically immunoprecipitated by a monoclonal antibody to an allotypic determinant on the membrane form of PC‐1, and resides on a slightly smaller polypeptide than membrane PC‐1. Biosynthetic studies revealed a single, mono‐meric, endoglycosidase‐H‐sensitive membrane PC‐1. precursor, which was gradually converted to a disulphide‐bonded, endoglycosidase‐H‐resistant form over a period of about 2 h. Soluble PC‐1 was first detectable in the supernatant after about 2 h. A distinct intracellular form of soluble PC‐1 was not seen. The soluble form of PC‐1 does not appear to arise by proteolytic cleavage from the cell surface, although cleavage inside the cell remains a possibility. When taken together with the structure of the relevant portions of PC‐1 gene exons, the data suggest that the most likely site of cleavage is between Pro152 and Ala153.
Role of the rac1 p21‐GDP‐dissociation inhibitor for rho heterodimer in the activation of the superoxide‐forming NADPH oxidase of macrophages
Authors: PICK Edgar , GORZALCZANY Yara , ENGEL Sharon
Published: March 2005

Activation of the superoxide (O−2)‐generating NADPH oxidase of phagocytes requires the interaction of membrane‐associated cytochrome b559 with three cytosolic components; p47‐phox, p67‐phox and σ1. We proposed that σ1 was a heterodimer composed of proteins of 22 kDa and 24 kDa that were tentatively identified as the small GTP‐binding protein (G protein) rac1 p21 and GDP‐dissociation inhibitor for rho (rho GDI). We now describe a modified procedure for the rapid purification of σ1 and demonstrate that the NADPH‐oxidase‐activating capacity is associated, throughout the purification sequence, with a protein binding 35S‐labelled guanosine 5′‐[3‐O‐thio]tri‐phosphate. SDS/PAGE analysis confirmed the absolute association of σ1 activity with the presence of both the 22 kDa and 24 kDa proteins. Immunoblotting with a battery of antibodies against the small G proteins demonstrated that the 22‐kDa protein was only recognized by antibodies reacting with rac1 p21; no reaction was found with anti‐(rac2 p21), anti‐[v‐ras(H) p21] and anti anti‐(rap1 p21). Free rac1 p21 (not in complex with rho GDI) was not detected at any stage of cytosol fractionation. The proteins comprising the σ1 heterodimer could be separated by reverse‐phase chromatography and amino acid sequencing was performed on peptides derived by trypsin digestion of each of the isolated proteins. This demonstrated the identity of the 22‐kDa protein with rac1 p21 and that of the 24‐kDa protein with rho GDI. Purified heterodimeric σ1 did not require exogenous GTP for activity under conditions that assured the absence of free nucleotides. Treatment of the σ1 heterodimer with 1% sodium cholate, followed by gel filtration or anion‐exchange chromatography in the presence of 1% sodium cholate, effectively separated rac1 p21 from rho GDI. Monomeric rac1 p21, obtained by these procedures, was able to stimulate cell‐free O−2 generation. Artificial heterodimeric σ1, capable of NADPH oxidase activation, could be reconstituted in vitro by recombining purified monomeric rac1 p21 and rho GDI and removing the sodium cholate used to dissociate the native σ1 dimer. Monomeric rac1 p21 exhibited an almost absolute dependence on exogenous GTP following removal of the endogenous nuclcotide in low Mg2+ solution. Under similar conditions, heterodimeric σ1 was resistant to nucleotide exchange. We propose that rac1 p21 is present in cytosol exclusively as a heterodimer with rho GDI and that activation of NADPH oxidase involves the dissociation of the dimer and the subsequent interaction of free rac1 p21 with another component participating in the assembly of the enzyme complex.
Proof‐reading 3′→5′ exonucleases isolated from rat liver nuclei
Authors: BELYAKOVA Natalya V. , KLEINER Natalya E. , KRAVETSKAYA Tatyana P. , LEGINA Olga K. , NARYZHNY Stanislav N. , PERRINO Fred W. , SHEVELEV Igor V. , KRUTYAKOV Valery M.
Published: March 2005

Mammalian nuclear DNA polymerases α and β are known to be devoid of the editing 3′→5′ exonucleolytic activity. Presumably this activity could be effected by the exonucleases non‐associated covalently with DNA polymerases. Two 3′→5′ exonucleases of 40 kDa and 50 kDa (exo‐40 and exo‐50) have been isolated from rat liver nuclei and purified to near homogeneity. They are shown to excise mismatched nucleotides from poly[d(A‐T)] template, respectively, 10‐fold and 2‐fold faster than the matched ones. Upon addition of either of these exonucleases to the DNA polymerase α from rat liver or calf thymus, the fidelity of in‐vitro reproduction of the primed DNA from bacteriophage φX174 amber 3 is increased 5–10‐fold, levels of exonuclease and DNA‐polymerase activities being similar. Extrapolation of in‐vitro DNA‐replication fidelity to the cellular levels of activities of the exonucleases and the α‐polymerase suggests that exonucleolytic proofreading augments the accuracy of DNA synthesis by 2–3 orders of magnitude.
Molecular cloning and primary structure of Man9‐mannosidase from human kidney
Authors: BAUSE Ernst , BIEBERICH Erhard , ROLFS Andreas , VÖLKER Christof , SCHMIDT Bernhard
Published: March 2005

Man9‐mannosidase, a processing enzyme found in the endoplasmic reticulum (ER), catalyses the removal of three distinct mannose residues from peptide‐bound Man9‐GlcNAc2 oligosaccharides producing a single Man6 isomer [Bause, E., Breuer, W., Schweden, J., Roesser, R. & Geyer, R. (1992) Eur. J. Biochem. 208, 451–457]. We have isolated four Man9‐mannosidase‐specific clones from a human kidney cDNA library and used these to construct a full‐length cDNA of 3250 base pairs. A single open reading frame of 1875 nucleotides encodes a protein of approximately 71 kDa, consistent with data from immunological studies. Analysis of the coding sequence predicts that Man9‐mannosidase is a type II transmembrane protein consisting of a short cytoplasmic polypeptide tail, a single transmembrane domain acting as a non‐cleavable signal sequence and a large luminal catalytic domain. This domain architecture closely resembles that of other ER and Golgi‐located processing enzymes, pointing to common structural motifs involved in membrane insertion and topology. The protein sequence of the Man9‐mannosidase contains three potential N‐glycosylation sites of which only one site is used. The amino acid sequence of several peptide regions, including a calcium‐binding consensus sequence, bears striking similarities to an ER α‐1,2‐mannosidase from yeast, whereas, by contrast, no sequence similarity was detectable with rat liver ER α‐mannosidase and Golgi α‐mannosidase II. This finding may indicate that the mammalian α‐mannosidases, which differ significantly in their substrate specificity, are coded for by evolutionarily unrelated genes, providing an attractive means of regulation and fine‐tuning oligosaccharide processing, not only at the enzymic but also at the transcriptional level.