bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for a GTP‐binding protein coupling thrombin receptor to PIP2‐phospholipase C in membranes of hamster fibroblasts
Authors: Magnaldo Isabelle , Talwar Harvinder , Anderson Wayne B. , Pouyssegur Jacques
Published: July 2002

Two different methods were used to study directly α‐thrombin modulation of polyphosphoinositide breakdown in membranes prepared from Chinese hamster lung (CHL) fibroblasts. In the first one we labelled the lipid pool by incubating the intact cells with myo‐[su3H]inositol prior to membrane isolation; in the other we used exogenous [3H]PIP2 with phosphatidylethanolamine (1:10) added as liposomes to freshly isolated membranes. A Ca2+‐dependent PIP2 and PIP phospholipase C activity was characterized by measuring the rate of formation of inositol tris‐ and bisphosphate. Basal phospholipase C activity was stimulated up to 3‐fold by GTP or GTP‐γ‐S. Of the two mitogens, α‐thrombin and EGF, known to stimulate DNA synthesis in Chinese hamster fibroblasts, only α‐thrombin is a potent activator of PIP2 breakdown in intact cells. Consistent with this observation, α‐thrombin but not EGF potentiated GTP‐γ‐S‐dependent phospholipase C activity in membrane preparations. These results strongly support the hypothesis that a GTP‐binding protein couples α‐thrombin receptor to PIP2 hydrolysis. Because both methods used to assay phospholipase C gave identical results, we conclude that the coupling is at the level of PIP2‐phosphodiesterase activity.
Retinal Schiff base position relative to the surfaces of photoreceptor disk
Authors: Abdulaev N.G. , Nabiev I.R. , Efremov R.G. , Chumanov G.D.
Published: July 2002

Surface‐enhanced Raman spectra of native and photobleached bovine rod outer segment disks as well as inside‐out (inverted) photoreceptor disks adsorbed on silver hydrosol have been analyzed. Surface‐enhanced spectra of inverted disks and disk‐monoclonal antibody complexes reveal the short‐range mechanism of enhancement. The distance between retinal Schiff base and the cytoplasmic side of native disk has been shown to be 5–10 Å.
The three‐dimensional similarity between a dimeric antiparallel extended structure and a β‐turn folded form of enkephalin
Authors: Doi Mitsunobu , Tanaka Masayuki , Ishida Toshimasa , Inoue Masatoshi
Published: July 2002

The three‐dimensional similarity between two fundamental conformations, a dimeric antiparallel extended structure and a type I'β‐turn folded form, of enkephalin was examined by computer graphic and empirical energy calculation methods. By the rotation of Tyr and Phe side chains, one half of the former structure could mimic the three‐dimensional form of the latter without a large loss of conformational energy. This result provides a new idea for considering the conformation of enkephalin suitable for the multiple opioid receptors. The active conformation of enkephalin for μ‐ and δ‐opioid receptors is discussed in the light of the present study.
Is the acetylcholine releasing protein mediatophore present in rat brain?
Authors: Israe¨l Maurice , Lesbats Bernard , Morel Nicolas , Manaranche Robert , Le Gal la Salle Gildas
Published: July 2002

Mediatophore is a protein purified from the nerve terminal membranes ofTorpedo electric organ. It confers to artificial membranes a calcium‐dependent mechanism that translocates acetylcholine. When similar reconstitution experiments are applied to rat brain synaptosomal membranes they reveal the presence of mediatophore activity with properties close to those described for theTorpedo protein (extractability, sensitivity to calcium, and effect of the drug cetiedil). The acitivity was more abundant in synaptosomal membranes than in mitochondrial or myelinic membranes and in cholinergic areas as compared to cerebellum.
A partial structure for the γ‐aminobutyric acid (GABAA) receptor is derived from the model for the nicotinic acetylcholine receptor The anion‐exchange protein of cell membranes is related to the GABAA receptor
Authors: Kosower Edward M.
Published: July 2002

Based on the nicotinic acetylcholine receptor model [(1987) Eur. J. Biochem. 168, 431–449], a partial model is constructed for the exobilayer portion of the GABAA receptor, an approach justified by the superfamily relationship of the two receptors [(1987) Nature 328, 221–227]. The model predicts succesfully the excess positive charge on interior strands which constitute the ligand‐responsive portion of the receptor. Binding to GABA expands the exobilayer portion of the receptor, opening a pathway to a chloride channel. Separate binding sites for antianxiolytics (benzodiazepines) and hypnotics (barbiturates) are suggested, with prolongation of chloride entry projected as a consequence of stabilization of the open form. The anion‐exchange protein (AEP) of membranes (band 3 of red blood cell membranes) is similar in some respects to the γ‐aminobutyric acid (GABAA) receptor. Both proteins are inhibited and labeled by diisocyanatostilbenedisulfonate (DIDS), both transport Cl− and HCO−
3, and both are membrane proteins. Starting with the lysines known to be labeled in band 3 protein, searches of the amino acid sequences of the GABAA receptor α‐ and β‐subunits reveal at least 4 reasonably homologous sequences. The relationship between AEP and GABAA receptor leads to the idea that the chloride/bicarbonate channel may be the ancestor of all ligand‐gated channels, with ligand gating by γ‐aminobutyric acid and acetylcholine arising later in evolution.
Evidence for a Ca2+‐independent association between calpain II and phospholipid vesicles
Authors: Garret Christophe , Cottin Patrick , Dufourcq Jean , Ducastaing André
Published: July 2002

Possible interactions between calpain II and phospholipids such as phosphatidylinositol, phosphatidylserine and phosphatidylcholine were studied using fluorescence and gel filtration techniques. Changes in fluorescence intensity of purified calpain II show that the enzyme strongly interacts with phosphatidylinositol and phosphatidylserine and to a lesser extent with phosphatidylcholine. These results are corroborated by the gel filtration technique which permits the isolation of the enzyme/phospholipid complex. Association between calpain II and various phospholipid vesicles can occur in the absence of calcium. Such binding occurs without any observable change of the molecular mass of the two subunits on SDS‐polyacrylamide gel electrophoresis.
Sequence of the gene for ribosomal protein L23 from the archaebacterium Methanococcus vannielii
Authors: Köpke Andreas K.E. , Wittmann-Liebold Brigitte
Published: July 2002

The N‐terminal sequence of HPLC‐purified protein L23 from the Methanococcus vannielii ribosome has been determined by automated liquid‐phase Edman degradation. Using the N‐terminal amino acid sequence, an oligonucleotide probe complementary to the 5′‐end of the gene was synthesized. The 26‐mer oligonucleotide, containing two inosines, was used for hybridization with digested M. vannielii chromosomal DNA. The hybridizing band from HpaII‐digested genomic DNA was ligated into pUC18 to yield plasmid pMvaZl containing the entire gene of protein L23. The nucleotide sequence complemented the partial amino acid sequence, and the gene codes for a protein of 9824 Da. The amino acid sequence of protein L23 form M. vannielii was compared to that of ribosomal proteins from other archaebacteria as well as from eubacteria and eukaryotes. The number of identical amino acids is highest when the M. vannielii protein is compared to the homologous protein from yeast and lowest vs that from tobacco chloroplasts. Interestingly, the secondary structures of the proteins as predicted by computer programs are more conserved than the primary structures.
125I‐d(CH2)5[Tyr(Me)2,Tyr(NH2)9]AVP: Iodination and binding characteristics of a vasopressin receptor ligand
Authors: Elands Jack , Barberis Claude , Jard Serge , Lammek Bernard , Manning Maurice , Sawyer Wilbur H. , de Kloet E.Ronald
Published: July 2002

A radioiodinated vasopressin antagonist, d(CH2)5[Tyr(Me)2,Tyr(NH2)9]AVP has been prepared. Iodination was carried out at the phenyl moiety of the tyrosylamide residue at position 9, followed by HPLC purification. Non‐radiolabelled monoiodinated antagonist was used as a reference for identification. 125I‐d(CH2)5[Tyr(Me)2,Tyr(NH2)9]AVP binding appeared to take place with a dissociation constant of 0.28±0.09 nM (K
d±SD) to V1 vasopressin receptors on rat liver membranes.
Titin: Quantitative mass measurements by scanning transmission electron microscopy and structural implications for the sarcomere matrix of skeletal muscle
Authors: Hainfeld James F. , Wall Joseph S. , Wang Kuan
Published: July 2002

Scanning transmission electron microscopy has been used to investigate mass and linear mass density of native titin‐2, a large soluble fragment of intact titin, from rabbit skeletal muscle. Dark field images of unstained, freeze‐dried titin‐2 appeared as either compact globules or looser and larger balls of string. Direct mass measurements indicated that the compact forms have an average mass of 2.40±0.50 × 106 Da. The mass to length ratio, determined from well‐spread portions of titin strands (3–5 nm wide) from the ball of string forms, averaged 2.7±0.9 kDa/nm. Thus a single native intact titin molecule has a calculated contour length of well above ∼1 μm, sufficient to span unidirectionally between the Z line and M line region in a resting‐length sarcomere.
DNA polymerase α‐DNA primase from human placenta Immunoaffinity purification and preliminary characterization
Authors: Podust V.N. , Lavrik O.I. , Nasheuer H.-P. , Grosse F.
Published: July 2002

Highly purified DNA polymerase α‐DNA primase from normal human tissue (human placenta) has been prepared by immunoaffinity purification on immobilized anti‐human DNA polymerase α monoclonal antibody SJK 287‐38. According to data from SDS electrophoresis this preparation consists of subunits of 180, 160, 145, 140 kDa (a cluster of DNA‐polymerizing subunits), 73 kDa (function unknown) and 59, 52 kDa (corresponding to primase). Three active enzyme forms of 270, 460 and 575 kDa have been revealed using native electrophoresis followed by detection of DNA polymerase activity.
Microtubule‐associated protein tau Identification of a novel peptide from bovine brain
Authors: Iqbal Khalid , Smith Alan J. , Zaidi Tanweer , Grundke-Iqbal Inge
Published: July 2002

The microtubule‐associated protein tau isolated from bovine brain was cleaved with CNBr and the 3 largest peptides of approx. 21, 19 and 18 kDa were obtained. Dephosphorylation of the CNBr digest of tau with alkaline phosphatase changed the electrophoretic mobility of these peptides to 19, 18 and 17 kDa. Amino acid sequencing of the total CNBr digest of tau revealed at least 3 sequences, two of which were highly homologous to previously published mouse and human tau sequences derived from cDNAs. A third amino acid sequence of 17 residues with heterogeneity at position 11 showed no homology with the cDNA‐derived tau sequences. These studies suggest that the amino acid sequences of mammalian tau predicted from their cDNAs might be incomplete.
Characterisation of enkephalinase (EC 3.4.24.11) activity on various leukemic cells expressing the common acute lymphocytic leukemia antigen (CALLA)
Authors: Arock Michel , Gros Claude , Devillier Philippe , Guillosson Jean-Jacques , Schwartz Jean-Charles
Published: July 2002

The deduced amino acid sequences of CALLA, a cell surface marker of human acute lymphocytic leukemia, and human enkephalinase (neutral endopeptidase, EC 3.4.24.11) were recently reported to be almost identical. We show that membranes of CALLA+ cells of the REH lymphoblastic cell line as well as blast cells derived from the blood or bone marrow of patients with acute lymphocytic leukemia display high enkephalinase activity. This activity was abrogated by several enkephalinase inhibitors at concentrations closely similar to those required to inhibit pure human enkephalinase. However, these compounds did not significantly modify the rate of REH cell proliferation in vitro. Hence, the functional role, if any, of the high peptidase activity in lymphoblastic cells remains to be established.
Presence of a basic amino acid residue at either position 66 or 122 is a condition for enzymic activity in the ribonuclease superfamily
Authors: Beintema Jaap J.
Published: July 2002

Some members of the ribonuclease superfamily differ at more than 50% of the amino acid positions. Although the three‐dimensional structures probably are very similar and the active‐site residues have been conserved, other substrate‐binding regions have changed considerably. Several proteins in the superfamily are active ribonucleases while others exhibit practically no enzymic activity. The presence of a basic residue at either position 66 or 122 appears to be a condition for ribonuclease activity.
Synthesis of alkylating oligonucleotide derivatives containing cholesterol or phenazinium residues at their 3′‐terminus and their interaction with DNA within mammalian cells
Authors: Boutorin A.S. , Gus'kova L.V. , Ivanova E.M. , Kobetz N.D. , Zarytova V.F. , Ryte A.S. , Yurchenko L.V. , Vlassov V.V.
Published: July 2002

5′‐[32P]‐labelled alkylating decathymidylate [4‐(N‐2‐chloroethyl)N‐methylaminobenzyl]‐5′‐phosphamide derivatives containing cholesterol or phenazinium residues at their 3′‐termini were synthesized and used for alkylation of DNA within mammalian cells. The uptake of the cholesterol derivative by the cells and the extent of DNA alkylation are about two orders of magnitude higher than those of a similar alkylating derivative lacking the groups at the 3′‐termini. The presence of the phenazinium residue at the 3′‐terminus of the oligonucleotide reagent does not improve the reagent uptake by the cells but drastically increases the DNA modification efficiency.
Effect of the different dimeric forms of the platelet‐derived growth factor on cellular responses in mouse Swiss 3T3 fibroblasts
Authors: Sturani E. , Brambilla R. , Morello L. , Cattaneo M.G. , Zippel R. , Alberghina L.
Published: July 2002

PDGF consists of two polypeptide chains, A and B, and all three possible dimers have been isolated from different sources. Human PDGF, essentially AB, porcine PDGF (BB) and recombinant PDGF‐AA were tested on Swiss 3T3 fibroblasts for their ability to stimulate mitogenesis, phosphoinositide turnover and tyrosine phosphorylation of the PDGF receptor. When used in saturating amounts, the three isoforms were equally active in inducing mitogenesis. However, PDGF‐AA was less active than AB and BB to induce the phosphorylation of the receptor and the turnover of phosphoinositides (30% and 50%, respectively). These findings suggest that, in Swiss 3T3 fibroblasts, PDGF receptors of the α‐type are present in a slightly lower amount than β‐type. In addition, the two types of receptor appear to have similar physiological functions.
Negative regulation of human insulin gene expression by the 5′‐flanking region in non‐pancreatic cells
Authors: Takeda Jun , Ishii Shunsuke , Seino Yutaka , Imamoto Fumio , Imura Hiroo
Published: July 2002

We have examined the effect of the 5′‐flanking region of the human insulin gene on its expression in non‐pancreatic cells. The presence of the region containing the insulin gene enhancer (−339 to −169 bp) markedly repressed the promoter activity of the insulin gene. This suppressive phenomenon was restored by the addition of forskolin or dibutyryl cAMP, suggesting that this region alone is not sufficient to repress completely insulin gene expression in the presence of extracellular stimuli which increase the intracellular cAMP level. The hypervariable region (HVR) located at −365 bp also repressed the promoter activity. These results show negative regulation of human insulin gene expression in non‐pancreatic cells by these regions.
Crystallization of proteins under microgravity
Authors: Erdmann Volker A. , Lippmann Corinna , Betzel Christian , Dauter Zbyszek , Wilson Keith , Hilgenfeld Rolf , Hoven Julia , Liesum Alexander , Saenger Wolfram , Müller-Fahrnow Anke , Hinrichs Winfried , Düvel Martina , Schulz Georg E. , Müller Christoph W. , Wittmann Heinz G. , Yonath Ada , Weber Gabriela , Stegen Karin , Plaas-Link Andreas
Published: July 2002

For the crystallization of proteins under microgravity conditions, a Chinese re‐entry system was used, in which 101 experiments of 25 different biological macromolecules were accommodated. From the results obtained we conclude that under microgravity conditions crystal growth can only be expected under those crystallization conditions which also permit crystal growthon earth. A number of space‐grown crystals were larger in size and of a better quality in their ability to diffract X‐rays than the corresponding ground control crystals grown at the Chinese launch site. However, the space‐grown crystals have not reached the X‐ray diffraction quality of the crystals obtained under optimal conditions in the home laboratories.
The selectivity filter of a ligand‐gated ion channel
Authors: Hucho Ferdinand , Hilgenfeld Rolf
Published: July 2002

Acetylcholine receptor; Ion channel; Selectivity filter; Molecular modeling
Detection of the monocarboxylate transporter from pea mitochondria by means of a specific monoclonal antibody
Authors: Vivekananda Jeevalatha , Oliver David J.
Published: July 2002

The monocarboxylate (pyruvate) transporter from pea (Pisum sativum) mitochondria was identified by means of a specific monoclonal antibody. The antibody blocked pyruvate‐dependent oxaloacetate metabolism without interfering with the metabolism of malate, α‐ketoglutarate, or glycine. The antibody also blocked the pyruvate/pyruvate exchange reaction of the partially purified transporter reconstituted into phospholipid membranes. Using the specific monoclonal antibody, the transporter was identified on Western blots as a minor 19 kDa protein.
Expression of protein kinase C I in NIH 3T3 cells increases its growth response to specific activators
Authors: Cuadrado Antonio , Molloy Christopher J. , Pech Michael
Published: July 2002

In order to investigate the effects of protein kinase C (PKC) expression on cellular growth and morphology, we established mouse fibroblast cell populations which expressed the rat pkc‐γ gene under the control of a retroviral promoter. NIH 3T3 stable transfectants displayed a three‐fold increase in total PKC levels. These cells appeared morphologically unaltered but exhibited a stronger mitogenic response to 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) and cardiolipin (CL) as well as enhanced growth in semisolid medium in the presence of TPA. Thus, at these enzyme levels, PKC conferred growth advantages to NIH 3T3 cells only in response to specific activators.
The N intermediate of bacteriorhodopsin at low temperatures: Stabilization and photoconversion
Authors: Balashov S.P. , Imasheva E.S. , Litvin F.F. , Lozier R.H.
Published: July 2002

In aqueous suspensions of purple membranes (pH 10.2, 0.4 M KCl) an intermediate having an absorption maximum at 570–575 nm (at −196°C) was produced by first heating the M intermediate up to −30°C and then stabilizing it by subsequent cooling to −60°C. We suggest that this species is the intermediate N (or P or R) found and characterized earlier near room temperature. Upon illumination at −196°C N is transformed into a bathochromically absorbing species K
n
which has an absorption maximum near 605 nm and an extinction 1.35 times that of N. This light reaction is photoreversible. The quantum yield ratio for the forward and back reaction is 0.18 ± 0.02. The maximum photo steady state concentration of K
n
is about 0.24. The N intermediate was also trapped in water suspensions of purple membranes at neutral pH and low salt concentration by illumination at λ > 620 nm during cooling. In addition to N another intermediate absorbing in the red (maximum at 610–620 nm) was accumulated in smaller amounts. It is not photoactive at 196°C and apparently is the O intermediate or a photoproduct of N.
Regulation of cytochrome P‐450 CYPIA1 gene expression and protooncogene expression by growth factors in primary hepatocytes
Authors: Höhne Martin , Becker-Rabbenstein Volker , Kahl Georg F. , Taniguchi Hisaaki
Published: July 2002

The effect of growth factors on the cytochrome P‐450 (CYPIA1) gene expression was studied in primary mouse hepatocytes. Of the three growth factors used, i.e. epidermal growth factor (EGF), transforming growth factor α (TGFα) and insulin, only EGF or TGFα completely blocked CYPIA1 expression in the presence of the CYPIA1 inducer 3‐methylcholanthrene (3‐MC). This repression was not linked to cell cycle progression of the hepatocyte because insulin was active to induce ‘early immediate genes’ and DNA replication as well as EGF/TGFα but failed to suppress CYPIA1 expression. A specific EGF/TGFα receptor‐mediated function may repress CYPIA1 gene expression and contribute to the acquisition of a xenobiotic drug resistance phenotype.
The protein kinase inhibitor, K‐252a, decreases elicitor‐induced Ca2+ uptake and K+ release, and increases coumarin synthesis in parsley cells
Authors: Conrath Uwe , Jeblick Wolfgang , Kauss Heinrich
Published: July 2002

An elicitor preparation from fungal cell walls known to induce coumarin synthesis in suspension‐cultured parsley cells also elicits a rapid and transient Ca2+ uptake, K+ release and external alkalinization, and increases uptake of 45Ca2+ into the cells. The latter three responses were inhibited by the protein kinase inhibitor K‐252a at 0.2 μM. Elicitor‐induced coumarin synthesis, a process which requires gene activation, was greatly enhanced by K‐252a. These results suggest that protein phosphorylation might be involved in the initial steps of signal transduction as well as in the long‐term induction of coumarin synthesis.
Biogenesis of the yeast vacuole (lysosome) Active site mutation in the vacuolar aspartate proteinase yscA blocks maturation of vacuolar proteinases
Authors: Rupp Steffen , Hirsch Hans H. , Wolf Dieter H.
Published: July 2002

The activation process of vacuolar (lysosomal) proteinases in the yeast Saccharomyces cerevisiae is initiated by the PRAI (PEP4) gene product, proteinase yscA. To elucidate the activation process of proteinase yscA the catalytically active amino acid Asp294 of the enzyme was exchanged with Ala294 using site directed mutagenesis. The resulting proteinase yscA‐Ala294 showed no proteolytic activity against the substrate hemoglobin. The mutant protein did not undergo processing in vivo. This phenomenon is in good agreement with the hypothesis that maturation of proteinase yscA is due to self‐processing or pro‐proteinase yscA. Furthermore, proteinase yscA‐Ala294 is not able to convert the zymogens pro‐proteinase yscB and pro‐carboxypeptidase yscY to the mature enzymes.
Site‐directed mutants of human myeloperoxidase A topological approach to the heme‐binding site
Authors: Jacquet Alain , Deleersnyder Virginie , Garcia-Quintana Lida , Bollen Alex , Moguilevsky Nicole
Published: July 2002

Two site‐directed mutants of human promyeloperoxidase, MPO(His416→Ala) and MPO(His502→Ala), have been expressed in Chinese hamster ovary cells and purified. Overall purification yields and apparent molecular masses of the mutant proteins were similar to those of the wild‐type enzyme. Both mutant species were analyzed spectroscopically to check the presence of the hemic iron in the proteins and were assayed for peroxidasic activity. The data show that substitution of His502 leads to the loss, or to an inappropriate configuration, of the heme together with the loss of activity, suggesting that this residue could be the proximal His involved in the binding to the iron centers. On the other hand, substitution of His416 by alanine had no effect on either of the studied parameters.
Class IV alcohol dehydrogenase (the gastric enzyme) Structural analysis of human σσ‐ADH reveals class IV to be variable and confirms the presence of a fifth mammalian alcohol dehydrogenase class
Authors: Parés Xavier , Cederlund Ella , Moreno Alberto , Saubi Narcis , Hóóg Jan-Olov , Jörnvall Hans
Published: July 2002

Human gastric alcohol dehydrogenase (σσ‐ADH) was submitted to peptide analysis at picomole scale. A total of 72 positions were determined in the protein chain, providing information on three aspects of alcohol dehydrogenase structures in general. First, the data establish the presence of a unique class of the enzyme, now confirmed as class IV, expressed in gastric tissue and separate from another novel class. now termed class V. Second, the class IV gastric enzyme has active site relationships compatible with an ethanol‐active, zinc‐containing alcohol dehydrogenase. Third, this enzyme class is of the variable type, like that for the ‘variable’, classical liver alcohol dehydrogenase of class I, and in contrast to that for the ‘constant’ class III enzyme. Known human alcohol dehydrogenase structures now prove the presence of at least seven human genes for the enzyme and nine for the whole protein family.
Immunological characterization of the endoproteases PC1 and PC2 in adrenal chromaffin granules and in the pituitary gland
Authors: Kirchmair Rudolf , Gee Patricia , Hogue-Angeletti Ruth , Laslop Andrea , Fischer-Colbrie Reiner , Winkler Hans
Published: July 2002

Specific antisera against synthetic Fragments of the endoproteases, PC1 and PC2, were used to characterize these proteins. In one‐dimensional immunoblots these antisera labelled components of 85 kDa for PC1 and of 70 kDa for PC2 in purified bovine chromaffin granules and anterior and posterior pituitary of ox and rat. In membranes of bovine chromaffin granules glycoprotein H was identified as the major PC2 immunoreactive spot. A major part of these endoproteases appeared membrane bound.
Interleukin‐8, a chemotactic and inflammatory cytokine
Authors: Baggiolini Marco , Clark-Lewis Ian
Published: July 2002

Interleukin‐8 (IL‐8) belongs to a family of small, structurally related cytokines similar to platelet factor 4. It is produced by phagocytes and mesenchymal cells exposed to inflammatory stimuli (e.g., interleukin‐1 or tumor necrosis factor) and activates neutrophils inducing, chemotaxis, exocytosis and the respiratory burst. In vivo, IL‐8 elicits a massive neutrophil accumulation at the site of injection. Five neutrophil‐activating cytokines similar to IL‐8 in structure and function have been identified recently. IL‐8 and the related cytokines are produced in several tissues upon infection, inflammation, ischemia, trauma etc., and are thought to be the main cause of local noutrophil accumulation.
Mode of action of the Spiroplasma CpG methylase M.SssI
Authors: Renbaum Paul , Razin Aharon
Published: July 2002

The cytosine DNA methylase from the wall‐less prokaryote, Spiroplasma strain MQ1 (M.SssI) methylates completely and exclusively CPG‐containing sequences, thus showing sequence specificity which is similar to that of mammalian DNA methylases. M.SssI is shown here to methylate duplex DNA processively as judged by kinetic analysis of methylated intermediates. The cytosine DNA methylases, M.Hpall and M.Hhal, from other prokaryotic organisms, appear to methylate in a non‐processive manner or with a very low degree of processivity. The Spiroplasma enzyme interacts with duplex DNA irrespective to the presence of CpG sequences in the substrate DNA. The enzyme proceeds along a CpG‐containing DNA substrate molecule methylating one strand of DNA at a time.
Isolation and characterization of a high molecular weight type IV collagenase isolated from human carcinoma tissue
Authors: Tsuda Tomi T. , Kodama Akira , Yamamura Masaichi , Matsuzaki Shohei , Tsuda Michio
Published: July 2002

A proform of high molecular weight type IV collagenase was isolated and purified 1230‐fold from human metastatic carcinoma tissue. Like matrix metalloproteinases (MMPs), the enzyme was activated by trypsin and degraded type IV collagen and gelatin at a neutral pH, the activity was inhibited by EDTA and o‐phenanthroline. However, the molecular weight was much higher than MMPs which degraded type IV collagen, gelatinase A (MMP‐2; 72 kDa gelatinase/type IV collagenase) (EC 3.4.24.24), gelatinase B (MMP‐9; 92 kDa gelatinase/type IV collagenase) (EC 3.4.24.35), stromelysin‐1 (MMP‐3; 57 kDa) (EC 3.4.24.17) and stromelysin‐2 (MMP‐10; 57 kDa) (EC 3.4.24.22). The other significant difference from MMPs was that the enzyme was not activated by 4‐aminophenylmercuric acetate nor inhibited by TIMP. Taking together these results, this high molecular weight type IV collagenase might be a newly found enzyme different from MMPs or might have the same configuration as MMPs already reported.
Properties of engineered antifreeze peptides
Authors: Warren Gareth J. , Hague Charles M. , Corotto Loren V. , Mueller Gunhild M.
Published: July 2002

Eight antifreeze‐like peptides were produced by cleavage from engineered chimeric proteins. One was homologous to an antifreeze peptide of the winter flounder; the others differed in length and/or sequence. The homologous peptide and all those of equal or greater length were able to inhibit recrystallization. The longer peptides were so hydrophobic that their identification required modification of the usual protocols for high pressure liquid chromatography. Their elution positions were correlated to their hydrophobicities and their lengths. Additional naturally occurring antifreezes may be identifiable with this knowledge.
Molecular cloning and expression of a cDNA encoding a receptor for pituitary adenylate cyclase activating polypeptide (PACAP)
Authors: Morrow J.A. , Lutz E.M. , West K.M. , Fink G. , Harmar A.J.
Published: July 2002

We have cloned and sequenced a novel cDNA (RPR7) encoding a receptor for pituitary adenylate cyclase activating polypeptide (PACAP). RPR7 was identified by PCR of rat pituitary cDNA, and full‐length clones were isolated from a rat olfactory bulb cDNA library. When expressed in COS cells, RPR7 was functionally coupled to increases in intracellular cyclic adenosine monophosphate (cAMP) in response to stimulation by PACAP‐38, PACAP‐27, vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI). The order of potency of these ligands was PACAP‐38 ~ PACAP‐27 > VIP > PHI, suggesting that the receptor corresponds to the pharmacologically characterised PACAP Type I receptor.
Similarity between serine hydroxymethyltransferase and other pyridoxal phosphate‐dependent enzymes
Authors: Pascarella Stefano , Schirch Verne , Bossa Francesco
Published: July 2002

A structural homology of the pyridoxal‐5'‐phosphate (PLP)‐dependent enzyme serine hydroxymethyltransferase (SHMT) with aspartate aminotransferase (AAT) is proposed. Although the two sequences are very dissimilar, a reasonable alignment was obtained using the profile analysis method. Sequences of AAT and dialkylglycine decarboxylase (DGD), for which crystal structure data are available, have been aligned on the basis of their structure superposition. A profile was then calculated and SHMT sequence aligned to it. Three of the four residues conserved in all aminotransferases (including the PLP‐binding lysine) are matched. A profile search with DGD‐AAT‐SHMT profile is more selective and sensitive than individual sequence profiles for PLP‐dependent enzyme detection. Potential homologies with the eryCl gene product involved in erythromycin biosynthesis and with amino acid decarboxylases were observed. Homology with AAT will be used as a guideline for planning site‐directed mutagenesis experiments on SHMT.
Expression and targeting of a 47 kDa integral peroxisomal membrane protein of Candida boidinii in wild type and a peroxisome‐deficient mutant of Hansenula polymorpha
Authors: Sulter G.J. , Waterham H.R. , Vrieling E.G. , Goodman J.M. , Harder W. , Veenhius M.
Published: July 2002

A 47 kDa integral peroxisomal membrane protein (PMP47) of Candida boidinii was expressed in wild type (WT) and a temperature‐sensitive (Ts6) peroxisome‐deficient (per) mutant of Hansenula polymorpha. The subcellular location of PMP47 appeared to be dependent on the level of expression. At low expression levels PMP47 was sorted to the peroxisomal membrane; however, in Ts6 cells grown at restrictive temperatures (which lack intact peroxisomes) PMP47 was solely located in small cytosolic aggregates, together with homologous H. polymorpha PMP's. At enhanced expression levels, however, part of the protein also became incorporated into mitochondria, both in transformed WT and Ts6 cells.
Peptide sequencing identifies MSS1, a modulator of HIV Tat‐mediated transactivation, as subunit 7 of the 26 S protease
Authors: Dubiel Wolfgang , Ferrell Katherine , Rechsteiner Martin
Published: July 2002

Subunit 7 is an integral component of the human erythrocyte 26 S protease. Peptide sequence analysis reveals that 22 amino acids from the N‐terminus of subunit 7 correspond exactly to the N‐terminus of MSS1, a modulator of HIV gene expression. Additional internal peptides from subunit 7 obtained by CNBr cleavage also match 100% with the deduced amino acid sequence of MSS1. Based on the fact that directly sequenced peptides from subunit 7 are identical to more than 12% of the hypothetical translation product of MSS1, and the fact that the molecular weight of subunit 7 (49 kDa) corresponds to the predicted molecular weight of MSS1 (48,633 Da), we conclude that subunit 7 is MSS1.
Binding of alanine‐substituted peptides to the MHC class I protein, Kd
Authors: Ojcius David M. , Abastado Jean-Pierre , Godeau François , Kourilsky Philippe
Published: July 2002

for binding to Kd.
The local information content of the protein structural database
Authors: Rao Srikar , Zhu Qing-Lin , Vajda Sandor , Smith Temple
Published: July 2002

A simple study of the information available within proteins of known structure reveals both the limits of structure prediction based on overall statistical correlations between sequence and structure, and the areas where there is still a possibility for further structural database exploitation.
Distribution profiles of GC content around the translation initiation site in different species
Authors: Mizuno Masahiko , Kanehisa Minoru
Published: July 2002

We have analyzed the distribution of guanine—cytosine (GC) content around the translation initiation site in genomic DNA sequences of different species. A set of sequences belonging to one species is aligned at the translation initiation site, and the average GC content is calculated for 100 base windows over a range of 500 bases each for upstream and downstream region. Consistent with previous observations that coding regions are more GC‐rich than non‐coding regions, we observe a jump in the GC content at the translation initiation site, except for vertebrate sequences. It was also found that the overall shape of the GC content profile is similar within each organism group even though the average GC contents can be very different. Furthermore, by examining different profiles for different species, we have found a negative correlation between the average GC content and the jump size at the translation initiation site. Apparently, more AT‐rich genomes, which tend to lack macroscopic mosaic structures, exhibit more marked differences in the GC content at the microscopic level.
The role of gravity in chick embryogenesis
Authors: Suda Tatsuo , Abe Etsuko , Shinki Toshimasa , Katagiri Takenobu , Yamaguchi Akira , Yokose Satoshi , Yoshiki Shusaku , Horikawa Hiroshi , Cohen Glen W. , Yasugi Sadao , Naito Mitsuru
Published: July 2002

Thirty fertilized chick eggs preincubated for 0, 7 and 10 days on earth (10 eggs each) were flown in the space shuttle ‘Endeavour’ and further incubated for 7 days under microgravity. Twenty out of thirty eggs ( ten‐day‐old; 10/10 seven‐day‐old; zero‐day‐old) were recovered alive after landing. The only living embryo of the zero‐day‐old group died 24 days after launch, and was comparable to a 16‐day‐old embryo. The high mortality of the 0‐day‐old eggs appeared to be related to the specific inner structure of the egg. Simulation experiments performed on earth indicated that when yolk stayed in the albumen for more than 2 days, most of the embryos died. The subtle difference in specific gravity between the yolk (1.029) and albumen (1.040) plays a critical role in early chick embryogenesis.
Functional analysis of human PACE4‐A and PACE4‐C isoforms: identification of a new PACE4‐CS isoform
Authors: Zhong Mei , Benjannet Suzanne , Lazure Claude , Munzer Scott , Seidah Nabil G.
Published: July 2002

There are seven known subtilisin/kexin‐like proprotein convertases responsible for the processing of numerous precursors at either pairs or specific single basic residues. Three members, PACE4, PC4 and PC5, exhibit alternative splicing of their RNAs resulting in the generation of multiple isoforms differing in their C‐ or N‐terminal segments. In this study we examined the biosynthesis, functional activity and cellular localization of two of these isoforms, namely the full length PACE4‐A and the C‐terminally truncated PACE4‐C which lacks 11 amino acids at the end of its chaperone‐like P‐domain. We report the existence of a new isoform, termed PACE4‐CS, which is a C‐terminally shortened version of PACE4‐C. Cellular expression results demonstrated that PACE4‐A codes for a functional secretable enzyme capable of cleaving pro7B2 into 7B2. In contrast, PACE4‐CS is not secreted since it remains in the endoplasmic reticulum as an inactive zymogen form, thereby emphasizing the importance of the integrity of the P‐domain. Microsequencing of the intracellular PACE4‐CS protein in two cell lines revealed that it is proPACE4‐CS with an N‐terminal trimming reminiscent of the action of a dipeptidylpeptidase recognizing the motifs X‐Ala and X‐Pro.
Effect of radiation on serum glycoproteins and glycosidases in patients with cervical carcinoma
Authors: Bhuvarahamurthy V. , Balasubramanian N. , Vijayakumar S. , Govindasamy S.
Published: July 2002

Objective: The serum glycan moieties of glycoproteins and the activities of glycosidases were analyzed in order to elucidate the metabolism of glycoprotein in uterine cervical cancer. Methods: Serum was collected from patients at different stages of cervical cancer before and after treatment. The results were analyzed using Student's t‐test. Results: The glycan moieties such as sialic acid and fucose were significantly elevated even in the early stages of carcinoma, whereas a significant elevation in hexose and hexosamine levels was observed only in the advanced stages. Among the investigated lysosomal glycohydrolases, β‐d‐galactosidase, N‐acetyl‐β‐d‐glucosaminidase and α‐l‐fucosidase activities were elevated significantly at all stages except for stage I. The altered levels of glycoproteins and glycosidases were reduced to near‐normal levels after radiotherapy. Conclusion: The levels of glycan moieties and the activities of glycosidases can be used as diagnostic markers to asses the stage of cancer and can be used as prognostic markers during therapy.
Adolescent health: a Middle East and North African perspective
Authors: Özcebe H. , Akin A.
Published: July 2002

Adolescence is a period of physical, psychological and social transition from childhood to adulthood. At present there are 95 million young people aged between 10 and 24 years in the Middle East and North Africa. Female education is increasing in these regions, but secondary school enrollment rates for girls lag behind those of their male counterparts. Average age at first marriage varies between 17.9 and 24.3 years, but teenage marriage is quite common. Total fertility rates are higher than the world average. Pregnancy at an early age often translates into a high fertility rate and high risks for women and their children. Female children's share of food, health care, work options and general care is often less than that of boys, because they are perceived to be less valuable than boys in some countries.
In vitro induction of radial‐like cells by leptomeningeal and cortical astroglial conditioned media. Effect of protease inhibitors
Authors: Colombo Jorge A. , Napp Mónica I.
Published: July 2002

A population of subcultured astroglia from rat fetal cortex was transformed into radial‐like cells after exposure to cerebral cortex astroglial conditioned medium in vitro. Such changes were also induced by basal medium modified by fetal leptomeningeal subcultures, but not by postnatal leptomeninges nor by fetal skin fibroblasts. The radializing effects of astroglial conditioned medium were inhibited by previous heat treatment. The addition of protease inhibitors to the basal medium did not cause spontaneous radialization of subcultured cortical astroglia, but increased the length of cell processes and incidence of radial‐like forms when added to cortical astroglial conditioned medium. It is concluded that cortical astroglia and leptomeningeal cells share the capability of synthesizing and releasing diffusible molecules into the culture medium which act as morphogenetic inducers in vitro. Based on the present results, it is suggested that such effects would depend on the presence of instructive factor(s) in the conditioned medium which are able to induce rearrangement of the cytoskeleton, rather than on secreted molecules able to modify cell adhesion to the substrate.
Binding specificity of siglec7 to disialogangliosides of renal cell carcinoma: possible role of disialogangliosides in tumor progression
Authors: Ito Akihiro , Handa Kazuko , Withers Donald A. , Satoh Makoto , Hakomori Sen-itiroh
Published: July 2002

Previous studies indicate that expression of higher gangliosides in renal cell carcinoma (RCC) is correlated with metastatic potential, particularly in the lung. Out of five major gangliosides in RCC, three disialogangliosides (disialogalactosylgloboside, IV3NeuAcIII6NeuAcLc4, and IV4GalNAcIV3NeuAcIII6NeuAcLc4) bind strongly to siglec7, which is expressed highly in monocytes and natural killer cells. Out of other gangliosides tested, 2→6 sialylparagloboside, GD3, GD2, and GT1b, but not other lacto‐ or ganglio‐series gangliosides, showed clear binding to siglec7. In view of preferential metastasis of RCC to the lung, and binding of RCC cell line TOS‐1 to lung tissue sections as shown in our previous study, we examined expression of siglec7 in the lung. siglec7 is expressed highly in resident blood cells, but not in parenchymatous cells. TOS‐1 cells aggregate together strongly through adhesion with peripheral blood mononuclear cells to form large clumps. This suggests the possibility that such aggregates may form embolisms of microvasculature, particularly in the lung, which initiate metastasis. Other possible roles of higher gangliosides in RCC in promoting metastasis and tumor progression are discussed.
C‐terminal truncation of thymosin β10 by an intracellular protease and its influence on the interaction with G‐actin studied by ultrafiltration
Authors: Huff Thomas , Müller Christian S.G. , Hannappel Ewald
Published: July 2002

Two β‐thymosins are expressed in most mammalian tissues. We detected small amounts of a third peptide in extracts of rabbit spleen. The portion of this peptide increased when the tissue was first frozen and then thawed at 4°C. Small amounts of the peptide are also present in cells from suspension cultures homogenized immediately in diluted perchloric acid. By means of amino acid analysis and MALDI‐mass spectroscopy this peptide was identified to be a C‐terminally truncated form of thymosin β10. Having studied the formation in more detail we found that after a 4‐h thaw at 4°C all thymosin β10 was truncated to thymosin β10
1‐41, which was further degraded during the next 20 h. On the other hand, thymosin β4
Ala, the second β‐thymosin being present in rabbit spleen, was not truncated or degraded even after 22 h. It might be possible that in vivo a truncated form of thymosin β10 is formed by a carboxydipeptidase while thymosin β4
Ala is rather stable against proteolytic modification. By using a newly designed ultrafiltration assay, we determined the dissociation constants of the complexes of G‐actin and these three β‐thymosins to be 0.28, 0.72, and 0.94 μM for thymosin β4
Ala, β10, and thymosin β10
1‐41, respectively. The complex with β4
Ala is unambiguously more stable than the complex with β10 or β4 (0.81 μM). The change in the dissociation constant generated by the truncation of the two C‐terminal amino acid residues of β10 is small but statistically significant. This demonstrates that even the very last amino acid residues at the C‐terminus of β‐thymosins are involved in the interaction with G‐actin.
The in vitro DNA binding properties of NDP kinase are related to its oligomeric state
Authors: Mesnildrey Sébastien , Agou Fabrice , Véron Michel
Published: July 2002

Genetic and biochemical evidences suggest that the enzymatic activity of NDP kinase is necessary but not sufficient for its biological function. While the human NDPK‐B binds specifically single‐strand polypyrimidines sequences, the hexameric enzyme from Dictyostelium does not. We demonstrated by electrophoretic mobility shift assay and filter binding assay that a dimeric mutant from Dictyostelium binds to an oligodesoxynucleotide while the wild‐type does not. These data suggest that the differences in the DNA binding properties of several eucaryotic NDP kinases might be correlated to the differences in the stability of their hexameric structure.
Expression of B‐type endothelin receptor gene during neural development
Authors: Tsaur Meei-Ling , Wan Yi-Chiou , Lai Fang-Pin , Cheng Hwei-Fang
Published: July 2002

Mutations of the B‐type endothelin receptor (ETRB) gene have been found to cause defects in the development of enteric neurons, which resulted in aganglionic megacolon in rodents and humans. To determine the distribution of ETRB mRNA during neural development, mainly in the CNS, in situ hybridization was applied at various developmental stages of rat. ETRB gene was abundantly expressed prenatally in the ventricular and subventricular zones, as well as postnatally in the ependymal and subependymal cells. ETRB mRNA was also strongly detected prenatally in the dorsal root ganglia, as well as postnatally in the cerebellar Bergmann glial cells and epithelial cells of choroid plexus. Our data suggest that ETRB acts as a regulator in the differentiation, proliferation, or migration of neural cells during development.
Differential expression of the protein kinase A regulatory subunit (RIα) in pancreatic endocrine cells
Authors: Arava Yoav , Adamsky Konstantin , Belleli Adina , Shaltiel Shmuel , Walker Michael D
Published: July 2002

A PCR‐based subtractive cloning procedure was used to identify genes expressed at higher levels in the pancreatic beta cell line βTC1, as compared to the pancreatic alpha cell line αTC1. One of the clones isolated by this procedure corresponded to the regulatory subunit (RIα) of protein kinase A (PKA). Using antibodies directed against RIα, we now demonstrate both by immunoblot and immunofluorescence that RIα protein is present at higher levels in cultured beta cells as compared to alpha cells. In vitro PKA assays revealed high basal PKA activity in αTC1 extracts, which changed little on addition of exogenous cAMP. On the other hand, extracts from beta cells showed very low basal activity of PKA, which was elevated upon addition of cAMP. A similar trend was observed in vivo using transfected luciferase constructs bearing multiple copies of a CRE element: in αTC1 cells, no induction by forskolin was observed, whereas in βTC1 cells, forskolin produced a 9‐fold increase in activity. Therefore, the results indicate that RIα of PKA is selectively expressed in pancreatic beta cells as compared to alpha cells: this selective expression is associated with major differences in the properties of the PKA signal transduction pathway. Differential expression of the regulatory subunit may play a role in determining the patterns of gene expression and signal transduction characteristic of alpha and beta cells.