bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin‐stimulating protein from human plasma
Authors: Arakaki Naokatu , Oribe Toshiya , Ueno Akemichi , Inoue Hideo , Takeda Yoshiro
Published: July 2002

An insulin‐stimulating protein was isolated from human plasma by a procedure involving Sephadex G‐100 chromatography and reverse‐phase HPLC. The isolated material gave a single band on SDS‐polyacrylamide gel electrophoresis. This protein itself had no insulin‐like activity, but enhanced fatty acid synthesis from glucose in rat adipose tissue explants in the presence of suboptimal concentrations of insulin. It also stimulated the effect of insulin on CO2 liberation from glucose by isolated rat adipocytes and increased the maximal response to insulin.
GTPγS‐induced solubilization of actin and myosin from rabbit peritoneal neutrophil membrane
Authors: Huang Chi-Kuang , Devanney James F.
Published: July 2002

Addition of guanosine 5'‐(3‐O‐thio)triphosphate (GTPγS) to the membrane fraction isolated from rabbit peritoneal neutrophils results in the solubilization of several proteins from the membrane. The major proteins are of 180 kDa (myosin) and 43 kDa (actin). The effect is observed with a half‐maximum GTPγS concentration of 70 μM. The potencies of various nucleotides are compared: GTPγS ⪢ GtP ATP ⪢ GDP, GMP, cGMP, cAMP. The effect does not require calcium and is not inhibited by using membranes perpared from cells that have been pretreated with pertussis toxin.
Thyrotropin and norepinephrine stimulate the metabolism of phosphoinositides in FRTL‐5 thyroid cells
Authors: Philp Nancy J. , Grollman Evelyn F.
Published: July 2002

Hormone‐induced changes in phospholipid metabolism were examined in a functioning rat thyroid cell line (FRTL‐5). Stimulation of FRTL‐5 cells, prelabeled with 32P, with TSH or NE resulted in a rapid decrease in the radioactivity of both phosphatidylinositol 4,5‐bisphosphate (PIP2) and phosphatidylinositol 4‐mono‐phosphate (PIP). The effects of TSH on phospholipid metabolism and calcium mobilization are independent of those on adenylate cyclase. This suggests that the TSH receptor may be unique in that it activates enzyme cascades involved in cAMP production and Ca2+ mobilization.
High‐level production and isolation of human recombinant α1‐proteinase inhibitor in yeast
Authors: Hoylaerts M. , Weyens A. , Bollen A. , Harford N. , Cabezón T.
Published: July 2002

The cDNA coding for mature human α1‐proteinase inhibitor (α1‐PI) has been inserted into a variety of yeast expression vectors. Yeast cells transformed with these plasmids were then assayed for the production of mature, unglycosylated α1‐PI. The production level is optimal when the recombinant plasmid carries the TDH promoter, the complete 2μ and the leu2D selection marker. Biologically active recombinant α1‐PI can be purified either analytically, by affinity chromatography using a monoclonal antibody, or on a large scale, by a procedure involving precipitation of high‐M
r, yeast material with polyethylene glycol 3300 followed by successive chromatography on DEAE‐agarose, Zn‐chelate agarose, κ‐chain agarose, heparin‐agarose and aminohexyl‐agarose.
Archaebacterial and eukaryotic ribosomal subunits can form active hybrid ribosomes
Authors: Altamura Sergio , Cammarano Piero , Londei Paola
Published: July 2002

Purified ribosomal subunits from the extremely thermoacidophilic archaebacterium Sulfolobus solfataricus are able to recognize ribosomal subunits from the yeast Saccharomyces cerevisiae forming hybrid monosomes that can be revealed by sucrose gradient analysis and are active in peptide bond formation. Both reciprocal combinations (archaebacterial 30 S + eukaryotic 60S and archaebacterial 50 S + eukaryotic 40 S) are functional. In contrast, no hybrid couples are formed between subunits of yeast and Escherichia coli ribosomes. These results indicate that ribosomes of at least one archaebacterial species share specific structural features with those of the lower eukaryote S. cerevisiae.
cAMP reduces the affinity of Ca2+‐triggered secretion in platelets
Authors: Collazos José Maria , Sanchez Ana
Published: July 2002

Prostacyclin and other related compounds known to increase intracellular cAMP levels inhibit platelet responses. The mechanisms involved are only partially known, especially those concerning the complex relations between Ca2+ and cAMP as opposite intracellular mediators. Here, we have investigated aggregation and secretion in quin2‐loaded platelets under conditions in which Ca2+ and cAMP are the only intracellular mediators. Our results show that cAMP inhibits aggregation and secretion in ionophore‐treated cell without modifying their intracellular Ca2+ levels. This result suggests that the inhibition takes place on some intracellular target for Ca2+.
Splicing of group II introns in mRNAs coding for cytochrome b6 and subunit IV in the liverwort Marchantia polymorpha chloroplast genome Exon specifying a region coding for two genes with the spacer region
Authors: Fukuzawa Hideya , Yoshida Tadashi , Kohchi Takayuki , Okumura Takeshi , Sawano Yasuji , Ohyama Kanji
Published: July 2002

Two genes, petB and petD, encoding cytochrome b
6 and subunit IV of the cytochrome b
6/ƒ complex, respectively, of the chloroplast genome of Marchantia polymorpha, a liverwort, have been mapped between the psbH and rpoA genes and each of the petB and petD genes has been predicted to contain a group II intron in its coding sequence [(1986) Nature 322, 572–574]. Primer extension analysis of the liverwort chloroplast RNA using two synthetic oligodeoxyribonucleotides, complementary to parts of the coding sequences of the petB and petD transcripts, as primers has provided evidence that the two genes are co‐transcribed to a precursor mRNA which is then precisely spliced at the predicted sites.
On an Mg2+‐dependent interaction of actin with glyceraldehyde‐phosphate dehydrogenase The fundamental role of KCl in the organization of F‐actin
Authors: Lanzara Vincenzo , Grazi Enrico
Published: July 2002

In the presence of Mg2+, the formation of actin filaments is hindered by glyceraldehyde‐3‐phosphate dehydrogenase. This effect, which increases with the square of Mg2+ concentration, is counteracted by 0.15 M KCl. Thus KCl, at concentrations found in the intracellular compartment, appears to be strictly required for the correct formation of actin filaments in all tissues in which the glyceraldehyde‐phosphate dehydrogenase concentration is high.
Evidence of an α2‐macroglobulin‐like molecule in plasma of Salamandra salamandra Structural and functional similarity with human α2‐macroglobulin
Authors: Sallenave Jean-Michel , Bellot Ricardo
Published: July 2002

A high‐Mr (M
r 750 000) α1 ‐macroglobulin, obtained from Salamandra salamandra, is described. Salamander α1‐macroglobulin is composed of two monomers of equal M
r, which are composed of two polypeptide chains, each of M
r 180 000, linked by disulfide bonds. The molecular parameters of this protein, its binding to trypsin and inactivation by methylamine suggest that salamander α1‐macroglobulin is closely related to human α2‐macroglobulin and to other related proteins described in the animal kingdom.
Expression of human pancreatic polypeptide precursors from a dicistronic mRNA in mammalian cells
Authors: Boel Esper , Berkner Kathleen L. , Nexø Bjørn A. , Schwartz Thue W.
Published: July 2002

The ability of eukaryotic ribosomes to reinitiate translation at downstream AUG codons on polycistronic mRNAs was used to select transfected CHO clones that secreted a precursor to the human pancreatic poly‐peptide (PP). In the in vitro constructed transcription unit, a viral promoter directed the synthesis of a dicistronic mRNA. The PP cDNA was placed in the 5′‐part of this transcript, while a DHFR cDNA was placed 3′ to the PP. This dicistronic expression unit was transfected into CHO cells, and methotrexate‐resistant colonies were selected. RNA‐blots verified that the PP precursor and DHFR were expressed from the same dicistronic mRNA. The CHO cells synthesized the hormone precursor and secreted it through the constitutive secretory pathway.
DNA methylases separated through the HeLa cell cycle methodology show allosteric properties
Authors: Delfini C. , Crema A.L. , Alfani E. , Lo Presti E. , Eremenko T. , Volpe P.
Published: July 2002

Two DNA methylases (DNAmets) can be separated through the cell cycle. The first appears as a minor peak in G1, the second as a major peak in S. Both enzymes protect from Hpall a plasmid (H31), constructed with the pBR322 vector (4.3 kbp) and the inverted Aγ, fragment of the human globin gene (3.5 kbp), inserted at its HindIII site (the vector carries several HpaII sites, the insert only one HpaII site). DNAmets G1 and S show distinct K
m values and different kinetics vs the ionic strength of the medium, while their Michaelis‐Menten and Lineweaver‐Burk plots are sigmoidal and hyperbolical curves, respectively. This is the first suggestion about the allosteric nature of the eukaryotic DNAmet system.
Irreversible inactivation of calcium‐dependent proteinases from rat liver by biological disulfides
Authors: Di Cola Domenico , Sacchetta Paolo
Published: July 2002

The effect of low‐molecular‐mass biological disulfides and their related reduced compounds on the activity of two calcium‐dependent neutral proteinases (calpains) from rat liver has been investigated. L‐Cystine and L‐cystamine bring about the inactivation of both enzymes, while the related reduced compounds L‐cysteine and L‐cysteamine are without effect. Calpain II is more sensitive to the inactivating effect of glutatione disulfide in comparison with calpain I. The inactivation rates of both calpains depend on the concentration of glutathione disulfide. Reduced glutathione, added at physiological concentration (5 mM), neither affects the proteinase activities nor protects the enzymes from the inactivating effect of glutathione disulfide. The enzymes inactivated by biological disulfides cannot be restored by a large excess of a reducing thiolic compound (dithiothreitol). It is suggested that calcium‐dependent proteinases might be inactivated also in vivo by enhanced level of glutathione disulfide.
Isolation of <Glu‐Gly‐Leu‐Arg‐Trp‐NH2 (Antho‐RWamide II), a novel neuropeptide from sea anemones
Authors: Graff D. , Grimmelikhuijzen C.J.P.
Published: July 2002

Using a radioimmunoassay for the peptide sequence Arg‐Phe‐NH2 (RFamide), a novel peptide has been purified from acetic acid extracts of the sea anemone Anthopleura elegantissima. This peptide has the structure <Glu‐Gly‐Leu‐Arg‐Trp‐NH2, and was named Antho‐RWamide II. Antho‐RWamide II is a neuropeptide. Its structure is closely related to an earlier characterized neuropeptide from Anthopleura <Glu‐Ser‐Leu‐Arg‐Trp‐NH2 (Antho‐RWamide I).
Phosphohistidine is found in basic nuclear proteins of Physarum polycephalum
Authors: Pesis Karen H. , Wei Yingfei , Lewis Matthew , Matthews Harry R.
Published: July 2002

Nuclear extracts of the true slime mold, Physarum polycephalum, show protein histidine kinase activity towards exogenous histones [(1985) J. Biol. Chem. 260, 16106–16113]. Physarum microplasmodia were labeled with [32P]phosphate in vivo and two basic proteins containing alkali‐stable phosphate were detected. The labeled proteins comigrated with Physarum histones H1 (approximately) and H2A and phosphoamino acid analysis showed that each protein contained [32P]phosphohistidine. The H2A‐like protein was also labeled in isolated nuclei incubated with [35S]thio‐ATP. We conclude that some Physarum nuclear proteins contain phosphohistidine.
1H‐NMR studies on nucleotide binding to the catalytic sites of bovine mitochondrial F1‐ATPase
Authors: Garin Jérôme , Vignais Pierre V. , Gronenborn Angela M. , Clore G.Marius , Gao Zhan , Baeuerlein Edmund
Published: July 2002

The conformation of adenine nucleotides bound to bovine mitochondrial F1‐ATPase was investigated using transfer nuclear Overhauser enhancement measurements. It is shown that all nucleotides investigated adopt a predominantly anti conformation when bound to the catalytic sites. Furthermore, the experiment suggests that 8‐azido‐ADP and 8‐azido‐ATP, which are predominantly in the syn conformation in solution, are in the anti conformation when bound to F1 catalytic sites.
Phorbol esters have a dual action through protein kinase C in regulation of proliferation of FRTL‐5 cells
Authors: Takada Kaoru , Amino Nobuyuki , Tetsumoto Toru , Miyai Kiyoshi
Published: July 2002

In quiescent rat thyroid (FRTL‐5) cells, phorbol‐12‐myristate‐13‐acetate (PMA) inhibited DNA synthesis induced by a combination of insulin and thyrotropin (TSH) or dibutyryl cyclic AMP (Bt2cAMP). This inhibitory effect of PMA was observed even when PMA was added after addition of these growth factors, and was maximal when PMA was added 2–4 h after the growth factors (late in the G1‐phase of the cell cycle). On the other hand, PMA alone induced DNA synthesis and also enhanced that induced by Bt2cAMP or insulin in these quiescent cells. 1‐Oleoyl‐2‐acetylglycerol mimicked these effects of PMA, but 4α‐phorbol‐12,13‐didecanoate had no effect. These data demonstrate that in FRTL‐5 cells protein kinase C has a stimulatory effect on the G0 to G1 transition and an inhibitory effect on the G1 to S transition in the cell cycle.
Demonstration of N‐acetylchondrosine‐degrading β‐glucuronidase in rabbit liver
Authors: Nakamura Toshiya , Takagaki Keiichi , Majima Mitsuo , Endo Masahiko
Published: July 2002

N‐Acetylchondrosine was incubated at pH 4.0 with a rabbit‐liver crude enzyme extract. Gel filtration of the reaction products on Sephadex G‐15 revealed the presence of monosaccharide liberated from the disaccharide. The monosaccharide fraction was analyzed by gas‐liquid chromatography, and identified as a mixture of glucuronic acid and N‐acetylgalactosamine. These results indicate the presence of β‐glucuronidase, which degrades N‐acetylchondrosine, in rabbit liver. The discovery of the presence of this enzyme may help to establish the complete degradation process of chondroitin sulfates.
Inaccurate protein synthesis in a mutant of Salmonella typhimurium defective in transfer RNA pseudouridylation
Authors: Negre D. , Cortay J.C. , Donini P. , Cozzone A.J.
Published: July 2002

Protein synthesis was studied comparatively in a wild‐type strain of Salmonella typhimurium and in hisT mutant cells defective in the pseudouridylation of transfer RNA. From a quantitative point of view, no significant difference between the two types of strain was observed when measuring the rate of protein synthesis during either exponential growth or starvation for histidine. In contrast, the qualitative analysis of proteins by two‐dimensional gel electrophoresis showed that histidine‐starved hisT cells mistranslate the genetic program at a higher frequency than exponentially growing hisT cells or either starved or unstarved hisT
+ cells.
Long‐term ingestion of ammonium inhibits lysosomal proteolysis in rat liver
Authors: Felipo Vicente , Miñana María-Dolores , Wallace Ruth , Grisolía Santiago
Published: July 2002

A standard diet was supplemented with ammonium acetate (20%, w/w). The effect on liver protein degradation of oral administration of the ammonium diet to rats for 6 weeks has been studied. It is shown that lysosomal proteolysis is markedly decreased (by 62%) while non‐lysosomal proteolysis is inhibited by 11%. This is the first report showing that ammonium ingestion inhibits liver proteolysis.
Modes of action of uncouplers in thylakoids
Authors: Schönfeld Mordechay , Schickler Hedva
Published: July 2002

The pH dependence of electron transport in isolated lettuce chloroplasts can be simulated by a model involving four ionizable groups: two facing the inside and two facing the outside of the thylakoids. The pH dependence can be resolved into separate pH profiles for the outside and the inside of the thylakoids, with optima at 7.1 and 5.6, respectively. A ΔpH of 3 units across the thylakoid membrane allows only partial overlapping of the two profiles and relatively low rates of electron transport. Uncouplers stimulate electron transport by reducing the ΔApH, thereby improving the overlapping of the two pH profiles.
Oscillating enzyme‐bound NADH in glycolysis
Authors: Yuan Z. , Müller S.C. , Hess B.
Published: July 2002

In glycolyzing cell‐free cytoplasmic medium extracted from yeast Saccharomyces cerevisiae, the action spectrum of oscillation has an absorption maximum around 335 nm, nearly coinciding with that of the yeast alcohol dehydrogenase (ADH)‐NADH complex due to its bound NADH. Our approximate calculations based on the amount of this enzyme and coenzyme NADH present in the extract suggest that the ADH‐NADH complex alone can account for 90% or more of the total absorbance change.
Muscarinic receptor modulation of glucose‐induced electrical activity in mouse pancreatic B‐cells
Authors: Santos Rosa M. , Rojas Eduardo
Published: July 2002

Acetylcholine (1–10 μM) depolarized the membrane and stimulated glucose‐induced bursts of electrical activity in mouse pancreatic B‐cells. The acetylcholine effects were mimicked by muscarine while nicotine had no effect on membrane potential. Pirenzepine, an antagonist of the classical M1‐type muscarinic receptors, but not gallamine (1–100 μM), an antagonist of the classical M2‐type receptors, antagonized the acetylcholine action on glucose‐induced electrical activity (IC50 = 0.25 μM). Bethanechol, an agonist of the classical M2‐type muscarinic receptors, was approximately 100 times less effective than acetylcholine in stimulating the electrical activity. In addition, acetylcholine (1 μM) induced a marked increase (25%) in input resistance to the B‐cell membrane. The results indicate that acetylcholine exerted its effects on the B‐cell membrane by inhibiting K+ conductance via activation of a muscarinic receptor subtype distinct from the classical M2‐type receptor.
Genetic code preferentially conserves long‐range interactions among the amino acids
Authors: Sitaramam V.
Published: July 2002

The physical properties of amino acids were investigated in order to evaluate their possible relationship to the assignment of codons for amino acids in the genetic code. A comparison of the interconversion probability between amino acids and the distances between the amino acids for individual physical properties revealed a striking hierarchy among the physical properties. Surprisingly, it is the long‐range/solvent interactions and not the short‐range/stereochemical properties which are preferentially conserved in the genetic code.
Inhibition of reverse transcription in rat liver intracisternal A‐particles by thymidine derivatives
Authors: Pyrinova Galina B. , Kuzminova Elena A. , Salganik Rudolf I. , Krayevsky Alexander A. , Kukhanova Marina M.
Published: July 2002

The thymidine derivatives araAzT, dTTP(3′N3), TTP(3′NH2), and araTTP(3′N3), were studied as inhibitors of the reverse transcription taking place within endogenous retroviral A‐type particles, where retroviral RNAs served as templates and primers. dTTP(3′N3) was shown to be the most efficient inhibitor of retroviral particle reverse transcription. Termination of DNA chain elongation is the basic mechanism of the inhibitory action of dTTP(3′N3). The compound has a very low inhibitory effect on mammalian DNA‐dependent DNA polymerases α, β and γ.
Lipoxin formation in human nasal polyps and bronchial tissue
Authors: Edenius Charlotte , Kumlin Maria , Björk Thure , Änggård Anders , Lindgren Jan Åke
Published: July 2002

Chopped human nasal polyps and bronchial tissue produced lipoxin A4 and isomers of lipoxins A4 and B4, but not lipoxin B4, after incubation with exogenous leukotriene A4. In addition, these tissues transformed arachidonic acid to 15‐hydroxyeicosatetraenoic acid. The capacity per gram of tissue to produce lipoxins and 15‐hydroxyeicosatetraenoic acid was 3–5‐times higher in the nasal polyps. Neither tissue produced detectable levels of lipoxins or leukotrienes after incubation with ionophore A23187 and arachidonic acid. Co‐incubation of nasal polyps and polymorphonuclear granulocytes with ionophore A23187 led to the formation of lipoxins, including lipoxins A4 and B4. The results indicate the involvement of an epithelial 15‐lipoxygenase in lipoxin formation in human airways.
Irreversible inhibition of D‐3‐aminoisobutyrate‐pyruvate aminotransferase by gabaculine
Authors: Kaneko Masae , Fujimoto Shigeko , Kikugawa Mariko , Tamaki Nanaya
Published: July 2002

Gabaculine, 5‐amino‐l,3‐cyclohexadienylcarboxylate, is an analogue of GABA and a potent irreversible inhibitor of GABA aminotransferase. However, D‐3‐aminoisobutyrate‐pyruvate aminotransferase for which GABA was neither a substrate nor an inhibitor was also inactivated by gabaculine. The K
i for D‐3‐aminoisobutyrate‐pyruvate aminotransferase was 8.3 × 10−6 M, and the K
cat fot its turnover was 0.31 min−1 at 25°C. β‐Alanine protected the enzyme from inactivation by gabaculine, but GABA did so to much a lesser extent.
Control and recognition of anionic ligands in myoglobin
Authors: Cutruzzolà Francesca , Allocatelli Carlo Travaglini , Ascenzi Paolo , Bolognesi Martino , Sligar Stephen G. , Brunori Maurizio
Published: July 2002

Equilibrium and kinetic experiments on site‐directed mutants of a synthetic sperm whale myoglobin (Mb) gene have been performed. Results on the reactivity on both ferric and ferrous wild type and mutants Mb's are presented. Analysis of ligand binding to His(E7) Val and His(E7) Val‐Thr(E10) Arg mutants compared to wild‐type sperm whale, horse and Aplysia limaelna Mb's, shows that the introduction of an arginyl residue at the topological position E10 greatly enhances the stability of the various Mb:heme ligand adducts. Alternative mechanisms of ligand stabilization may therefore be operative in Mb's lacking the distal histidine.
Photoaffinity labeling of dog pancreas microsomes with 8‐azido‐ATP inhibits association of nascent preprolactin with the signal sequence receptor complex
Authors: Zimmermann Richard , Zimmermann Maria , Mayinger Peter , Klappa Peter
Published: July 2002

Transport of bovine preprolactin into dog pancreas microsomes involves a microsomal protein which is sensitive to photoaffinity labeling with azido‐ATP and which is distinct from the ATP‐binding protein, immunoglobulin heavy chain binding protein. Here we addressed the question of what stage of preprolactin transport is affected. Thus a nascent presecretory protein which is related to preprolactin, termed ppl‐86mer, was employed. Here we show that the nascent preprolactin did not become associated with the α‐subunit of the signal sequence receptor complex after photoaffinity labeling of microsomes with azido‐ATP. Therefore, we conclude that the microsomal protein which is sensitive to photoaffinity labeling with azido‐ATP acts prior to the signal sequence receptor complex.
The effect of glycosyl inositol‐phosphate on cAMP production in isolated rat fat‐cells is transduced by a pertussis toxin sensitive G‐protein
Authors: Martiny Laurent , Dib Karim , Haye Bernard , Corrèze Claude , Jacquemin Claude , Lambert Bernard
Published: July 2002

In fat‐cells, insulin increases synergistically the inhibitory effects of adenosine and prostaglandin E2 on adenylyl cyclase activity. When the endogenous production of these feedback inhibitors is suppressed, insulin is no more active in conditions where glycosyl inositol‐phosphate which is a putative mediator of its action, is always efficient. Moreover, glycosyl inositol‐phosphate signal is transduced by a G protein sensitive to IAP intoxication.
Colicin A and colicin E1 lysis proteins differ in their dependence on secA and secY gene products
Authors: Cavard Danièle
Published: July 2002

The export of colicin A and of colicin E1 is not equally affected in both secA and secY mutants of E. Echerichia coli: release of colicin A occurs slowly while that of colicin E1 is blocked. Processing and functioning of Cal, the colicin A lysis protein, seem to be slightly or not at all modified in these mutants, whereas synthesis and assembly of CelA, the colicin E1 lysis protein, are highly inhibited. These variations observed in the dependence of the two lysis proteins on secA and secY gene products are interpreted as being either the cause or the consequence of the differences observed in their rate of biogenesis.
A comparative study of the relationship between thermostability and function of phenylalanyl‐tRNA synthetases from Escherichia coli and Thermus thermophilus
Authors: Bobkova Ekaterina V. , Stepanov Victor G. , Lavrik Ol'ga I.
Published: July 2002

The relationship between thermostability and functional activities of phenylalanyl‐tRNA synthetases (EC 6.1.1.20) from E. coli and Thermus thermophilus has been studied. In the case of the E. coli enzyme, the activity decreased after the 43°C treatment, both in the [32P]PPi‐ATP exchange reaction and the overall aminoacylation reaction, due to thermo‐inactivation of the phenylalanyl‐tRNA synthetase, whereas tRNAPhe preserved its native structure. In the Th. thermophilus system, the enzyme showed extreme thermostability (up to 90°C), and the reduction in the tRNA aminoacylation rate after the 78°C treatment was ascribed to denaturation of the tRNAPhe. Since the enzyme did not lose the [32P]PPi‐ATP exchange activity up to 80°C, the observed lower thermo‐resistance of the tRNA is evidence that the native structure of ribonucleic acids should be one of the most difficult to stabilize at high temperatures.
Tryptophan fluorescence quenching by alkaline pH and ternary complex formation in human β1β1 and horse EE alcohol dehydrogenases
Authors: Ehrig Torsten , Muhoberac Barry B. , Hurley Thomas D. , Bosron William F.
Published: July 2002

The horse EE and human β1β1 alcohol dehydrogenase isoenzymes have almost identical protein backbone folding patterns and contain 2 tryptophans per subunit (Trp‐15 and Trp‐314). Tyr‐286, which had been proposed to quench the fluorescence of Trp‐314 by resonance energy transfer at alkaline pH in EE, is substituted by Cys in β1β1. The proposed role of Tyr‐286 in pH‐dependent quenching of EE is confirmed by our observation that tryptophan fluorescence of β1β1 is not substantially quenched at alkaline pH. Tyr‐286 had also been implicated in the quenching of Trp‐314 upon formation of the EE‐NAD+‐trifluoroethanol ternary complex. However, β1β1 exhibits the same extent of tryptophan fluorescence quenching as EE upon complexation, which strongly suggests that Tyr‐286 is not involved in ternary complex quenching.
Tobacco chloroplast ribosomes contain a homologue of E. coli ribosomal protein L28
Authors: Yokoi Fumiaki , Sugiura Masahiro
Published: July 2002

The genes ror ribosomal proteins L28 and L33 constitute an operon (rpmBG) in E. coli, but in plant chloroplasts L33 is encoded by the chloroplast DNA and L28 seems to be encoded by the nuclear genome. A 15 kDa protein was isolated from the 50 S subunit of tobacco chloroplast ribosomes and its N‐terminal amino acid sequence was determined. A cDNA for this protein was cloned and analyzed. The cDNA encodes a 151 amino acid protein consisting of a predicted transit‐peptide of 74 amino acids and a mature protein of 77 amino acids. The mature protein is homologous to E. coli L28, hence we named it chloroplast L28 (CL28). This is the first report on the presence of an E. coli L28‐like protein in another organism.
No genetic differences between affected and unaffected members of a German family with Leber's hereditary optic neuropathy (LHON) with respect to ten mtDNA point mutations associated with LHON
Authors: Gerbitz K.-D. , Paprotta A. , Obermaier-Kusser B. , Rietchel M. , Zerres K.
Published: July 2002

In order to investigate possible synergistic influences of different mtDNA mutations on penetrance and severity of Leber's hereditary optic neuropathy (LHON), a large German LHON pedigree is characterized with respect to 10 different mutations associated with LHON. All members of the family carry three different mtDNA mutations (at nucleotide 4,216, 11,778 and 13,708) in a homoplasmic form, regardless of whether or not they are clinically affected. Testing for another 7 mutations reveals negative results in all family members. Hence, the variable disease expression of the family members cannot be explained by varying combinations of LHON‐associated mtDNA mutations.
The neuropeptide, neuromedin C, activates a potassium current in mouse macrophages
Authors: Ichinose Mitsuyuki , Hara Nobumasa , Sawada Masashi , Maeno Takashi
Published: July 2002

Neuromedin C (NmC) induced an outward current (I
o(NmC)) in macrophanges. Reversal potentials were dependent on external K+ concentrations ([K+]o) and independent of [Cl−]o. Tetraethylammonium (TEA) and quinidine effectively suppressed I
o(NmC). Charybdotoxin (ChTX) and apamin had little effect. I
o(NmC) was abolished in Ca2+‐free EGTA‐containing solution. These results suggest that NmC activates a Ca2+‐dependent K+ current (I
K.Ca) and can modulate activities in macrophages.
The DsbA‐DsbB system affects the formation of disulfide bonds in periplasmic but not in intramembraneous protein domains
Authors: Whitley Paul , von Heijne Gunnar
Published: July 2002

The DsbA and DsbB proteins of Escherichia coli are involved in facilitating the formation of disulfide bonds in periplasmic proteins. Here, we show that the rate of formation of a disulfide bond in the periplasmic domain of the inner membrane protein leader peptidase is reduced in dsbA and dsbB strains, whereas the rate of formation of a disulfide bond engineered into the membrane embedded domain of the same protein is completely unaffected by these mutations. We conclude that the Dsb proteins do not facilitate the formation of intramembraneous disulfides.
Modulation of interleukin 2 high affinity binding to human T cells by a pyrimidodiazepine insect metabolite
Authors: Boyle Peter H. , Borchert Monika , Davis Anthony P. , Heaney Frances M. , Ziegler Irmgard
Published: July 2002

An insect metabolite containing the little known pyrimido[4,5‐b][l,4]diazepine ring system has been found to act as an effective mimic of tetrahydrobiopterin in its ability to modulate the affinity of interleukin 2 (IL‐2) for its receptors on human T cells. Semi‐empirical molecular orbital calculations reveal that while tetrahydrobiopterin has considerable flexibility, the pyrimidodiazepine has rather few conformational options and offers a useful model for exploring the nature of the pterin binding site.
Effect of neutral and acidic phospholipids on mitochondrial ATP synthase secondary structure
Authors: Sala Federica Dabbeni , Loregian Arianna , Lippe Giovanna , Bertoli Enrico , Tanfani Fabio
Published: July 2002

The secondary structure of delipidated and egg phosphatidylcholine or asolectin reconstituted mitochondrial ATP synthase complex from beef heart was investigated by Fourier transform infrared spectroscopy. Upon reconstitution, the infrared spectra of ATP synthase revealed an increase in turns and a concomitant decrease in β‐sheet content which occurred to a larger extent in the presence of asolectin rather than in the presence of egg phosphatidylcholine. These data correlate with kinetic data showing a higher ATPase activity of the asolectin reconstituted enzyme protein than the egg phosphatidylcholine reconstituted or delipidated enzyme complexes.
In vivo overexpression and purification of Escherichia coli tRNAser
Authors: Borel Franck , Härtlein Michael , Leberman Reuben
Published: July 2002

DNA fragments corresponding to the sequences of Escherichia coli tRNA2
ser and amber suppressor tRNAser, were synthesized from overlapping oligonucleotides. These were interposed between a strong promotor and a synthetic transcriptional terminator to ensure the production of a transcript of the correct size. The genes of promotor, fragment and terminator were cloned into a conditional runaway replication plasmid. At temperatures below 37°C this vector has a low copy number but, following a temperature shift to 42°C, the copy number is no longer regulated. Using these constructs an overexpression of tRNAser of about 20 times the level of the wild‐type pool could be obtained (corresponding e.g. to 200 times the expression tRNA2
ser). From these systems 10 mg quantities of tRNAsers could be isolated with a serine acceptance of 1,100 pmol/A
280 unit.
Developmental changes in the expression of HMG 2a protein
Authors: Oka Tatsuzo , Sasakawa Tkayo , Komori Naomi , Miyamoto Ken-ichi , Suzuki Isao , Sassa Toshihiro , Natori Yasuo
Published: July 2002

The levels of HMG 2a chromosomal protein and its mRNA change during the post‐hatched development of chicks were investigated. The contents of both HMG 2a and 2b proteins of liver, heart, brain, muscle and gizzard were abundant in the newly hatched chicks but their contents decreased significantly in those tissues of the 70‐day‐old chicks. The HMG 2a mRNA levels of liver, heart and brain in 70‐day‐old chick decreased to about 40% of those mRNA in the newly hatched chicks while the HMG 2a mRNA levels of muscle and gizzard in the 70‐day‐old chicks increased 5‐ and 3‐fold, respectively. These results suggest that the decrease in the HMG 2a protein contents of the muscle and gizzard in the 70‐day‐old chicks may be largely due to the stimulation of HMG 2a protein degradation or the reduction of HMG 2a mRNA translation.
Characterization of the zinc‐metalloprotein nature of rat spermatidal protein TP2
Authors: Kundu Tapas Kumar , Rao Manchanahalli R.Satyanarayana
Published: July 2002

Spermatidal transition protein, TP2, was purified from rat testes by Hg‐affinity chromatography. The present study reports the details of the zinc‐metalloprotein nature of TP2 by employing the 65Zn‐blotting technique. Chemical modification of cysteine by iodoacetic acid, and histidine by diethylpyrocarbonate, resulted in a near complete inhibition of 65Zn‐binding to TP2. The 65Zinc‐binding was localized to the V8 protease‐derived N‐terminal two‐third polypeptide fragment. Circular dichroism spectroscopy studies of TP2 (zinc pre‐incubated) and its V8 protease‐derived polypeptide fragments revealed that the N‐terminal fragment has a Type I‐β‐turn spectrum, while the C‐terminal fragment has a small but significant α‐helical structure. EDTA altered the circular dichroism spectrum of TP2 and the N‐terminal fragment (zinc binding domain) but not that of the C‐terminal fragment.
Regulatory effects of galactose on galactose‐1‐phosphate uridyltransferase activity on human hepatoblastoma HepG2 cells
Authors: Davit-Spraul A. , Pourci M.L. , Heng Ng K. , Soni T. , Lemonnier A.
Published: July 2002

Galactose‐1‐phosphate uridyltransferase (GALT) deficiency results in galactosemia in man. We have studied the regulation of the GALT gene expression on the HepG2 cell line by growing the cells in glucose or galactose medium. No difference of Km values was observed in glucose or galactose media but the V
max value with galactose was 50% higher than that with glucose. Also in galactose medium, an increased GALT specific activity was detected suggesting the production of more enzyme proteins. Yet, slot dot quantification of GALT mRNA revealed a decreased amount of these transcripts in cells cultured with galactose or inosine while Northern blot analysis revealed the normal 1.4 kb transcript in all culture media used. Finally, IEF gel analysis displayed different isozymic patterns for the GALT enzyme in cells grown in glucose, galactose or inosine media. With glucose‐free media, the major band of GALT corresponds to that found in human liver. Altogether, these results suggest that the control of GALT gene expression in HepG2 cells is located at the post‐transcriptional level and correlated to the growth rate of the cell.
Induction of vascular endothelial growth factor expression in synovial fibroblasts by prostaglandin E and interleukin‐1: a potential mechanism for inflammatory angiogenesis
Authors: Ben-Av Pazit , Crofford Leslie J. , Wilder Ronald L. , Hla Timothy
Published: July 2002

Inflammatory mediators such as prostaglandin E2 (PGE2) and interleukin‐1 (IL‐1) induce angiogenesis by yet undefined mechanisms. We demonstrate that PGE2 and IL‐1 induces the expression of vascular endothelial growth factor (VEGF), a selective angiogenic factor by rheumatoid synovial fibroblast cells. Transcripts for the EP1 and EP2, subtypes of PGE receptors are expressed in synovial fibroblasts. Activators of protein kinase A pathway stimulated the expression of VEGF whereas down‐regulation of protein kinase C did not influence the PGE effect, suggesting that signalling from the EP2 receptor via the protein kinase A pathway is important. The induction of VEGF expression by PGE2 and interleukin‐1α a may be an important mechanism in inflammatory angiogenesis.
Cloning and tissue expression of two cDNAs encoding the peroxisomal 2‐enoyl‐CoA hydratase/3‐hydroxyacyl‐CoA dehydrogenase in the guinea pig liver
Authors: Caira Françoise , Cherkaoui-Malki Mustapha , Hoefler Gerald , Latruffe Norbert
Published: July 2002

The 2‐enoyl‐CoA hydratase/3‐hydroxyacyl‐CoA dehydrogenase (HD) is the second enzyme of the peroxisomal β‐oxidation pathway. In human and rat, only one HD mRNA has been so far detected in the liver. This paper reports for the first time in a mammal species, the guinea pig, the cloning and sequencing of two cDNAs encoding an HD. The 3,274 nucleotide‐cDNA is a strictly identical but longer copy of the 2,494 nucleotide‐form. A 2,178 by‐open reading frame encodes a protein of 726 amino acids (M
r 79.3 kDa) with the peroxisomal‐targeting signal (tripeptide SKL) at the carboxyterminus. Northern blot analysis of HD mRNA identified three mRNAs of respective sizes 3.5, 2.6 and 1.6 kb in the guinea pig liver and kidneys.
Comparative studies of gamma‐interferon receptor‐like proteins of variola major and variola minor viruses
Authors: Seregin Sergei V , Babkina Irina N , Nesterov Andrei E , Sinyakov Aleksander N , Shchelkunov Sergei N
Published: July 2002

To study specific properties of the human gamma‐interferon (γ‐IFN) receptor‐like proteins of the highly virulent and low virulent strains of variola (smallpox) virus (VAR) recombinant plasmids determining synthesis of these proteins in E. coli cells have been constructed. The recombinant viral γ‐IFN receptor‐like proteins have been found to have high interferon‐neutralising activity with regards to human γ‐IFN but not murine γ‐IFN and human α‐IFN. The variola major and variola minor proteins under study do not differ in the efficiency of human γ‐IFN antiviral activity inhibition.
Characterization of a short unique sequence in the yeast HO gene promoter that regulates HO transcription in a SIN1 dependent manner
Authors: Yona Eyal , Bangio Haim , Friedman Yael , Shpungin Sally , Katcoff Don J.
Published: July 2002

Recently it has become clear that general chromatin proteins as well as sequence‐specific DNA binding proteins are important in the control of gene expression. SIN1 in Saccharomyces cerevislae is a chromatin component that regulates the transcription of a family of genes. Previously, we identified a 32 bp unique sequence (here termed XBS) in the promoter of one of those genes, HO, which specifically binds a protein that interacts with SIN1. We also found that this sequence can function as a weak UAS in a heterologous promoter that is dependent on the presence of SIN1. Here we report a relationship between the level of HO expression and the presence of the short sequence in situ in the HO gene. By comparing the expression of HO from wild type or XBS deleted HO promoters, we concluded that XBS serves as a weak UAS in situ in the HO gene, that it influences HO transcription via the SWI/SNF complex, and that sequences other than the XBS mediate the affect of SIN1 on HO transcription. In addition, we show that a portion of the SIN1 protein that has sequence similarity to mammalian HMG1 preferentially binds the XBS.
Dephosphorylated but not phosphorylated microtubule associated protein MAP1B binds to microfilaments
Authors: Pedrotti Barbara , Islam Khalid
Published: July 2002

We have reported that purified native MAP1B interacts with microtubules but not with microfilaments [Pedrotti and Islam, Cell Motil. Cytoskel. (1995) 30, 301–309]. However, MAP1B can be phosphorylated at multiple sites by casein kinase II (CKII) and proline‐directed protein kinases (PDPK) and immunoblotting studies show that purified native MAP1B is phosphorylated at least at two CKII sites and at one PDPK site [Pedrotti et al., Biochemistry (1996) 35, 3016–3023]. We now show that phosphorylation affects the in vitro binding of MAP1B with microfilaments. Native MAP1B does not bind to microfilaments but after treatment with alkaline phosphatase the dephosphorylated MAP1B binds and cosediments with microfilaments. Dephosphorylation kinetics suggest that the PDPK site, but not CKII sites, may negatively regulate the interaction with F‐actin. The ability of dephosphorylated MAP1B to crosslink microfilaments was also examined and showed that MAP1B exhibits only a weak crosslinking of F‐actin when compared with MAP2.
Baculovirus‐infected cells do not produce the amyloid peptide of Alzheimer's disease from its precursor
Authors: Essalmani R. , Guillaume J.M. , Mercken L. , Octave J.N.
Published: July 2002

The amyloid peptide (Aβ) of Alzheimer's disease (AD) is produced by proteolytic cleavage of a larger precursor, the amyloid peptide precursor or APP. The discovery of pathogenic mutations in the APP gene provides strong evidence for the hypothesis that APP metabolism is involved in the etiology of AD. To study the metabolism of the protein, human APP has been expressed in several mammalian cell types. Insect cells, infected by a recombinant baculovirus carrying the human APP sequence, also provide an interesting expression system because these cells do not produce endogenous APP. Baculovirus‐infected cells synthesize very high amounts of extracellular soluble APP, after cleavage of the transmembrane protein, as described for mammalian cells. However, we demonstrate here that insect cells do not produce Aβ from APP. These results suggest that while the enzymatic activity needed for the production of soluble APP is conserved between insect and mammalian cells, the enzymes required for the production of Aβ from APP are only expressed in mammalian cells.
Monoamine neurotransmitter transport mediated by the polyspecific cation transporter rOCT1
Authors: Busch A.E , Quester S , Ulzheimer J.C , Gorboulev V , Akhoundova A , Waldegger S , Lang F , Koepsell H
Published: July 2002

The polyspecific cation transporter rOCT,1 which is localized in the basolateral membrane of rat renal proximal tubules and in sinusoidal membranes of hepatocytes, was analyzed for transport of monoamine neurotransmitters. In voltage‐clamp experiments with rOCT1‐expressing Xenopus oocytes, superfusion with dopamine, serotonin, noradrenaline, histamine and the permanent cation acetylcholine induced saturable inwardly directed currents with apparent K
m values ranging from 20 to 100 μM. Transport of dopamine was also demonstrated by uptake measurements in oocytes and in the mammalian cell line (HEK 293) which was permanently transfected with rOCT1. The high uptake rates measured in rOCT1‐expressing oocytes and in transfected HEK 293 cells suggest that rOCT1 is a high capacity transporter which mediates the first step in the excretion of monoamine neurotransmitters.
The C‐terminus of human glutaminase L mediates association with PDZ domain‐containing proteins1
Authors: Olalla Lucı́a , Aledo J.Carlos , Bannenberg Gerard , Márquez Javier
Published: July 2002

The enzyme glutaminase in brain is responsible for the synthesis of neurotransmitter glutamate. We used the two‐hybrid genetic selection system in yeast to look for interactors of glutaminase in human brain. We have identified two proteins containing PDZ domains, α1‐syntrophin and a glutaminase‐interacting protein, named GIP, that showed association with human glutaminase L, as deduced from specificity test of the two‐hybrid system. The complete GIP cDNA clone has 1315 nucleotides with a 372‐base open reading frame encoding a 124‐amino acids protein. Glutaminase associates with both PDZ proteins through its C‐terminal end; mutagenesis of single amino acids revealed the sequence ‐ESXV as essential for the interaction. These data suggest the possibility that PDZ domain‐containing proteins are involved in the regulation of glutaminase in brain.
Conserved amino acids near the carboxy terminus of bacterial tyrosyl‐tRNA synthetase are involved in tRNA and Tyr‐AMP binding
Authors: Salazar J.C , Zuñiga R , Lefimil C , Söll D , Orellana O
Published: July 2002

Bacterial tyrosyl‐tRNA synthetases occur in two large subfamilies, TyrRS and TyrRZ, that possess about 25% amino acid identity. Their amino‐terminal region, the active site domain, is more conserved (>36% identity). The carboxy‐terminal segment of these enzymes includes the tRNA binding domain and contains only few conserved residues. Replacement of three of these residues in Acidithiobacillus ferrooxidans TyrRZ revealed that S356 and K395 play roles in tRNA binding, while H306, a residue at the junction of the catalytic and tRNA binding domains, stabilizes the Tyr‐AMP:TyrRZ complex. The replacement data suggest that conserved amino acids in A. ferrooxidans TyrRZ and Bacillus stearothermophilus TyrRS play equivalent roles in enzyme function.
Real time fluorescence analysis of the RecA filament: implications of base pair fluidity in repeat realignment
Authors: Sen Subhojit , Krishnamoorthy G , Rao Basuthkar J
Published: July 2002

During recombination, when Escherichia coli RecA mediates annealing across DNA repeats, Watson–Crick chemistry can only specify the complementarity of pairing, but not the most optimal frame of alignment. We describe that although stochastic alignments across poly(dA) and poly(dT) can lead to sub‐optimally annealed duplexes containing ssDNA gaps/overhangs, the same are realigned into an optimal frame by a putative motor activity of RecA [Sen et al. (2000) Biochemistry 39, 10196–10206]. In the present study, we analyze the nature of realignment intermediates in real time, by employing a fluorescent probe, 2‐aminopurine (2AP), which can not only report the status of RecA on the unstacked duplex, but also the fluidity of bases in such a filament. Although known to display a lower affinity for duplex DNA, RecA seems to remain functionally associated with these sub‐optimally aligned repeat duplexes, until the realignment approaches completion. Moreover, a comparison of 2AP fluorescence in repeat versus mixed sequences indicates that bases in a RecA repetitive DNA filament exhibit higher degrees of freedom that might mediate a ‘non‐planar hydrogen bonding cross talk’ across the bases on either strand. We discuss a model to explain the mechanistic basis of realignment and its implications in signaling the end of homology maximization, which triggers RecA fall off.
Selectivity of 4,5,6,7‐tetrabromobenzotriazole, an ATP site‐directed inhibitor of protein kinase CK2 (‘casein kinase‐2’)
Authors: Sarno Stefania , Reddy Helen , Meggio Flavio , Ruzzene Maria , Davies Stephen P. , Donella-Deana Arianna , Shugar David , Pinna Lorenzo A.
Published: July 2002

The specificity of 4,5,6,7‐tetrabromo‐2‐azabenzimidazole (TBB), an ATP/GTP competitive inhibitor of protein kinase casein kinase‐2 (CK2), has been examined against a panel of 33 protein kinases, either Ser/Thr‐ or Tyr‐specific. In the presence of 10 μM TBB (and 100 μM ATP) only CK2 was drastically inhibited (>85%) whereas three kinases (phosphorylase kinase, glycogen synthase kinase 3β and cyclin‐dependent kinase 2/cyclin A) underwent moderate inhibition, with IC50 values one–two orders of magnitude higher than CK2 (IC50=0.9 μM). TBB also inhibits endogenous CK2 in cultured Jurkat cells. A CK2 mutant in which Val66 has been replaced by alanine is much less susceptible to inhibition by TBB as well as by another ATP competitive inhibitor, emodin. These data show that TBB is a quite selective inhibitor of CK2, that can be used in cell‐based assays.