bluuebunny/crossref_metadata_2025 huggingface dataset:
High extracellular Ca2+ and Mg2+ stimulate accumulation of inositol phosphates in bovine parathyroid cells
Authors: Brown Edward , Enyedi Peter , LeBoff Meryl , Rotberg Jane , Preston Joseph , Chen Chu
Published: July 2002

We examined the effects of the divalent cations Ca2+ and Mg2+ on inositol phosphate accumulation in bovine parathyroid cells prelabelled with [3H]inositol to determine whether the high extracellular Ca2+ and Mg2+‐evoked transients in cytosolic Ca2+ in these cells might result from increases in cellular IP3 levels. In the presence of Li+, both Ca2+ and Mg2+ produced rapid, 2–6‐fold increases in IP3 and IP2 and a linear increase in IP of 6–8‐fold at 30 min. Smaller (1.5–2‐fold) increases in IP2 and IP3 were evident within 7.5–15 s upon exposure to high (3mM) Ca2+ in the absence of Li+. The relative potencies of Ca2+ and Mg+ (Ca2+ 3‐fold more potent than Mg2+) in elevating inositol phosphates were similar to those for their effects in inhibiting PTH release. Fluoride (5 and 10 mM) also produced similar increases in inositol phosphate accumulation, presumably through activation of phospholipase C by a guanine nucleotide (G) protein‐dependent process. Thus, high extracellular Ca2+ and Mg2+‐induced spikes in cytosolic Ca2+ in bovine parathyroid cells may be mediated by increases in IP3, perhaps through a receptor‐mediated process linked to phospholipase C by a G‐protein.
Oncogenic ras protein induces meiotic maturation of amphibian oocytes in the presence of protein synthesis inhibitors
Authors: Allende Catherine C. , Hinrichs M.Victoria , Santos Eugenio , Allende Jorge E.
Published: July 2002

Microinjection of the activated ras oncogenic protein can induce the meiotic maturation of Xenopus laevis oocytes, a process that can also be triggered by progesterone or high concentrations of insulin. Cycloheximide and puromycin, well‐known inhibitors of protein synthesis, block the maturation process induced by progesterone and insulin but do not affect the maturation caused by H‐ras
lys 12 protein microinjection. Theophylline, an inhibitor of cAMP phosphodiesterase that also affects oocyte protein synthesis, does cause a partial inhibition of ras protein‐induced maturation. These findings indicate that ras protein acts on the oocyte maturation process at a point that is downstream of the protein synthesis requirement, a characteristic shared with the maturation promoting factor, an activity that appears in oocytes and mitotic cells at the onset of cell division.
Association of rabbit muscle glyceraldehyde‐3‐phosphate dehydrogenase and 3‐phosphoglycerate kinase The biochemical and electron‐microscopic evidence
Authors: Sukhodolets Maks V. , Muronetz Vladimir I. , Tsuprun Valery L. , Kaftanova Alla S. , Nagradova Natalia K.
Published: July 2002

Rabbit muscle glyceraldehyde‐3‐phosphate dehydrogenase covalently bound to Sepharose was shown to form a complex with soluble 3‐phosphoglycerate kinase. The strength of the association appeared to depend upon the functional state of both enzymes. The holoform of the dehydrogenase exhibited a lower affinity for the kinase than the enzyme‐3‐phosphoglycerol·NADH complex. The substrate‐free 3‐phosphoglycerate kinase associated much stronger with the acylated dehydrogenase than the kinase in complex with 1,3‐diphosphoglycerate. Electron‐microscopic evidence for the association of the soluble acyl‐glyceraldehyde‐3‐phosphate dehydrogenase·NADH complex and 3‐phosphoglycerate kinase was also obtained.
Phosphorylation of hepatic insulin receptor by casein kinase 2
Authors: Grande Jorge , Pérez Mercè , Itarte Emilio
Published: July 2002

Casein kinase 2 was able to phosphorylate the β‐subunit of hepatic insulin receptor in the presence of either ATP or GTP. Phosphorylation by casein kinase 2 was observed even in the absence of insulin, was inhibited by low heparin concentrations, and led to the incorporation of phosphate on serine and threonine residues. Casein kinase 2 phosphorylation of insulin receptor partially decreased its tyrosine kinase activity.
Chemical reactions within DNA duplexes Cyanogen bromide as an effective oligodeoxyribonucleotide coupling agent
Authors: Sokolova N.I. , Ashirbekova D.T. , Dolinnaya N.G. , Shabarova Z.A.
Published: July 2002

Cyanogen bromide was found to condense oligodeoxyribonucleotides on a complementary template in aqueous solution. Optimum conditions for this vigorous and effective reaction were developed. CNBr proved to be useful for incorporation of phosphoramidate or pyrophosphate internucleotide bonds in DNA duplexes.
Inhibition of cellular phosphatidylinosito, turnover by psi‐tectorigenin
Authors: Imoto Masaya , Yamashita Takashi , Sawa Tsutomu , Kurasawa Shogo , Naganawa Hiroshi , Takeuchi Tomio , Bao-quan Z. , Umezawa Kazuo
Published: July 2002

Psi‐tectorigenin, an isoflavonoid, was isolated from a culture filtrate of actinomycetes as an inhibitor of epidermal growth factor‐induced phosphatidylinositol turnover in cultured A431 cells. It inhibited phosphatidylinositol turnover with an IC50 of about 1 μg/ml; thus, its inhibitory activity was 6‐times stronger than that of genistein or orobol. When added to cultured A431 cells psi‐tectorigenin inhibited phosphatidylinositol turnover without inhibiting epidermal growth factor receptor tyrosine protein kinase. Thus, psi‐tectorigenin is a specific inhibitor of phosphatidylinositol turnover and may be a useful tool for the functional analysis of phosphatidylinositol turnover.
Primary structure of porcine muscarinic acetylcholine receptor III and antagonist binding studies
Authors: Akiba Isamu , Kubo Tai , Maeda Akito , Bujo Hideaki , Nakai Junichi , Mishina Masayoshi , Numa Shosaku
Published: July 2002

The complete amino acid sequence of porcine muscarinic acetylcholine receptor III has been deduced by cloning and sequencing the genomic DNA. The antagonist binding properties of muscarinic acetylcholine receptor III expressed from the cloned DNA in Xenopus oocytes correspond most closely to those of the pharmacologically defined M2 glandular (III) subtype.
Detection of calcium binding by photosystem II polypeptides immobilised onto nitrocellulose membrane
Authors: Webber Andrew N. , Gray John C.
Published: July 2002

Photosystem II calcium‐binding polypeptides have been detected by their ability to selectively bind 45Ca when immobilised onto nitrocellulose membrane following SDS‐polyacrylamide gel electrophoresis. Two calcium‐binding polypeptides of 26 kDa and 24 kDa are shown to be components of LHCII. The 24 kDa polypeptide was further characterised by N‐terminal amino acid sequence analysis and shown to be the product of a type II cab gene. A third polypeptide of 33 kDa bound calcium more weakly and was not positively identified.
Extracellular adenosine triphosphate activates phospholipase C and mobilizes intracellular calcium in primary cultures of sheep anterior pituitary cells
Authors: van der Merwe P.Anton , Wakefield Ian K. , Fine Jennifer , Millar Robert P. , Davidson James S.
Published: July 2002

In primary cultures of sheep anterior pituitary cells extracellular ATP (ED50 0.4–0.8 μM) stimulated efflux of 45Ca2+ from a slow‐turnover intracellular pool. ADP was also effective whereas AMP and adenosine were not. The ATP effect was not due to cell permeabilization as 100 μM ATP did not elicit efflux of 2‐deoxy[3H]glucose metabolites. This 45Ca2+ mobilization may be mediated by inositol trisphosphate, since ATP (ED50 1 μM) stimulated inositol phosphate generation. These results demonstrate P2‐purinoceptors in sheep anterior pituitary cells which are coupled to phospholipase C activation and intracellular Ca2+ mobilization, and raise the possibility of a regulatory role for extracellular ATP in the anterior pituitary.
Direct evidence that pyrophosphate: Fructose‐6‐phosphate phosphotransferase can act as a glycolytic enzyme in plants
Authors: Hatzfeld Wolf-Dieter , Dancer Jane , Stitt Mark
Published: July 2002

Experiments were carried out to provide direct evidence that pyrophosphate: fructose‐6‐phosphate phosphotransferase (PFP) can operate as a glycolytic enzyme in some circumstances. A large increase in the rate of glycolysis was produced by adding uncoupler and alkalinising the medium of heterotrophic Chenopodium rubrum cells. Initially, a marked decrease in phosphoenolpyruvate, 3‐phosphoglycerate and hexose phosphates occurred, but no change in fructose 2,6‐bisphosphate or inorganic pyrophosphate was observed. However, a gradual increase in the rate of O2 uptake during the subsequent 5 min was accompanied by an increase in fructose 2,6‐bisphosphate and a decrease in pyrophosphate, providing evidence that activation of PFP contributes to the increase in rate of glycolysis. This was accompanied by partial recovery of the adenine nucleotide energy status.
Isolation from human cerebrospinal fluid of a new insulin‐like growth factor‐binding protein with a selective affinity for IGF‐II
Authors: Roghani Monireh , Hossenlopp Paul , Lepage Pierre , Balland Alain , Binoux Michel
Published: July 2002

In biological fluids IGF‐I and IGF‐II are bound to specific, high‐affinity binding protein (BPs). Two human BPs have been isolated, one from serum, which is GH‐dependent, the other from amniotic fluid (AF BP), and their cDNAs have recently been cloned. We report here the isolation of another, new species from cerebrospinal fluid (CSF) where this BP predominates. The protein was purified to homogeneity by a four‐step procedure: gel filtration, chromatofocusing, hydrophobic‐interaction chromatography and reverse‐phase chromatography. Thereafter, SDS‐polyacrylamide gel electrophoresis gave an M
r of 34 000 (non‐reduced), chromatofocusing gave an isoelectric point of 5.0, and its affinity for IGF‐II (3 × 1010 M−1) was 10 times that for IGF‐I. The N‐terminal amino acid sequence of the first 15 residues determined in a BP preparation from the CSF of children was Leu‐Ala‐Pro‐Gly‐(/)‐Gly‐Gln‐Gly‐Val‐Gln‐Ala‐Gly‐Ala‐Pro‐Gly. A similar sequence was found for adult CSF, apart from residues 12 and 13 (‐Leu‐Leu‐). These are highly analogous with the sequences starting from residue 69 of the GH‐dependent BP, and from residue 61 of the AF BP. The new BP isolated is therefore related to, but distinct from, the other human BPs.
Kinetic evidence for the function of Z in isolated photosystem II reaction centers
Authors: Mathis P. , Satoh K. , Hansson Ö.
Published: July 2002

(D1,D2) Photosystem II reaction centers were studied by flash absorption spectroscopy with microsecond resolution. Without addition, absorption transients in the 660–850 nm region are due to a triplet state, probably of P‐680, with a decay half‐time of 700 μs (no oxygen) or 40 μs (under air). With the addition of DBMIB, presumably acting as electron acceptor, new kinetic and spectral features appear, which are attributed to P‐680. A 5 μs phase of decay is present, which is pH‐dependent and is attributed to donation from Z to P‐680+.
Correlation between dissipative fluorescence quenching at photosystem II and 50 μs recombination luminescence
Authors: Schreiber Ulrich , Neubauer Christian
Published: July 2002

Charge recombination luminescence in the 50 μs time range was measured in isolated chloroplasts under identical conditions as modulated chlorophyll fluorescence. In this way the relationship between fluorescence quenching, as analysed by the saturation pulse method, and charge recombination was investigated. Various treatments known to slow down the donation rate of water splitting, were found to induce correlated increases in 50 μs recombination luminescence and fluorescence quenching, with both photochemical and non‐photochemical quenching components being enhanced. It is concluded that charge recombination at photosystem II provides a mechanism for rapid dissipation of excitation energy.
Functional expression and sites of gene transcription of a novel α subunit of the GABAA receptor in rat brain
Authors: Malherbe P. , Sigel E. , Baur R. , Persohn E. , Richards J.G. , Möhler H.
Published: July 2002

Two α subunits of the gabaa receptor in rat brain have been identified by molecular cloning. The deduced polypeptide sequences share major characteristics with other chemically gated ion channel proteins. One polypeptide represents the rat homologue of the α3 subunit previously cloned from bovine brain [14], while the other polypeptide is a yet unknown subunit, termed α5. When coexpressed with the β1 subunit in Xenopus oocytes the receptors containing the α5 subunit revealed a higher sensitivity to GABA than receptors expressed from α1 + β1 subunits or α3 + β1 subunits (K
a = 1 μM, 13 μM and 14 μM, respectively). The α5 subunit was expressed only in a few brain areas such as cerebral cortex, hippocampal formation and olfactory bulb granular layer as shown by in situ hybridization histochemistry. Since the mRNA of the α5 subunit was colocalized with the αl and α3 subunits only in cerebral cortex and in the hippocampal formation the α5 subunit may be part of distinct GABAA receptors in neuronal populations within the olfactory bulb.
Firefly luciferase synthesizes P1,P4‐bis(5'‐adenosyl)tetraphosphate (Ap4A) and other dinucleoside polyphosphates
Authors: Guranowski Andrzej , Sillero Maria A.Günther , Sillero Antonio
Published: July 2002

The synthesis of P1,P4‐bis(5'‐adenosyl)tetraphosphate (Ap4A) has been considered, for a long time, to be catalyzed mainly by some aminoacyl‐tRNA synthetases [Brevet et al. (1989) Proc. Natl. Acad. Sci. USA 86, 8275‐8279]. Recently, yeast Ap4A phosphorylase, acting in reverse (Guranowski et al. (1988) Biochemistry 27, 2959‐2964), was shown to synthesize Ap4A, too. In the case of the synthetases, the intermediate complex E‐aminoacyl‐AMP may serve as donor of AMP to ATP, yielding Ap4A. Here we demonstrate that firefly luciferase (EC 1.13.12.7) which forms the E‐luciferin‐AMP intermediate also synthesizes Ap4A as well as other dinucleoside polyphosphates. We suggest moreover that: other enzymes (mainly synthetases and some transferases), which catalyze the transfer of a nucleotidyl moiety, via nucleotidyl‐containing intermediates and releasing PPi may produce dinucleoside polyphosphates.
Transfer of tightly‐bound tritium from the chloroplast membranes to CF1 is activated by the photophosphorylation process
Authors: Zolotareva E.K. , Gasparyan M.E. , Yaguzhinsky L.S.
Published: July 2002

Isolated thylakoids were incubated for 14–16 h in the buffer containing 3‐6 mCi T2O/ml and then resedimented and suspended in non‐radioactive medium. It was found that illumination of thylakoids induced an increase in radioactivity level in CF1 isolated from these thylakoids. Such effect was observed only if photophosphorylation substrates (ADP and phosphate or arsenate) were added to the medium during illumination. The light‐induced ADP and arsenate‐dependent incorporation of tritium into CF1 was suppressed by DCCD and inhibited by low gramicidin concentrations to the same extent as photophosphorylation.
Human tRNAGlu genes: their copy number and organisation
Authors: Gonos Efstathios S. , Goddard John P.
Published: July 2002

The tRNAGlu gene copy number, determined by genomic blot analysis of human plaeental DNA, is approximately thirteen. These studies, using several probes and DNA digested with several restriction enzymes singly or in combination, show that most of these tRNAGlu genes are flanked by DNA of very similar sequence for at least 5 kb. This conclusion is supported by the close similarity of the restriction maps of two λ Charon‐4A recombinants of human genomic DNA containing two different tRNAglu genes.
RNA sequence and the nature of the CuA‐binding site in cytochrome c oxidase
Authors: Covello Patrick S. , Gray Michael W.
Published: July 2002

For cytochrome c oxidase subunit II (COXII), DNA and protein sequences suggest that Met‐207 (bovine numbering) is conserved in all species except plants. Sequencing of plant mitochondrial COXII mRNAs now indicates that Met‐207 is also conserved among plants as a result of a C‐to‐U type of RNA editing. Considering the strict evolutionary conservation of Met‐207 and the homology of COXII to type I (blue) copper proteins and nitrous oxide reductase, we propose a model in which Met‐207 is associated with the CuA‐binding site (along with Cys‐196, Cys‐200 and His‐204) and plays a role in determining its reduction potential and stability.
Interaction of α‐actinin and nebulin in vitro
Authors: Nave Rüdiger , Fürst Dieter O. , Weber Klaus
Published: July 2002

Nebulin is a high molecular weight polypeptide (mass 0.6–0.8 million) which accounts for 3% of the myofibrillar mass in skeletal muscle. Due to its resistance to extraction under native conditions, relatively little is known about the biochemistry of the molecule. Here we report in vitro binding of α‐actinin (a major Z‐line protein) to nebulin. After solubilization with sodium dodecylsulfate myofibrillar polypeptides separated by gel electrophoresis were blotted on nitrocellulose and probed with 125I‐labelled α‐actinin, Nebulin is the only polypeptide decorated by α‐actinin. This result gives biochemical support for the hypothesis, based on recent immunoelectron micrographs, that nebulin could form in skeletal muscle a fourth filament system, possibly extending to the Z‐line.
Metal ion co‐ordination in the DNA binding domain of the yeast transcriptional activator GAL4
Authors: Povey Jane F. , Diakun Gregory P. , Garner C.David , Wilson Stephen P. , Laue Ernest D.
Published: July 2002

The structure of the DNA binding domain of the yeast transcriptional activator GAL4 was investigated by extended X‐ray fine structure (e.x.a.f.s.). Two samples of GAL4 were studied, one containing cadmium as a structural probe (Cd(II)GAL4) and the other containing the ‘native’ zinc (Zn(Il)‐GAL4). The results suggest that the structure of the DNA binding domain of GAL4 contains a two metal ion cluster distinguishing it from the ‘zinc finger’ proteins typified by the Xenopus laevis transcription factor TFIIIA.
Stathmin phosphorylation patterns discriminate between distinct transduction pathways of human T lymphocyte activation through CD2 triggering
Authors: le Gouvello Sabine , Chneiweiss Herve , Tarantino Nadine , Debre Patrice , Sobel Andre
Published: July 2002

CD2 triggering of human T lymphocyte activation has been associated with the activation of different interacting protein kinases, including protein kinase C (PKC). However the precise roles of its phosphorylated substrates are still unknown. We show here that PKC‐dependent and ‐independent pathways arc responsible for the CD2‐induced phosphorylation of stathmin, a ubiquitous soluble phosphoprotein, most likely acting as a general intracellular relay integrating various second messenger pathways. The phosphorylated variants of stathmin provide a fingerprint reflecting the second messenger pathway(s) stimulated. The respective roles of both PKC and stathmin in the regulation of T lymphocyte proliferation are discussed.
Light‐induced D1 protein degradation is catalyzed by a serine‐type protease
Authors: Virgin Ivar , Salter A.Hugh , Ghanotakis Demetrios F. , Andersson Bertil
Published: July 2002

Light‐induced degradation of the D1 protein in isolated spinach photosystem II core preparations was studied after addition of various protease inhibitors. The degradation was selectively inhibited by several serine protease inhibitors in particular diisopropylfluorophosphate. The results demonstrate that the D1 protein is degraded by a serine‐type of proteolytic activity that is an integral part of photosystem II.
The 5–55 single‐disulphide intermediate in folding of bovine pancreatic trypsin inhibitor
Authors: Darby N.J. , van Mierlo C.P.M. , Creighton T.E.
Published: July 2002

An analogue of the BPTI folding intermediate that contains only the disulphide bond between Cys‐5 and Cys‐55 has been prepared by mutation or the other four Cys residues to Ser. On the basis of its circular dichroism and 1H‐nuclear magnetic resonance spectra and its electrophoretic mobility, this intermediate is shown to be at least partially folded at low temperatures. This probably accounts for several of the unique properties of this intermediate observed during folding.
Shuffled domains in extracellular proteins
Authors: Bork Peer
Published: July 2002

A comprehensive list of domains in extracellular mosaic proteins is presented. About 40 domains were distinguished by consensus patterns. A subsequent sequence database search recognized these domains in more than 200 extracellular proteins. The results point to a structural network, which may also represent the molecular basis for a complex coordination of various functions within the world of extracellular proteins.
A cytosolic protein tyrosine kinase in rat adipocytes
Authors: Shisheva Assia , Shechter Yoram
Published: July 2002

Previous studies suggested that insulin receptor tyrosine kinase (IRTK) is the sole tyrosine kinase in rat adipocytes. We now report that this cell type also contains a cytosolic soluble protein tyrosine kinase (CytPTK) which is not related to IRTK. The enzyme phosphorylated PolyGlu4Tyr whith high efficiency at a rate of 20 ± 2 pmoI PTyr/20μg PolyGlu4 Tyr/20 min/μg cytosolic protein. Upon gel filtration chromatography the enzyme activity was eluted as a single peak corresponding to a molecular mass of 53 ± 3 kDa. Unlike IRTK, CytPTK activity was supported by Co2+ rather than by Mn2+, and it was not inactivated by N‐ethylmaleimide. The enzyme was extremely sensitive to inhibition by staurosporine (ID50 = 3 nM) as opposed to IRTK (ID50 = 8μM). In addition, CytPTK (but not IRTK) was largely activated by vanadate ions. Agents which affect the serine/threonine phosphorylation state of cell proteins did not alter CytPTK activity when subjected to intact adepocytes. In a cell‐free system CytPTK activity was largely reduced by pretreatment with immobilized alkaline phosphatase at physiological pH. The possibility that CytPTK participates in insulin‐independent regulation of glucose metabolism is suggested.
Induction of zinc metallothionein by calcium ionophore in vivo and in vitro
Authors: Xiong Xiaoyan , Arizono Koji , Garrett Scott H. , Brady Frank O.
Published: July 2002

The calcium ionophore, A23187, can induce rat hepatic metallothionein (MT) when administered in vivo (5.8‐fold, 5.0 μM, 11 h) and rat hepatocyte MT when administered in vitro (10.70‐fold, 1.0 μM, 24 h). Several rat hepatoma cell lines (2M, 4.55‐fold; JM2, 12.29‐fold; EC3, 14.12‐fold; HTC, 7.99‐fold) and a normal rat liver cell line (Clone 9, 39.67‐fold) were tested for their inducibility of MT mRNA by Cd2+ (10 μM, 8 h). Quantitatively, JM2 and 2M made the most MT mRNA, while HTC made the least. A23187 (0.1–7.0 μM) was studied as an inducer of MT mRNA in these cell lines (except for HTC) and in HeLa. A variety of responses and tolerances were seen with inductions ranging up to 32.11‐fold. Quantitatively, the best responding cell lines were EC3 and 2M. A combination induction experiment, using TPA, a protein kinase C activator, and A23187 in EC3 cells revealed an additive effect of the two inducers on MT mRNA levels: TPA (10 nM), 11.71‐fold; A23187 (3.0μM), 6.71‐fold; and TPA + A23187, 20.00‐fold. These studies have implicated perturbations in cytosolic calcium ion concentrations, caused by the ionophore A23187, as being involved in the complicated signaling systems which can lead to induction of MT mRNA and protein.
Activation of nucleoside diphosphate kinase by mastoparan, a peptide isolated from wasp venom
Authors: Kikkawa Satoshi , Takahashi Katsunobu , Takahashi Koh-ichi , Shimada Nobuko , Ui Michio , Kimura Narimichi , Katada Toshiaki
Published: July 2002

We have previously reported that GDP‐bound αβγ‐trimeric GTP‐binding (G) proteins can be converted into the active GTP‐bound form with nucleoside diphosphate (NDP) kinase and ATP, although its exact activation mechanism still remains to be resolved. In the present study, we investigated whether NDP kinase activity was modified by mastoparan, a wasp venom peptide that is known to activate G proteins as an agonist‐receptor complex. The activity of NDP kinase measured by the formation of GTP from ATP and GDP was markedly stimulated, when the kinase was incubated with mastoparan. The concentration of mastoparan required for the activation was much lower than that observed for the peptidc‐induced activation of G proteins under similar assay conditions. There was also an increase in the phosphorylated intermediate of NDP kinase as well as the catalytic activity upon its incubation with mastoparan. These results suggest that mastoparan not only activates G proteins directly via guanine nucleotide exchange reaction but also stimulates NDP kinase activity.
Characterization of the Lactococcus lactis pepN gene encoding an aminopeptidase homologous to mammalian aminopeptidase N
Authors: Tan Paris S.T. , van Alen-Boerrigter Ingrid J. , Poolman Bert , Siezen Roland J. , de Vos Willem M. , Konings W.N.
Published: July 2002

The nucleotide sequence of the pepN gene from Lactococcus lactis encoding a zinc‐metallo aminopeptidase has been determined. The open reading frame of 2,538 base pairs encodes a protein with a calculated M
r of 95,368, which agrees with the apparent M
r of 95,000 of the gene product which was identified by polyclonal antibodies raised against the purified aminopeptidase. The amino acid sequence of the aminopeptidase of L. lactis was found to be similar to the corresponding enzymes of human, rat and mouse, with almost 30% of the residues identical. Also, a highly conserved area was identified which has similarity with the active site of thermolysin. A zinc‐binding site, as well as the catalytic site for PepN, is predicted to lie within this conserved stretch. Putative promoter regions upstream of PepN were confirmed by primer extension analysis.
Expression of c‐fos, c‐jun, jun‐B, metallothionein and metalloproteinase genes in human chondrocyte
Authors: Zafarullah Muhammad , Martel-Pelletier Johanne , Cloutier Jean-Marie , Gedamu Lashitew , Pelletier Jean-Pierre
Published: July 2002

Normal and osteoarthritic (OA) human articular cartilage chondrocytes, released enzymatically in the presence of 0.5% fetal calf serum, display constitutive expression of early response activating protein (AP‐1) genes; c‐fos, c‐jun and jun‐B. Among the late AP‐1 responsive genes, total matallothionein (MT) and stromelysin mRNAs were expressed at high levels in both normal and OA chondrocytes, while collagenase and hMT‐IIA mRNA levels were elevated only in OA individuals. Despite the common AP‐1 sequences present in their promoter regions, the three late genes were differentially expressed.
Does biocatalysis involve inhomogeneous kinetics?
Authors: Demchenko Alexander P.
Published: July 2002

Biocatalytic reactions can occur according to two very different mechanisms: homogeneous, which is described by standard transition state theory (TST) and its modifications, and inhomogeneous (polychromatic), which is characteristic for some of the charge‐transfer reactions in liquids and amorphous solids. While most data published on enzyme reactions are interpreted on the basis of homogeneous kinetics, the important recent findings suggest the involvement of inhomogeneous kinetics mechanism.
Expression and binding properties of two isoforms of the human growth hormone receptor
Authors: Sobrier Marie-Laure , Duquesnoy Philippe , Duriez Bénédicte , Amselem Serge , Goossens Michel
Published: July 2002

Two isoforms of the human growth hormone receptor mRNA, one containing exon 3 (encoding an extracellular domain of the receptor), hGHR, and one excluding exon 3, hGHRd3, have been described. To study the cellular distribution of the two types of messengers we have analysed a panel of tissues. Both isoforms were expressed independently or simultaneously depending on the tissue studied. To investigate the binding properties of hGHRd3 we have cloned its cDNA in a eukaryotic expression vector; transient expression in COS‐7 cells showed that the receptor without exon 3 was expressed on the plasma membrane and was able to bind human growth hormone (hGH) with the same high affinity as hGHR. Human lactogen (hCS) removed 125I‐hGH bound to the full‐length and exon 3 ‐ excluding receptors to the same extent. These results show that hGHR and hGHRd3 have tissue‐specific expression and share identical binding properties for hGH and hCS and leave open the possibility that exon 3 might influence receptor signalling.
Human γ‐interferon expression in the mammary gland of transgenic mice
Authors: Dobrovolsky V.N. , Lagutin O.V. , Vinogradova T.V. , Frolova I.S. , Kuznetsov V.P. , Larionov O.A.
Published: July 2002

Transgenic mice carrying a hybrid gene consisting of ovine β‐lactoglobulin gene sequences and human γ‐interferon (hIFN‐g) cDNA were produced. hIFN‐g expression in the mammary gland of two lactating transgenic founder females was found. The concentration of active hIFN‐g in the milk was estimated as being ca. 1800 IU/ml. The hIFN‐g ability to express in the mammary gland was found in the progeny of transgenic founder male.
Differential contribution of Mr 120 kDa rasGTPase‐activating protein and neurofibromatosis type 1 gene product during the transition from growth phase to arrested state in human fibroblasts accompanied by a unique rasGTPase‐activating active
Authors: Kobayashi Manko , Hashimoto Naohiro , Hoshino Masato , Hattori Seisuke , Iwashita Shintaro
Published: July 2002

Using octyl glucoside‐solubilized cell extracts from human fibroblasts during growth phase to G0/G1 arrest state, we found that while the number of M
r, 120 kDa rasGTPase‐activating protein (p120GAP) molecules per cell decreases to half its original levels, the amount of neurofibromatosis type 1 gene product (NF1, neurofibromin) remains constant during the transition. The contribution of pl20GAP to the total rasGTPase‐activating (rasGA) activity in growing cells was found to be larger than that observed in arrested cells (84% vs 53%). On the other hand, NF1 contributes less than 15% of the total rasGA activity in either extract. These results indicate that the qualitative changes occur in the contributors to rasGA activity during transition. They also suggest that a unique rasGA activity exists in the arrested cells, which was obtained separatedly from both pl20GAP and NF1 by heparin‐Sepharose column chromatography.
Biosynthesis of vitamin B12
Authors: Scott A.I. , Roessner C.A. , Stolowich N.J. , Spencer J.B. , Min C. , Ozaki S.-I.
Published: July 2002

In the vitamin B12 biosynthetic pathway the enzymes responsible for the conversion of precorrin‐3 to precorrin‐4 have been identified as the gene products of cobG and cobJ from Pseudomonas denitrificans. CobG catalyzes the oxidation of precorrin‐3 to precorrin‐3x (a hydroxy lactone) whereas CobJ is a SAM‐dependent C‐17 methyl transferase and is necessary for ring contraction. A mechanism for ring contraction is proposed.
Molecular basis for the different binding properties of benzodiazepines to human and bovine peripheral‐type benzodiazepine receptors
Authors: Farges Roseli , Joseph-Liauzun Evelyne , Shire David , Caput Daniel , Le Fur Gerard , Loison Gerard , Ferrara Pascual
Published: July 2002

The 18 kDa peripheral benzodiazepine receptor (PBR) can be labelled by benzodiazepines, such as Ro5‐4864, and isoquinoline carboxamides such as PK11195. These two compounds are reversible competitive inhibitors of each other. However, while the binding affinity of Ro5‐4864 varies enormously across species, PK11195 always displays high affinity, suggesting that their binding domains are overlapping but not identical. We report here that recombinant human and bovine PBR produced in yeast, a microorganism devoid of endogenous PBR, can be labelled with [3H]PK11195, but only the human receptor can be labelled with [3H]Ro5‐4864. Furthermore, we identified, through the binding analysis of human‐bovine chimaeric receptors, a region near the C‐terminal end of the PBR, with only five non‐conserved amino acids between human and bovine sequences, as responsible for the difference in high affinity binding of Ro5‐4864 to the two receptors.
Kinetic analysis on the substrate specificity of 3‐isopropylmalate dehydrogenase
Authors: Miyazaki Kentaro , Kakinuma Katsumi , Terasawa Hiroaki , Oshima Tairo
Published: July 2002

Substrate specificity of 3‐isopropylmalate dehydrogenase is analyzed using a series of synthetic (2R,3S)‐3‐alkylmalates. Each analog with hydrogen, methyl, ethyl, isopropyl, isobutyl, tert‐butyl, and isoamyl group on C‐3 functions as a substrate, implying a broad substrate specificity of the enzyme toward alkylmalates. The incremental binding energy of the isopropyl group of 3‐isopropylmalate to the enzyme is estimated to be 3.55 kcal/mol, the rather small value supporting the broad specificity. Although the enzyme shows a broad specificity toward the alkylmalates, it does not show activity with isocitrate which has a negatively charged carboxymethyl group instead of the alkyl groups.
Tyr‐139 in Thermus thermophilus 3‐isopropylmalate dehydrogenase is involved in catalytic function
Authors: Miyazaki Kentaro , Oshima Tairo
Published: July 2002

The role of Tyr‐139, which is thought to be located at the active site of Thermus thermophilus HB8 3‐isopropylmalate dehydrogenase, has been investigated by site‐specific replacement with phenylalanine. The replacement scarcely affected the Michaelis constant (K
m) for 3‐isopropylmalate, but caused a 13‐fold decrease of that for NAD. The catalytic constant (k
cat) showed a 14‐fold decrease. Accordingly, the catalytic efficiency (k
cat/K
m) decreased for 3‐isopropylmalate but not for NAD. The results suggest that Tyr‐139 is involved in the catalytic function through interaction with 3‐isopropylmalate.
A change in soybean agglutinin binding patterns of bovine milk fat globule membrane glycoproteins during early lactation
Authors: Ujita Minoru , Furukawa Kiyoshi , Aoki Naohito , Sato Takeshi , Noda Akihiro , Nakamura Ryo , Greenwalt Dale E. , Matsuda Tsukasa
Published: July 2002

Milk fat globule membrane (MFGM) glycoproteins were prepared from bovine milk at different stages of early lactation. Western blot analyses using several lectins revealed that reactivity of MFGM glycoproteins, especially 47K and 80K bands, to soybean agglutinin (SBA) remarkably increased during the lactation, while no change was observed for Ricinus communis agglutinin‐I (RCA‐I) binding. Sialidase treatment of MFGM glycoproteins revealed that the number of SBA‐positive bands and the amount of SBA‐positive oligosaccharides in these bands are increased during the lactation. Since SBA binds N‐acetylgalactosamine terminated oligosaccharides, the results indicated that N‐acetylgalactosaminylation of bovine MFGM glycoproteins is stimulated during the lactation.
New isoforms of multifunctional calcium/calmodulin‐dependent protein kinase II
Authors: Mayer Peter , Möhlig Matthias , Schatz Helmut , Pfeiffer Andreas
Published: July 2002

Calcium/calmodulin dependent protein kinase II (CaM kinase II) seems to act as an important regulator of intracellular signal transmission. Four subtypes, termed α to δ, have been cloned; some of them can exist as different splicing variants. All these isoforms share a great overall homology, and they contain 3 areas of low homology. We have identified 5 new variants of subtype delta so that the total number of different isoforms now adds up to 12. These variants are probably a result of different splicing and show several deletions in regard to subtype delta. The deletion sites do exactly match regions of low homology between the subtypes. This suggests a functional division of the CaM kinase II molecule into homologous and variable domains. The homologous domains are highly conserved. Therefore, it might be the case that the constitution of the variable domains is more significant for a certain isoform than its belonging to one of the 4 subtypes α to δ.
The effect of local anaesthetics on the ryanodine receptor/Ca2+ release channel of brain microsomal membranes
Authors: Martin Cecile , Ashley Richard , Shoshan-Barmatz Varda
Published: July 2002

The effects of various local anaesthetics (LAs) on ryanodine binding of the sheep brain ryanodine receptor were tested. Tetracaine and dibucaine inhibit the binding with half‐maximal inhibition (CI50) of 0.12 mM and 0.7 mM, respectively. Lidocaine and its analog QX‐314, on the other hand, stimulate the binding up to 3‐fold with half‐maximal stimulation occurring with about 2 mM of the drugs. Lidocaine increases both the receptor affinity for ryanodine by about 5‐fold and the rate of ryanodine association with its binding site by about 6‐fold. Tetracaine and lidocaine also interact with the purified brain ryanodine receptor and produce inhibitory and stimulatory effects similar to those obtained with the membranebound receptor. The interaction of the LAs with the brain ryanodine receptor, as well as with the skeletal muscle receptor [J. Memb. Biol. 133 (1993) 171‐182], suggest that ryanodine receptor possesses intrinsic binding site(s) for LAs.
Methyl green
Authors: Kim Seog K. , Nordén Bengt
Published: July 2002

Interaction and binding geometries of complexes of Methyl green with poly(dA‐dT)2, poly(dA) · poly(dT), and triplex poly(dA) · 2poly(dT) complexes have been studied by linear dichroism. For both of the complexes with double helical DNAs, the z symmetry axis of Methyl green is found to be approximately parallel to the DNA bases while the x symmetry axis lies at 40–44° relative to the local DNA helix axis, in agreement with a groove binding mode. However, in contrast to minor‐groove binders (such as DAPI and Hoechst 33258) Methyl green is found to be excluded from binding to the triple helical poly(dA) · 2poly(dT) in which the major groove is filled by the third strand. While most so far studied groove‐binding dyes bind in the minor groove of DNA, Methyl green thus appears to be an exception.
Vitamin D binding protein is produced by human monocytes
Authors: Sabbatini A. , Petrini M. , Mattii L. , Arnaud P. , Galbraith R.M.
Published: July 2002

The expression of the DBF (vitamin D binding protein) gene was investigated in monocytes and in peripheral blood lymphocytes. DBF message was amplified through 35 cycles of PCR amplification using specific oligonucleotide primers. PCR products of the expected size were further identified by Southern blotting using a specific DBF probe. No expression of the DBF gene could be detected in peripheral blood lymphocytes, nor in the monocyte‐derived U 937 cell line. In contrast, message for DBF was identified in monocytes activated with lipopolysaccharide when analyzed between 6 and 10 h following stimulation. These results suggest that the temporal expression of the DBF gene could play a major role in the activation of monocytes by 1‐25(OH)2D3.
Site‐specific incorporation of photofunctional nonnatural amino acids into a polypeptide through in vitro protein biosynthesis
Authors: Hohsaka Takahiro , Sato Ken , Sisido Masahiko , Takai Kazuyuki , Yokoyama Shigeyuki
Published: July 2002

Nonnatural amino acids with photofunctional groups were incorporated site‐specifically into a polypeptide by using in vitro protein synthesizing system. The nonnatural amino acids were attached to tRNACCU through chemical misacylation method, and added to the in vitro system with a mRNA containing a single AGG codon. l‐p‐Phenylazophenylalanine, l‐2‐anthrylalanine, l‐1‐naphthylalanine, l‐2‐naphthylalanine and l‐p‐biphenylalanine were successfully incorporated into a polypeptide, but l‐1‐pyrenylalanine was not. The polypeptides containing the nonnatural amino acids showed photofunctionalities.
Temperature‐dependent change of aerological specificity of Candidaalbicans NIH A‐207 cells cultured in yeast extract‐added Sabouraud liquid medium: disappearance of surface antigenic factors 4, 5, and 6 at high temperature
Authors: Okawa Yoshio , Takahata Takashi , Kawamata Masaki , Miyauchi Masayoshi , Shibata Nobuyuki , Suzuki Akifumi , Kobayashi Hidemitsu , Suzuki Shigeo
Published: July 2002

The cells of Candida albicans NIH A‐207 strain (A‐strain) cultivated in YSLM at high temperatures (37 and 40°C) did not undergo agglutination with the factor sera 4, 5, and 6 in a commercially available factor serum kit, ‘Candida Check’, and formed a grape‐like shape. The mannans isolated from the cells had lost their reactivity against the factor sera in ELISA. It was also revealed by 1H NMR analysis that the mannans contained neither a phosphate group nor a β‐1,2‐linked mannopyranose unit, although these mannans increased the non‐reducing terminal α‐1,3‐linked mannopyranose unit. The cells and the mannans prepared by cultivation at such high temperatures followed by 27°C in the same medium entirely recovered the reactivity with the factor sera.
Isolation and characterization of a high molecular weight cytochrome from the sulfate reducing bacterium Desulfovibrio gigas
Authors: Chen Liang , Pereira Manuela M. , Teixeira Miguel , Xavier António V. , Le Gall Jean
Published: July 2002

A high molecular weight c‐type cytochrome (Hmc) was purified and characterized from Desulfovibrio gigas. The molecular weight was estimated to be 67 kDa by SDS‐PAGE and its N‐terminus is homologous to those of the 16 hemes containing high molecular weight cytochrome c from Desulfovibrio vulgaris strains Hildenborough and Miyazaki. The purified hemoprotein shows c‐type cytochrome absorption spectrum with ε553(red) = 368 mM−1 · cm−1. A band at 640 nm, characteristic of high‐spin hemes, was detected. The EPR spectra show the presence of two high‐spin heme species, plus several non‐equivalent low‐spin hemes. The heme reduction potentials, at pH 7.6, range from −50 mV to −315 mV. In contrast to what has been described for D. vulgaris Hmc, the protein isolated from D. gigas directly accepts electrons from hydrogenase and further reduces other redox proteins.
Isolation and characterization of a cDNA clone encoding the pokeweed antiviral protein II from Phytolacca americana and its expression in E. coli
Authors: Poyet Jean-Luc , Radom Jean , Hoeveler Arnd
Published: July 2002

Three distinct ribosome‐inactivating proteins (RIPs) were isolated from pokeweed Phytolacca americana We identified and sequenced for the first time a complete cDNA encoding the pokeweed antiviral protein II (PAP II), which is expressed in the late summer leaves of pokeweed. The cDNA of PAP II consists of 1, 187 nucleotides and encodes a mature protein of 285 amino acids. Its predicted amino acid sequence is only 33% similar to PAP and PAP‐S. The NH2 terminal extrapeptide (25 amino acid residues) was similar but not identical to that of PAP's extrapeptide. The cDNA of PAP II was expressed in E. coli. The growth of the transformants was strongly inhibited after induction of the gene. Furthermore, PAP II, which was produced in E. coli, inhibited protein synthesis in a rabbit reticulocyte translation system. Thus, recombinant PAP II would appear to be as functional as native PAP in inhibiting protein synthesis in both prokaryotes and eukaryotes.
Neuromedin B receptor, expressed in Xenopus laevis oocytes, selectively couples to Gαq and not Gα11
Authors: Shapira Hagit , Way James , Lipinsky Dafna , Oron Yoram , Battey James F.
Published: July 2002

G‐proteins of the q family have been implicated as mediators of bombesin receptors action. We cloned Xenopus Gαq and Gα11 and specifically disrupted the synthesis of either protein with selective antisense oligonucleotides. Gαq antisense inhibited responses mediated by neuromedin B receptor (NMB‐R) by 74%, though not by gastrin‐releasing peptide receptor (GRP‐R). Gα11 antisense had little effect on either GRP‐R‐ or NMB‐R‐mediated responses. This suggests that NMB‐R couples to Gαq, and that GRP‐R and NMB‐R show distinct G‐protein coupling preferences in the Xenopus oocyte.
The N‐terminal region of the α‐subunit of the TRAP complex has a conserved cluster of negative charges
Authors: Hartmann Enno , Prehn Siegfried
Published: July 2002

The α‐subunit of the TRAP complex (TRAPα) is a single‐spanning membrane protein of the endoplasmic reticulum (ER) which is found in proximity of nascent polypeptide chains translocating across the membrane. Here, we demonstrate the widespread occurrence of TRAPα in eukaryotes as indicated by its existence in man, fish and plants. Despite the fact that the sequence homology is much lower than for other proteins in the translocation site, the overall topology, the location of the glycosylation sites and, most interestingly, the distribution of charges are conserved. These data indicate that the TRAP complex has a ubiquitous function.
Molecular cloning of Bac7, a proline‐ and arginine‐rich antimicrobial peptide from bovine neutrophils
Authors: Scocchi Marco , Romeo Domenico , Zanetti Margherita
Published: July 2002

Bac7 is a 7 kDa proline‐ and arginine‐rich antimicrobial peptide which was purified from bovine neutrophils. We have used PCR to clone the cDNA of Bac7 precursor, a polypeptide of 21,569 Da. This cDNA is highly conserved in the 5′ region, with respect to the corresponding region in the precursors of several other structurally unrelated myeloid antimicrobial peptides. Furthermore, a 148 nt non‐coding region at the 3′ end is 75% homologous to a corresponding region of the cDNA of the precursor of PR‐39, a porcine antibacterial peptide which is also proline‐ and arginine‐rich.
Tamoxifen stimulates phospholipase D activity by an estrogen receptor‐independent mechanism
Authors: Kiss Zoltan
Published: July 2002

The effects of tamoxifen (TAM), a widely used agent in the treatment of breast cancer, were examined on phospholipase D (PLD)‐mediated phospholipid hydrolysis. In drug‐sensitive MCF‐7 human breast carcinoma cells TAM, similar to several well‐established activators of PLD, had no effect on phospholipid hydrolysis. In an estrogen receptor‐deficient multidrug‐resistant subline of MCF‐7 cells, TAM preferentially stimulated the hydrolysis of phosphatidylethanolamine; two‐fold stimulation required 2.5 or 5 μM TAM in the absence or presence of serum, respectively. In NIH 3T3 fibroblasts significant (4‐ to 4.8‐fold) stimulation of phosphatidylethanolamine and phosphatidylcholine hydrolysis in the presence of serum required 10 μM TAM. These data establish that TAM can stimulate PLD activity by an estrogen receptor‐independent mechanism.
The hsc70 gene which is slightly induced by heat is the main virus inducible member of the hsp70 gene family
Authors: Sainis Ioannis , Angelidis Charalambos , Pagoulatos Gerassimos , Lazaridis Ioannis
Published: July 2002

We have found that SV40 infection of CV1 cells induces the synthesis of a 72 kDa protein that upon molecular cloning was shown to be the product of the hsc70 gene. The above gene product was found to be mainly virus inducible, in contrast to the hsp70 gene product which was mainly heat inducible. The two genes were found to be cell cycle regulated in a distinctively different manner.