bluuebunny/crossref_metadata_2025 huggingface dataset:
Biologically active ether lipids
Authors: Apte Shashank S. , Weber Nikolaus , Mangold Helmut K.
Published: July 2002

Biotransformation of rac‐1(3)‐O‐hexadecylglycerol by photomixotrophic rape (Brassica napus) cells in suspension culture leads to 1‐O‐hexadecyl‐2‐acyl‐sn‐glycero‐3‐phosphocholines and small proportions of other ether lipids, e.g. 1‐O‐hexadecyl‐2‐acyl‐sn‐glycero‐3‐phosphoethanolamines. Reaction of the hexadecylacyl‐glycerophosphocholines with ethanolamine in the presence of phospholipase D from Streptomyces chromofuscus yields additional hexadecylacylglycerophosphoethanolamines. Partial hydrolysis of the combined hexadecylacylglycerophosphoethanolamines followed by reacylation of the resulting lyso compound with palmitic anhydride gives 1‐O‐hexadecyl‐2‐palmitoyl‐sn‐glycero‐3‐phospho‐(N‐palmitoyl) ethanolamine, a nontoxic ether glycerophospholipid with antitumor activity. The corresponding 1‐O‐tetradecyl,1‐O‐octadecyl, and 1‐O‐[(Z)‐9'‐octadecenyl] derivatives are prepared similarly.
Biosynthesis of peptide precursors and protease inhibitors using new constitutive and inducible eukaryotic expression vectors
Authors: Johansen Teit Eliot , Schøller Marianne Skak , Tolstoy Susanne , Schwartz Thue W.
Published: July 2002

A series of expression vectors has been constructed as based on the pML derivative of pBR322. The eukaryotic transcription units employ various promoters followed by polycloning sites for 3–9 commonly used restriction enzymes and are completed by the SV40 polyadenylation sequence. In 4 of the vectors, designed for co‐transfection or transient expression studies, only a single transcription unit containing either a constitutive or an inducible promoter was incorporated. The human ubiquitin (UbC) promoter was used as a strong constitutive promoter, while the mouse metallothionein promoter and the promoter of the long terminal repeats of the mouse mammary tumor virus were used as inducible promoters. Another vector contained an additional transcription unit encoding a eukaryotic selection marker, the neomycin resistance encoding gene. The vectors were used in CHO cells and in neuroendocrine CA77 cells to synthesize peptide precursors, protease inhibitors and a protease. It is shown that these vectors are very efficient for the constitutive and inducible expression of nucleotide sequences in both transient and stable transfections of eukaryotic cells.
Molecular cloning of a cDNA encoding aldosterone synthase cytochrome P‐450 in rat adrenal cortex
Authors: Imai Michiyo , Shimada Hideo , Okada Yukiko , Matsushima-Hibiya Yuko , Ogishima Tadashi , Ishimura Yuzuru
Published: July 2002

Using an oligonucleotide probe designed on the basis of the N‐terminal amino acid sequence of purified rat aldosterone synthase cytochrome P‐450 [(1989) J. Biol. Chem. 264 10935] we have isolated from rat adrenal cDNA library a 2687 base pair cDNA that encodes a protein of 500 amino acid residues. The deduced amino acid sequence contained the regions well conserved among all cytochrome P‐450s sequenced to date, and also a portion (residues 25–44) which was identical to the N‐terminal peptide sequence of rat aldosterone synthase cytochrome P‐450. These results indicate that the cDNA encodes a precursor form of rat aldosterone synthase cytochrome P‐450.
Protein phosphatase 1 activity in Drosophila mutants with abnormalities in mitosis and chromosome condensation
Authors: Dombrádi Viktor , Axton J.Myles , Barker Hazel M. , Cohen Patricia T.W.
Published: July 2002

A Drosophila gene encoding a protein phosphatase 1(PPI) has been sequenced, and lethal mutations in this locus (87B) analysed. Two mutants (ck19
e211 and ck19
hs46), which disrupt mitosis, lack the 87B isoenzyme and express only ∼20% of wild type PPI activity. The Promoter region of the gene is deleted in the ck19
e11 mutant. A third mutant (ck19
e078), which shows suppression of position effect variegation, but has little effect on mitosis, possesses ∼35% of wild type PPI activity. The results indicate that the PPI 87B isoenzyme is involved in regulation of chromosome condensation at interphase as well as mitosis.
The platelet‐derived growth factor isomers, PDGF‐AA, PDGF‐AB and PDGF‐BB, induce contraction of vascular smooth muscle cells by different intracellular mechanisms
Authors: Sachinidis Agapios , Locher Rudolf , Hoppe Jürgen , Vetter Wilhelm
Published: July 2002

The effect of human recombinant platelet‐derived growth factor (PDGF) isoforms, (r)PDGF‐AA, PDGF‐AB and PDGF‐BB, on contractility of rat aortic rings as well as on intercellular free Ca2+ ([Ca2+]i), intracellular pHi (pHi) and thromboxane A2 (TXA2) formation in cultured vascular smooth muscle cells (VSMC) was examined. PDGF‐BB behaved similar to PDGF‐AB and both have features characteristic of conventional vasocontrictor‐agonists that directly increase [Ca2+]i, activate the Na+/H+ exchanger, stimulate the TXA2 formation, and induced contraction in VSMC whereas PDGF‐AA induced contraction without increasing of [Ca2+]i, pHi, and TXA2 formation.
Functional receptors for the insulinotropic hormone glucagon‐like peptide‐I(7–37) on a somatostatin secreting cell line
Authors: Fehmann Hans C. , Habener Joel F.
Published: July 2002

Glucagon‐like peptide‐I (7–37) [(GLP‐I(7–37)] is an instestinal peptide hormone that has potent insulinotropic activities in vivo in response to oral nutrients. In the isolated perfused pancreas, and in vitro in cultured B cells, GLP‐I(7–37) receptor binding and GLP‐I(7–37)‐induced cAMP generation and harmone secretion was studied using cell lines producing insulin/B cell (βTC‐1), glucagon/A cell (INR1G9) and somatostatin/D cell (RIN 1027‐B2). [125I]GLP‐I(7–37) bound specifically to both B and D cells but not to A cells. GLP‐I(7–37) induced cAMP‐formation in B and D cells with a maximum response at 10 nmol/l(B cells) or at 100 nmol/l (D cells). Insulin secretion from perifused B cells was stimulated by GLP‐I(7–37) (maximum at 10 nmol/l) and 10 nmol/l GLP‐I(7–37) released somatostatin from perifused D cells, GLP‐I(7–37) did not influence cAMP or glucagon secretion from A cells. These data indicate that pancreatic B and D cells, but not the A cells are influenced directly by GLP‐I(7–37) via binding to specific receptors. Our findings support a model of physiologic regulation of insulin secretion whereby GLP‐I(7–37) released from the intestine in response to oral nutrients potently stimulates insulin secretion via an endocrine mechanism that in turn may be dampened by a feed‐back suppression by the release of somatostatin. In addition, suppression of the secretion of glucagon, a hormone whose actions are counter‐regulatory to those of insulin, may occur by paracrine mechanisms involving GLP‐I(7–37)‐mediated stimulation of both insulin and somatostatin secretion.
[Ca2+]i oscillations from internal stores sustain exocytic secretion from the chromaffin cells of the rat
Authors: Malgaroli Antonio , Meldolesi Jacopo
Published: July 2002

A large (65%) fraction of in vitro cultured rat chromaffin cells exhibit spontaneous [Ca2+]i oscillations, and the rest can be recruited to oscillate by appropriate stimulations. Based on fura‐2 single cell [ca2+]i measurements, evidence is provided that these oscillation originate, via the activation of Ca2+‐induced Ca2+‐release, from intracellular Ca2+ stores in rapid equilibrium with extracellular Ca2+. By combining [Ca2+]i measurements with a specific plaque secretion assay we demonstrate that oscillating cells exhibit a spontaneous exocytic secretory activity whereas the cells with stable [Ca2+]i do not, [Ca2+]i oscillations appear therefore to account for the high unstimulated catecholamine release, an activity typical of the chromaffin cells of the rat.
The origin of matrix metalloproteinases and their familial relationships
Authors: Murphy George J.P. , Murphy Gillian , Reynolds John J.
Published: July 2002

New computer comparisons of the sequences of mammalian matrix metalloproteinases have established for the first time strong links with bacterial metalloproteinases. We also propose that there are five groups in the family of matrix metalloproteinases, although only three are as yet well‐characterized as proteins, and discuss their origin and relationships with other zinc containing proteases.
Lethal neurotoxicity in mice of the basic domains of HIV and SIV Rev proteins Study of these regions by circular dichroism
Authors: Mabrouk Kamel , Van Rietschoten Jurphaas , Vives Eric , Darbon Hervé , Rochat Hervé , Sabatier Jean-Marc
Published: July 2002

We have recently reported a basic domain‐mediated neurotoxic activity of HIV‐1 Tat [1991, J. Virol. 65, 961–965]. Here we have tested the neurotoxicity in vivo of several Rev‐related synthetic peptides and found that only those mimicking the basic regions of Rev from HIV‐1, HIV‐2 and SIV were lethal to mice. In contrast, the homologous domain of HTLV‐1 Rex was found to be inactive for lethal activity. Analysis of the tropism of these peptides for phospholipids has demonstrated a direct interaction of the basic domain‐containing peptides, except Rex, with acidic — but not neutral — phospholipids. As determined by circular dichroism, a possible correlation between the conformation of the basic regions and the toxicity is discussed.
The two forms of the pituitary adenylate cyclase activating polypeptide (PACAP (1–27) and PACAP (1–38)) interact with distinct receptors on rat pancreatic AR 4‐2J cell membranes
Authors: Robberecht Patrick , Woussen-Colle Marie-Claire , De Neef Philippe , Gourlet Philippe , Buscail Louis , Vandermeers André , Vandermeers-Piret Marie-Claire , Christophe Jean
Published: July 2002

The existence of specific receptors for the two PACAPs (Pituirary Adenylate Cyclase Activating Peptides of 27 and 38 amino acids) was previously demonstrated on membranes from the pancreatic acinar cell line AR 4‐2J (Buscail et al., FEBS Lett. 202, 77–81, 1990) by [125I]PACAP‐27 binding. Here we demonstrate, by comparing Scatchard analysis of saturation curves and competition binding curves obtained with [125I]PACAP‐27 and [125I]PACAP‐38 as radioligands, the coexistence of two classes of receptors : 1/ PACAP‐A receptors that recognize PACAP‐27 and PACAP‐38 with the same high affinity (K
d 0.3 nM) and 2/ PACAP‐B receptors that recognize PACAP‐38 with a high affinity (K
d 0.3 nM) and PACAP‐27 with a lower affinity (K
d 30 nM). These two receptors are coupled to adenylate cyclase but can be clearly distinguished by the ability of PACAP(6–27) to specifically inhibit PACAP‐27 adenylate cyclase activation.
Binding sites involved in the interaction of actin with the N‐terminal region of dystrophin
Authors: Levine B.A. , Moir A.J.G. , Patchell V.B. , Perry S.V.
Published: July 2002

Two actin‐binding sites have been identified on human dystrophin by proton NMR spectroscopy of synthetic peptides corresponding to defined regions of the polypeptide sequence. These are Actin‐Binding Site 1 (ABS1) located at residues 17–26 and Actin‐Binding Site 2 (ABS2) in the region of residues 128–156. Using defined fragments of the actin amino acid sequence, ABS1 has been shown to bind to actin in the region represented by residues 83–117 and ABS2 to the C‐terminal region represented by residues 350–375. These dystrophin‐binding sites lie on the exposed domain in the actin filament.
Inhibition of rat tissue kallikrein gene family members by rat kallikrein‐binding protein and α1‐proteinase inhibitor
Authors: Serveau C. , Moreau T. , Zhou G.X. , Chao J. , Gauthier F.
Published: July 2002

The regulation of tissue kallikrein activity by plasma serine proteinase inhibitors (serpins) was investigated by measuring the association rate constants of six tissue‐kallikrein family members isolated from the rat submandibular gland, with rat kallikrein‐binding protein (rKBP) and α1‐proteinase inhibitor (α1‐PI). Both these serpins inhibited kallikreins rK2, rK7, rK8, rK9 and rK101 with association rate constants in the 103–104 M−1·s−1 range, whereas only ‘true’ tissue kallikrein rK1 was not suceptible to α1‐PI. This results in slow inhibition of rK1 by plasma serpins, which could explain why this kallikrein is the ony member of the gene family identified so far that induces a transient decrease in blood pressure when injected in minute amounts into the circulation.
On the functional role of the Tyr‐639 residue of bacteriophage T7 RNA polymerase
Authors: Rechinsky V.O. , Kostyuk D.A. , Tunitskaya V.L. , Kochetkov S.N.
Published: July 2002

Substitution or Asp for a Tyr residue normally present at position 639 of the bacteriophage T7 RNA polymerase leads to a drastic drop in the enzymatic activity. This mutation does not affect the enzyme‐promoter interaction but decreases the ability of the RNA polymerase to discriminate between GTP and ATP molecules, resulting in a decrease in the rate of the incorporation of the nucleotide into the RNA chain.
cDNA encoding the chicken ortholog of the mouse dilute gene product Sequence comparison reveals a myosin I subfamily with conserved C‐terminal domains
Authors: Sanders Gabriele , Lichte Beate , Meyer Helmut E. , Kilimann Manfred W.
Published: July 2002

We report the cDNA‐deduced primary structure of the chicken counterpart of the murine dilute gene product, a member of the myosin I family. Comparison of the chicken and mouse sequences reveals a distinct pattern of domains of high and low sequence conservation. An internal deletion of 25 amino acids probably reflects differential mRNA processing. Compared with other myosin heavy chain molecules, sequence similarity is highest with the MYO2 gene product of Saccharomyces cerevisiae. The MYO2 protein, implicated in vectorial vesicle transport, is homologous to the dilute protein over practically its entire length. In addition, the C‐terminal domain of the dilute protein is highly similar to a putative glutamic acid decarboxylase sequence cloned from mouse brain. Alternatively, this closely related clone might represent an isoform of the dilute protein derived from a second gene, potentially involved in genetic conditions related to dilute.
Initiator‐promotor coupling of phospholipases D and A2 in platelets upon cholesterol incorporation
Authors: Kochhar N. , Kaul D.
Published: July 2002

Since phospholipases exist within a membrane lipid environment, it is not unreasonable to assume that cholesterol capable of changing the lipid environment can effect the coupling relationship among the signal transducing components. Our previous study showed that the ‘molecular switch’ through which membrane cholesterol modulates cyclic nucleotide levels and Na+/H+ exchange within human platelets is phospholipase A2. We demonstrate here that membrane cholesterol initiates the activation of phosphatidyl choline phospholipase D and phosphatidic acid thus generated promotes the activation of its phospholipase A2 in the presence of extraplatelet calcium. More important, inhibition of phospholipase D by zinc blocks the activation of phosphatidic acid phospholipase A2 in platelets upon cholesterol incorporation. Our result led us to postulate that membrane cholesterol induced initiation promotion coupling of phospholipases D and A2 in human platelets may be responsible for the hypersensitized state of platelets in hypercholesterolemic patients.
Human peripheral blood lymphocytes express D5 dopamine receptor gene and transcribe the two pseudogenes
Authors: Takahashi Nobuyuki , Nagai Yoshitaka , Ueno Satoshi , Saeki Yoshinaga , Yanagihara Takehiko
Published: July 2002

Sequential reverse transcription and polymerase chain reaction (RT‐PCR) of the mRNA were used to investigate the expression of dopamine receptors in human peripheral blood lymphocytes. The RT‐PCR products contained three types of sequences, each corresponding to those of the D5 dopamine receptor gene and the two related pseudogenes. The lymphocyte cDNA library also contained the clones encoding parts of the three genes. Binding profiles of dopaminergic ligands to the lymphocytes were similar to those for the native neuronal membranes.
Phospholipase A2 affects the activity of fusicoccin receptors
Authors: Aducci Patrizia , Ballio Alessandro , Donini Virgilio , Fogliano Vincenzo , Fullone Maria Rosaria , Marra Mauro
Published: July 2002

Biochemical properties of fusicoccin receptors are strongly influenced by the phospholipid environment. In this report we have studied the effect of different exogenous phospholipases on fusicoccin binding ability of both plasma membrane and solubilised receptors. Among the phospholipases tested only phospholipase A2; showed an inhibitory effect on fusicoccin binding. In particular, the influence of this enzyme on the time course and reversibility of the fusicoccin binding reaction was studied. The inhibitory effect of phospholipase A2 was the consequence of fatty acid release. The usual fatty acids of plasma membrane phospholipids were active in inhibiting the interaction of fusicoccin with its receptors. It is concluded that a phospholipid associated to the fusicoccin receptor might play a significant role in the modulation of binding.
Expression and regulation of the bovine vesicular monoamine transporter gene
Authors: Krejci Eric , Gasnier Bruno , Botton Damien , Isambert Marie-Françoise , Sagné Corinne , Gagnon Jean , Massoulié Jean , Henry Jean-Pierre
Published: July 2002

In monoaminergic cells, the neurotransmitter is accumulated into secretory or synaptic vesicles by a tetrabenazine‐ and reserpine‐sensitive transporter, catalyzing an H+/monoamine antiport. The major vesicular monoamine transporter from bovine chromaffin cells was cloned, using sequences common to adrenal medulla and brain rat vesicular monoamine transporters. Its identity was confirmed by peptide sequences, determined from the purified protein. Surprisingly, the bovine adrenal medulla sequence, bVMAT2, is more related to the transporter from human and rat brain than to that from rat adrenal medulla. PCR amplification showed that bVMAT2, is expressed in both adrenal medulla and brain, in contrast with the situation reported in rats, where distinct genes appear to be expressed in brain (SVAT or MAT, now renamed rVMAT2) and in the adrenal medulla (CGAT, now renamed rVMAT1). In bovine chromaffin cells, long‐term depolarization by KC1 resulted in an increase in the level of bVMAT2 mRNA, in agreement with the previously observed increase in the transporter binding sites, suggesting that a coupling between stimulation, secretion and synthesis changes the composition of the secretory granule membrane.
Evidence for a membrane skeleton in higher plants
Authors: Faraday Christopher D. , Spanswick Roger M.
Published: July 2002

A fraction enriched in plasma membranes was isolated from rice roots by differential centrifugation and aqueous polymer two‐phase partitioning. Analysis of the fraction by SDS‐PAGE showed the presence of several low mobility polypeptides (M
r> 100 kDa). One of these polypeptides (M
r ~ 230 kDa) was specifically recognized by polyclonal antibodies to human erythrocyte spectrin. This finding suggests that a higher plant spectrin‐based membrane skeleton may be preserved and studied using high‐purity plasma membrane fractions obtained by aqueous polymer two‐phase partitioning.
A comparative evaluation of the metabolic profiles of normal and inflammatory knee‐joint synovial fluids by high resolution proton NMR spectroscopy
Authors: Naughton Declan P. , Haywood Rachel , Blake David R. , Edmonds Sally , Hawkes Geoffrey E. , Grootveld Martin
Published: July 2002

High resolution 1H NMR spectroscopy has been employed to investigate the metabolic profile of heatlhy human knee‐joint synovial fluid (SF) and the biochemical data acquired have been compared with those of matched serum, and inflammatory knee‐joint SF samples. Results obtained indicate that the healthy human knee‐joint has a hypoxic status (high lactate level when expressed relative to that of paired serum) that is milder than that of the inflamed human knee‐joint. Moreover, normal SF differs from that of inflammatory SF in that it contains little or no NMR‐detectable lipoprotein‐associated fatty acids and ‘acute‐phase’ glycoproteins, an observation reflecting the limited passage of these macromolecules from plasma into the synovial space in healthy subjects.
Isoprenylation of Rab proteins possessing a C‐terminal CaaX motif
Authors: Joberty Gérard , Tavitian Armand , Zahraoui Ahmed
Published: July 2002

Rab proteins are small GTPases highly related to the yeast Ypti and Sec4 proteins involved in secretion. The Rab proteins were found associated with membranes of different compartments along the secretory and endocytic pathways. They share distinct C‐terminal cysteine motifs required for membrane association. Unlike the other Rab proteins, Rab8, Rab11 and Rab13 terminate with a C‐terminal CaaX motif similar to those of Ras/Rho proteins. This report demonstrates that Rab8 and Rab13 proteins are isoprenylated in vivo and geranylgeranylated in vitro. Rab11 associates in vitro geranylgeranylpyrophosphate and farnesylpyrophosphate. Our study shows that the CaaX motif is required for isoprenylation.
Purification and sequencing of cytochrome b from potato reveals methionine cleavage of a mitochondriallly encoded protein
Authors: Braun Hans-Peter , Schmitz Udo K.
Published: July 2002

Several mitochondrial genes from a large number of different fungi, mammals and plants have been sequenced but little is known about the corresponding translation products. We have affinity purified cytochrome c reductase from potato mitochondria and isolated the mitochondrially encoded cytochrome b protein. Amino‐terminal sequencing reveals that the polypeptide does not start with a methionine. Comparison of the amino acid sequence with the recently published sequence of the gene encoding the cytochrome b apoprotein suggests that the N‐fonnylmetnionine is removed. This result provides the first evidence for the presence of a deformylase and a methionine aminopeptidase in mitochondria.
Transcriptional activation of adrenocortical steroidogenic genes by high potassium or low sodium intake
Authors: Tremblay André , LeHoux Jean-Guy
Published: July 2002

We have previously shown that the long‐term alterations in the intake of sodium and potassium which stimulated aldosterone production in the rat adrenal significantly increased cytochrome P450scc (P450scc) and P450l11β (P45011β) mRNA's and also the mRNA of their electron donor adrenodoxin. In the present study run‐on analyses showed an accumulation of nascent RNA in isolated nuclei of zona glomerulosa cells in K+‐supplemented and Na+‐depleted rats for P450c (5‐ and 6‐fold), 3β‐HSD (3.6‐ and 2.0‐fold) and P45011β (6.0‐ and 6.1‐fold), but not for P450c21 (1.4‐ and 1.1‐fold). In contrast, that of adrenodoxin decreased (0.6‐fold) in high K
+ and remained near control (1.3‐fold) in low Na+ intake. Moderate variations in the rate of transcription of P450scc, P450c21, P45011β and adrenodoxin genes were observed in the zona fasciculata‐reticularis cells of the treated rats. Our results thus demonstrated that positive modulators of aldosterone such as long‐term K+ supplementation and Na+ restriction provoked an increase in transcription of the genes encoding key regulatory steroidogenic enzymes of aldosterone biosynthesis in the zona glomerulosa. The rates of transcription of the genes encoding 3β‐HSD and P450c21, two enzymes catalyzing intermediate steps in the aldosterone pathway, were moderately affected by such treatments. However, according to the known stimulation of adrenodoxin mRNA levels following these treatments, a decreased turnover of the adrenodoxin mRNA rather than initiation of transcription of its gene might be involved in the response to K+ ions, and partially so in the response to Na+ restriction. Finally, the effects of salt‐modified intake were mainly restricted to the zona glomerulosa cells, which are solely responsible for aldosterone production.
Amyloidogenicity of rodent and human βA4 sequences
Authors: Dyrks Thomas , Dyrks Elke , L. Masters Colin , Beyreuther Konrad
Published: July 2002

Previously we have shown that aggregation of the C‐terminal 100 residues (A4CT) of the βA4 amyloid protein precursor (APP) and also of βA4 itself depends on the presence of metal‐catalyzed oxidation systems [T. Dyrks et al. (1988) EMBO J. 7, 949‐957]. We showed that aggregation of the amyloidogenic peptides induced by radical generation systems requires amino acid oxidation and protein cross‐linking. Here we report that aggregation of A4CT and βA4 induced by radical generation systems involves oxidation of histidine, tyrosine and methionine residues. The roden βA4 sequence lacking the single tyrosine and one of the three histidine residues of human βA4 and a βA4 variant in which the tyrosine and the three histidine residues were replaced showed a reduced tendency for aggregation. Thus our results may explain why βA4 amyloid deposits could so far not been detected in the rodent brain.
Inhibition of the epidermal growth factor receptor tyrosine kinase activity by leflunomide
Authors: Mattar Thomas , Kochhar Kulvinder , Bartlett Robert , Bremer Eric G. , Finnegan Alison
Published: July 2002

The active metabolite of leflunomide, A77 1726 inhibits the proliferation of a variety of mammalian cell lines in culture. Epidermal growth factor (EGF)‐dependent proliferation is inhibited by A77 1726 at an effective dose of 30–40 μM. A77 1726 appears to directly inhibit the EGF receptor tyrosine‐specific kinase activity both in intact cells and purified EGF receptors at the same effective dose. These data suggest that leflunomide inhibits cellular proliferation by the inhibition of tyrosine‐specific kinase activities.
Subtypes of inositol 1,4,5‐trisphosphate receptor in human hematopoietic cell lines: Dynamic aspects of their cell‐type specific expression
Authors: Sugiyama Tomoyasu , Yamamoto-Hino Miki , Miyawaki Atsushi , Furuichi Teiichi , Mikoshiba Katsuhiko , Hasegawa Mamoru
Published: July 2002

Inositol 1,4,5‐trisphosphate (IP3)‐mediated Ca2+ signaling plays important roles in cellular responses to extracellular stimuli. We recently succeeded in cloning human counterparts of the three subtypes derived from separate genes. Using the cDNA sequences type‐specific to these subtype receptors, we here analyzed the expression profile of IP3R subtypes in stimulated and unstimulated human hematopoietic cell lines representing T cells, B cells, neutrophils, macrophages, erythrocytes and megakaryocytes. Northern and dot blot analysis showed that each IP3R subtype is expressed differently in these cells and that the expression profile in each cell is dynamically changed upon stimuli which induce differentiation. Moreover, most of these cells were found to simultaneously express at least two different subtype receptors.
Unphosphorylated and tyrosine‐phosphorylated forms of a focal adhesion protein, paxillin, are substrates for calpain II in vitro: Implications for the possible involvement of calpain II in mitosis‐specific degradation of paxillin
Authors: Yamaguchi Ryuji , Maki Masatoshi , Hatanaka Masakazu , Sabe Hisataka
Published: July 2002

Cell‐to‐substratum adhesion becomes weakened during mitosis of the cell cycle in fibroblasts. The level of one focal adhesion protein, paxillin, is greatly reduced in mitotic‐arrested cells. We show here the possible involvement of calpain II, known to be localized in focal adhesion plaques, in the degradation of paxillin. Paxillin is tyrosine‐phosphorylated during interphase of the cell cycle by protein tyrosine kinases (PTK) such as c‐Src and Csk, and becomes dephosphorylated during mitosis. Our data, however, indicate that tyrosine phosphorylation of paxillin does not affect the rate of paxillin degradation by calpain in vitro.
A phosphorothioate oligonucleotide blocks reverse transcription via an antisense mechanism
Authors: Boiziau Claudine , Moreau Serge , Toulmé Jean-Jacques
Published: July 2002

We have studied the inhibition by a phosphorothioate oligodeoxynucleotide (17PScap) of cDNA synthesis performed by either avian or murine reverse transcriptase. Three different mechanisms of inhibition were identified: at low concentrations (⊃100 nM), the cleavage of the RNA template by the retroviral RNase H at the level of the RNA/17PScap duplex accounted for most of the effect, whereas hybrid‐arrested cDNA synthesis by an RNase H‐independent mechanism marginally contributed to the inhibition. Both mechanisms were sequence‐specific. Above 100 nM, the overall cDNA synthesis was reduced in a non‐specific manner.
The GRB2/Sem‐5 adaptor protein
Authors: Downward Julian
Published: July 2002

GRB2/Sem‐5 is a 25‐kDa adaptor protein which contains a central Src homology type 2 (SH2) domain flanked by two Src homology type 3 (SH3) domains. GRB2/Sem‐5 was first identified due to the essential role of the sem‐5 gene product in the vulval induction pathway in Caenorhabditis elegans. The SH2 domain of GRB2/Sem‐5 binds to a number of tyrosine phosphorylated proteins, most notably the epidermal growth factor receptor, the insulin receptor substrate IRS‐1 and another putative adaptor protein, She. The SH3 domains bind to Sos, a guanine nucleotide exchange factor for Ras proteins. GRB2/Sem‐5 brings together Sos and tyrosine phosphoproteins into a complex and thereby may regulate the nucleotide exchange rate of Ras and hence its activation state.
Binding site‐shaped repeated sequences of bacterial wall peptidoglycan hydrolases
Authors: Ghuysen Jean-Marie , Lamotte-Brasseur Josette , Joris Bernard , Shockman Gerald D.
Published: July 2002

The non‐catalytic C‐terminal regions of the N‐acetylmuramidase (lysozyme) of Clostridium acetobutylicum and N‐acetylmuramoyl(d‐lactyl)‐l‐alanine amidases CwlA of Bacillus subtilis, ORFL3 and CwlL of Bacillus licheniformis were previously reported to have similarities with the amino acid sequence of the non‐catalytic N‐terminal module of the Streptomyces albus G Zn DD‐peptidase. This peptidase is a bipartite protein of known three‐dimensional structure. Its non‐catalytic N‐terminal module possesses, exposed at the surface, an elongated crevice which is defined by a loop‐helix‐loop‐helix motif that consists of two repeats, each 16 amino acid residues long, connected by a heptapeptide and whose design is compatible with its possible functioning as a substrate recognition and binding site. Amino acid alignments suggest that cavities nearly identical in shape to that present in the non‐catalytic module of the S. albus peptidase, are borne by the C‐terminal regions of the CwlA amidase (in one copy), the lysozyme and the ORFL3 and CwlL amidases (in two copies). Since a common feature of the five enzymes is their substrate, the bacterial cell wall peptidoglycan, we interpret the striking similarity of their non‐catalytic N‐ or C‐terminal modules to suggest that these modules are involved in the binding of these exocellular enzymes to their insoluble wall substrate.
Conformational rearrangements required of the V3 loop of HIV‐1 gp120 for proteolytic cleavage and infection
Authors: Johnson Michael E. , Lin Zhaolan , Padmanabhan Kaillathe , Tulinsky Alexander , Kahn Michael
Published: July 2002

HIV gp120 is specifically cleaved at a single site in the V3 loop between Arg315 and Ala316 by thrombin. Previous observations by others have indicated that binding to CD4 enhances the rate of V3 loop cleavage, and that this cleavage is a prerequisite for HIV infection. Other observations also suggest that the cleavage site is in a type II β‐turn centered at Pro313‐Gly314. However, our docking experiments indicate that this conformation cannot dock to thrombin and other trypsin‐like serine proteases. Thus, based on the thrombin‐bound conformation of peptide substrates, we propose that CD4 binding, at a site remote from the V3 loop, induces and stabilizes a trans to cis isomerization of the highly conserved residue Pro313, and that this conformational shift is a prerequisite for cleavage by a ‘thrombin‐like’ cellular pro tease and subsequent infection.
Structural rearrangements on HIV‐1 Tat (32–72) TAR complex formation
Authors: Metzger Armin U , Schindler Thomas , Willbold Dieter , Kraft Margot , Steegborn Clemens , Volkmann Andrea , Frank Rainer W , Rösch Paul
Published: July 2002

Expression of the early genes of the human immunodeficiency virus type‐1 (HIV‐1) genome is under the control of a trans‐activator (Tat) protein. HIV‐1 Tat action requires binding to TAR (trans‐activation responsive element), an RNA sequence located at the 5′‐end of all lentiviral mRNAs. We used various spectroscopic methods to investigate conformational changes on HIV‐1 TAR binding to the HIV‐1 (32–72) Tat peptide BP1. It comprises the RNA binding region and binds specifically to TAR. We conclude from our experiments that the regular A‐form of the TAR RNA is slightly distorted towards the B‐form when bound to BP1. Thus, the major groove is widened and the binding of BP1 facilitated. BP1 presumably adopts an extended conformation when binding to TAR and may fit well into the TAR major groove.
Molecular genetic strategies in Toxoplasma gondii: close in on a successful invader
Authors: Soldati Dominique
Published: July 2002

Toxoplasma gondii is an obligate intracellular parasite with an exceptional ability to invaDA, survive and replicate within nearly all nucleated cells. Upon differentation into an encysted form (bradyzoites), the parasites escape the host immune DAfenses and thus persist long enough in man and other hosts to ensure maintenance of transmission. This protozoan parasite has long been known to cause severe congenital infections in humans and animals but has recently gained additional notoriety as an opportunistic pathogen associated with AIDS. DAvelopment of a DNA transfection system for T. gondii has proviDAd a new tool for exploring molecular aspects of important processes such as invasion and differentiation. Additional strategies associated with genetic transfomation have been DAvised and elaboration of even more DAsirable molecular tools is in progress.
The aromatic domain 66YWYWW70 of subunit VIII of the yeast ubiquinol‐cytochrome c oxidoreductase is important for both assembly and activity of the enzyme
Authors: Lobo-Hajdu Gisele , van Gaalen Monique , Grivell Leslie A , Berden Jan A
Published: July 2002

The aromatic character of the region 66YWYWW70 of the 11‐kDa subunit VIII of ubiquinol‐cytochrome c oxidoreductase (bc
1 complex) of the yeast Saccharomyces cerevisiae has previously been demonstrated to be important for assembly of a functional complex [Hemrika et al. (1994) FEBS Lett. 344, 15–19]. Especially the aromatic nature of residue 66 appeared to be relevant, as the very low level (5%) of bc
1 complex in the mutant 66SASAA70 was restored to nearly 70% of the wild‐type level in a phenotypic revertant with the sequence 66FASAA70. In the present study, three other site‐directed mutants (66SAYAA70, 66SASAW70 and 66SWYWW70) were constructed and analysed. The data indicate that the presence of one aromatic residue is enough for a substantial level of assembly and that its position modulates the level of both assembly and electron transfer activity. The results also confirm the relevance of this region of subunit VIII for the formation of the Q
out
reaction domain, as reported by Hemrika et al. [(1993) Eur. J. Biochem. 215, 601–609]. It is further shown that the lowered specific activity of the mutant described by these authors is not due to the introduction of a cysteine in the sequence of subunit VIII.
Protonation changes during the photocycle of sensory rhodopsin II from Natronobacterium pharaonis
Authors: Engelhard M , Scharf B , Siebert F
Published: July 2002

The Fourier Transform Infrared (FTIR) spectra of photocycle intermediates of sensory rhodopsin II (pSRII) from Natronobacterium pharaonis were measured. The results of the FTIR experiments indicate considerable conformational movements of pSRII already at the stage of the early K‐like intermediate which persist at least during the lifetime of the long lived intermediate. These changes in the amide bond region are more intense than those described for sensory rhodopsin I (SRI) and are quite similar to those observed for rhodopsin. Concomitantly with the deprotonation of the Schiff base a carboxyl group located in a hydrophobic environment is protonated. In analogy to bacteriorhodopsin, this carboxyl group might arise from Asp‐75 which probably serves as counter ion to the Schiff base. The protonation reaction differs from the situation observed in SRI where the protonation is pH independent over the range of pH 5–8.
Diagnosis and management of malignant germ cell ovarian tumors in young females
Authors: Zalel Y. , Piura B. , Elchalal U. , Czernobilsky B. , Antebi S. , Dgani R.
Published: July 2002

Germ cell tumors (GCT) compose most of the preadolescent malignant ovarian tumors; dysgerminoma being the most common (48%), followed by endodermal sinus tumor, immature teratoma, mixed GCT and embryonal carcinoma. The percentage of malignant epithelial ovarian tumors rises with increasing age, while that of the GCT tumors declines. Of all tumor markers discussed, only AFP ang hCG are being routinely monitored. Their most effective use is in monitoring response to therapy and detecting recurrences early. The current therapeutic regimens are presented, among them bleomycin, etoposide and platinol (BEP) and other new regimens; their influence on the patients' fertility is discussed. Further improvement in the prognosis of these young patients will hopefully follow development of new surgical and chemotherapeutic approaches.
A POTENT INHIBITOR OF NEURITE OUTGROWTH THAT PREDOMINATES IN THE EXTRACELLULAR MATRIX OF REACTIVE ASTROCYTES
Authors: Canning David R. , Höke Ahmet , Malemud Charles J. , Silver Jerry
Published: July 2002

In a model of astrogliosis in vitro, cultured cortical astrocytes were triggered into a functionally reactive state by an immobilized fragment of the β‐amyloid peptide. Induced astrocytes produced an extracellular matrix that inhibited the outgrowth of embryonic CNS axons. Within the extracellular matrix deposited by reactive astrocytes, we found an overall increase in the deposition of chondroitin sulphate that accounted for the inhibition. Specifically, we have detected an increased biosynthesis of a small chondroitin/heparan sulphate proteoglycan that is a potent inhibitor of axon outgrowth. We further suggest that this proteoglycan, or related molecules yet to be discovered, may play a role in gliosis‐mediated regenerative failure of CNS axons.
Molecular cloning of fresh water and deep‐sea rod opsin genes from Japanese eel Anguilla japonica and expressional analyses during sexual maturation1
Authors: Zhang Huan , Futami Kunihiko , Horie Noriyuki , Okamura Akihiro , Utoh Tomoko , Mikawa Naomi , Yamada Yoshiaki , Tanaka Satoru , Okamoto Nobuaki
Published: July 2002

We have determined the complete cDNA sequences of fresh water rod opsin gene (fwo) and deep‐sea rod opsin gene (dso) from Japanese eel Anguilla japonica. The cDNA clones of fwo and dso consisted of 1437 and 1497 nucleotides, respectively. The predicted opsins of both genes consisted of 352 amino acid residues. Southern blot and PCR analyses of genomic DNA indicated that the Japanese eel genome contains only one fwo and one dso and they are intronless. Quantitative RT‐PCR analyses revealed that the expression of fwo decreases with sexual maturation while that of dso increases.
Homocysteine accelerates endothelial cell senescence
Authors: Xu Dong , Neville Richard , Finkel Toren
Published: July 2002

In this study we demonstrate that exposure of cultured endothelial cells to homocysteine significantly accelerates the rate of endothelial senescence. Examination of telomere length demonstrates that homocysteine increases the amount of telomere length lost per population doubling. The effects of homocysteine on both senescence and telomere length are inhibited by treatment with the peroxide scavenger catalase. Chronic exposure of endothelial cells to homocysteine also increases the expression of two surface molecules linked to vascular disease, intracellular adhesion molecule‐1 (ICAM‐1) and plasminogen activator inhibitor‐1 (PAI‐1). Interestingly, the level of expression of both ICAM‐1 and PAI‐1 correlates with the degree of endothelial senescence. Taken together, these results suggest that homocysteine accelerates the rate of cellular senescence through a redox‐dependent pathway. In addition, it suggests that chronic oxidative stress in the vessel wall may hasten the rate of senescence and that the senescent endothelial cell may in turn be pro‐atherogenic.
Detection of superoxide anion using an isotopically labeled nitrone spin trap: potential biological applications
Authors: Zhang Hao , Joseph Joy , Vasquez-Vivar Jeannette , Karoui Hakim , Nsanzumuhire Cline , Martásek Pavel , Tordo Paul , Kalyanaraman B
Published: July 2002

We describe the synthesis and biological applications of a novel nitrogen‐15‐labeled nitrone spin trap, 5‐ethoxycarbonyl‐5‐methyl‐1‐pyrroline N‐oxide ([15N]EMPO) for detecting superoxide anion. Superoxide anion generated in xanthine/xanthine oxidase (100 nM min−1) and NADPH/calcium‐calmodulin/nitric oxide synthase systems was readily detected using EMPO, a nitrone analog of 5,5′‐dimethyl‐1‐pyrroline N‐oxide (DMPO). Unlike DMPO‐superoxide adduct (DMPO–OOH), the superoxide adduct of EMPO (EMPO–OOH) does not spontaneously decay to the corresponding hydroxyl adduct, making spectral interpretation less confounding. Although the superoxide adduct of 5‐(diethoxyphosphoryl)‐5‐methyl‐pyrroline N‐oxide is more persistent than EMPO–OOH, the electron spin resonance spectra of [14N]EMPO–OOH and [15N]EMPO–OOH are less complex and easier to interpret. Potential uses of [15N]EMPO in elucidating the mechanism of superoxide formation from nitric oxide synthases, and in ischemia/reperfusion injury are discussed.
Genome‐wide protein interaction maps using two‐hybrid systems
Authors: Legrain Pierre , Selig Luc
Published: July 2002

Automated sequence technology has rendered functional biology amenable to genomic scale analysis. Among genome‐wide exploratory approaches, the two‐hybrid system in yeast (Y2H) has outranked other techniques because it is the system of choice to detect protein–protein interactions. Deciphering the cascade of binding events in a whole cell helps define signal transduction and metabolic pathways or enzymatic complexes. The function of proteins is eventually attributed through whole cell protein interaction maps where totally unknown proteins are partnered with fully annotated proteins belonging to the same functional category. Since its first description in the late 1980's, several versions of the Y2H have been developed in order to overcome the major limitations of the system, namely false positives and false negatives. Optimized versions have been recently applied at multi‐molecular and genomic scale. These genome‐wide surveys can be methodologically divided into two types of approaches: one either tests combinations of predefined polypeptides (the so‐called matrix approach) using various short‐cuts to speed up the process, or one screens with a given polypeptide (bait) for potential partners (preys) present in complex libraries of genomic or complementary DNA (library screening). In the former strategy, one tests what one knows, for example pair‐wise interactions between full‐length open reading frames from recently sequenced and annotated genomes. Although based on a one‐by‐one scheme, this method is reported to be amenable to large‐scale genomics thanks to multicloning strategies and to the use of small robotics workstations. In the latter, highly complex cDNA or genomic libraries of protein domains can be screened to saturation with high‐throughput screening systems allowing the discovery of yet unidentified proteins. Both approaches have strengths and drawbacks that will be discussed here. None yields a full proteome‐wide screening since certain proteins (e.g. some transcription factors) are not usable in Y2H. Novel two‐hybrid assays have been recently described in bacteria. Applications of these time‐ and cost‐effective assays to genomic screening will be discussed and compared to the Y2H technology.
Haemozoin formation in the midgut of the blood‐sucking insect Rhodnius prolixus
Authors: Oliveira Marcus F. , Silva José R. , Dansa-Petretski Marı́lvia , de Souza Wanderley , Braga Cláudia M.S. , Masuda Hatisaburo , Oliveira Pedro L.
Published: July 2002

Malaria parasites digest haemoglobin and detoxify the free haem by its sequestration into an insoluble dark‐brown pigment known as haemozoin (Hz). Until recently, this pigment could be found only in Plasmodium parasites. However, we have shown that Hz is also present in the midgut of the blood‐sucking insect Rhodnius prolixus [Oliveira et al. (1999) Nature 400, 517–518]. Here we show that Hz synthesis in the midgut of this insect is promoted by a particulate fraction from intestine lumen. Haem aggregation activity is heat‐labile and is inhibited in vitro by chloroquine (CLQ). Inhibition of Hz formation in vivo by feeding insects with CLQ leads to increased levels of haem in the haemolymph of the insect, which resulted in increased lipid peroxidation. Taken together, these results indicate that a factor capable of promoting Hz crystallisation is present in R. prolixus midgut and that this activity represents an important physiological defence of this insect against haem toxicity.
Activin A increases Pax4 gene expression in pancreatic β cell lines
Authors: Ueda Yoshitaka
Published: July 2002

Activin A, a member of the TGFβ superfamily, has many physiological and developmental functions. In pancreatic β cell cultures, activin promotes cell differentiation and insulin production. The author has found activin increases gene expression of the PAX4, one of the major transcription factors determining pancreatic β cell differentiation. This effect was mediated, at least in part, by the type IB activin receptor (ALK4). Moreover, the activity of human insulin promoter–reporter system was controlled by PAX4 and its isoform PAX4 Δ(G239–P251) in a unique fashion; positively by low concentrations, and negatively by high concentrations of these proteins. And the repression activities were different between these proteins. These findings confirm the importance of activin signal transduction in pancreatic β cell development and function.
Upregulation of vacuolar H+‐translocating pyrophosphatase by phosphate starvation of Brassica napus (rapeseed) suspension cell cultures
Authors: Palma David A. , Blumwald Eduardo , Plaxton William C.
Published: July 2002

The influence of phosphate (Pi) deprivation on the vacuolar H+‐translocating pyrophosphatase (PPiase) and ATPase in tonoplast vesicles from Brassica napus suspension cells was assessed. Pi starvation significantly elevated the ratios of PPi‐:ATP‐dependent H+ translocation rate and H+‐PPiase:H+‐ATPase hydrolytic activities. These increases were reversed 36 h following resupply of 2.5 mM Pi to the Pi‐starved cells. Immunoblotting indicated that Pi starvation also induced a two‐fold increase in the amount of H+‐PPiase protein, whereas the amount of H+‐ATPase remained unchanged. It is proposed that H+‐PPiase facilitates the conservation of limited ATP pools, and Pi recycling during Pi stress.
The AMP‐activated protein kinase prevents ceramide synthesis de novo and apoptosis in astrocytes
Authors: Blázquez Cristina , Geelen Math J.H , Velasco Guillermo , Guzmán Manuel
Published: July 2002

Fatty acids induce apoptosis in primary astrocytes by enhancing ceramide synthesis de novo. The possible role of the AMP‐activated protein kinase (AMPK) in the control of apoptosis was studied in this model. Long‐term stimulation of AMPK with 5‐aminoimidazole‐4‐carboxamide ribonucleoside (AICAR) prevented apoptosis. AICAR blunted fatty acid‐mediated induction of serine palmitoyltransferase and ceramide synthesis de novo, without affecting fatty acid synthesis and oxidation. Prevention of ceramide accumulation by AICAR led to a concomitant blockade of the Raf‐1/extracellular signal‐regulated kinase cascade, which selectively mediates fatty acid‐induced apoptosis. Data indicate that AMPK may protect cells from apoptosis induced by stress stimuli.