bluuebunny/crossref_metadata_2025 huggingface dataset:
Does microsomal glycerophosphate acyltransferase also catalyze the acylation of dihydroxyacetone phosphate?
Authors: Datta Nabanita S. , Hajra Amiya K.
Published: July 2002

Rat liver microsomal dihydroxyacetone phosphate acyltransferase, in contrast to the glycerophosphate acyltransferase, was found to be active at low pH (5.5), stable towards heat (55 °C, 15 min) and trypsin (in the absence of detergents) and was not inhibited by high concentrations of N‐ethyl maleimide. Dihydroxyacetone phosphate acyltransferase is only slightly and non‐competitively inhibited by sn‐glycerol‐3‐phosphate whereas glycerophosphate acyltransferase is strongly inhibited by dihydroxyacetone phosphate in a competitive manner. Kinetic analysis indicates that this competitive inhibition is not due to the competition of two common substrates for the same active center of one enzyme. These results demonstrate that microsomal glycerophosphate acyltransferase and dihydroxyacetone phosphate acyltransferase are two distinct and separate enzymes.
Kinetics of the reaction of intraerythrocytic haemoglobin by single cell microspectroscopy: effect of shape and osmolarity
Authors: Coletta Massimo , Giardina Bruno , Amiconi Gino , Gualtieri Paolo , Benedetti Pier A. , Brunori Maurizio
Published: July 2002

Microspectroscopy
Camel hemoglobin
Red blood cell
Diffusion
Osmolarity
Cell volume
Amino acid sequence of vitamin D‐dependent calcium‐binding protein from bovine cerebellum
Authors: Takagi Takashi , Nojiri Masanobu , Konishi Kazuhiko , Maruyama Kei , Nonomura Yoshiaki
Published: July 2002

The amino acid sequence of vitamin D‐dependent calcium‐binding protein from bovine cerebellum has been determined. It is composed of 260 amino acid residues and its N‐terminus is acetylated. The molecular mass is calculated to be 29 851 Da. The presence of six calcium‐binding sites (I–VI) has been proposed, two of them (sites II and VI) have lost their calcium‐binding function through amino acid replacements, and the other four are able to bind calcium. six calcium‐binding domains are supposed to be derived from two gene duplications of the two ancestral calcium‐binding domains. In comparison with the sequence of chick intestinal calcium‐binding protein deduced from a cDNA sequence [(1985) Nucleic Acids Res. 13, 8867‐8881], the bovine calcium‐binding protein is two amino acid residues shorter at the N‐terminus and the other parts show 78.5% identity.
Simultaneous separation of all lens crystallin subunits by chromatofocusing
Authors: Body P. , Bloemendal H.
Published: July 2002

The total population of water‐soluble lens crystallin subunits has been separated in one run on a single chromatofocusing column. Dissociation of the polymeric α‐ and β‐crystallin subunits occurred simultaneously on the column. Two components not described previously in the literature have been found in the γ‐crystallin region.
Ba2+‐sensitive K+ channels in luminal‐membrane vesicles from pars convoluta of rabbit proximal tubule
Authors: Jacobsen Christian , Røigaard-Petersen Hans , Sheikh M.I.
Published: July 2002

This paper describes properties of 86Rb+ fluxes through a novel K+ channel in luminal‐membrane vesicles isolated from pars convoluta of rabbit proximal tubule. The uptake of 86Rb+ into potassium salt loaded vesicles was specifically inhibited by Ba2+. The isotope accumulation is driven by an electrical diffusion potential as shown in experiments using these membrane vesicles loaded with anions of different membrane permeability and was as follows: gluconate > SO2−
4 > Cl−. Furthermore, the vesicles containing the channels show a cation selectivity with the order K+ > Rb+ > Li+ > Na+ = choline+.
Transcriptional activity is inducible in the cauliflower mosaic virus 35 S promoter engineered with the heat shock consensus sequence
Authors: Pietrzak Maciej , Burri Markus , Herrero Juan-Jose , Mosbach Klaus
Published: July 2002

Transcriptional activity of a strong constitutive plant promoter, the cauliflower mosaic virus (CaMV) 35 S promoter, could be modulated by addition of multiple synthetic oligonucleotides, carrying the heat shock promoter consensus sequence. The activity of this modified promoter increased up to 3‐fold under heat shock conditions. A similar construction using enhancerless 35 S promoter resulted in an engineered promoter, which showed an increased activity under both normal and heat conditions, without a significant induction by the heat shock. We did not observe a silencer effect of the heat shock elements in combination with either complete or enhancerless 35 S promoter, using transient expression assay to measure the transcription activity.
Electron paramagnetic resonance evidence that cellular oxygen toxicity is caused by the generation of superoxide and hydroxyl free radicals
Authors: Zweier Jay L. , Duke Scherer S. , Kuppusamy Periannan , Sylvester J.T. , Gabrielson Edward W.
Published: July 2002

Cells require molecular oxygen for the generation of energy through mitochondrial oxidative phosphorylation; however, high concentrations of oxygen are toxic and can cause cell death. A number of different mechanisms have been proposed to cause cellular oxygen toxicity. One hypothesis is that reactive oxygen free radicals may be generated; however free radical generation in hyperoxic cells has never been directly measured and the mechanism of this radical generation is unknown. In order to determine if cellular oxygen toxicity is free radical mediated, we applied electron paramagnetic resonance, EPR, spectroscopy using the spin trap 5,5′‐dimethyl‐1‐pyrroline‐N‐oxide, DMPO, to measure free radical generation in hyperoxic pulmonary endothelial cells. Cells in air did not give rise to any detectable signal. However, cells exposed to 100% O2 for 30 min exhibited a prominent signal of trapped hydroxyl radical, DMPO‐OH, while cell free buffer did not give rise to any detectable radical generation. This cellular radical generation was demonstrated to be derived from the superoxide radical since the observed signal was totally quenched by superoxide dismutase, but not by equal concentrations of the denatured enzyme. It was confirmed that the hydroxyl radical was generated since in the presence of ethanol the CH3·CH(OH) radical was formed. Loss of cell viability as measured by uptake of trypan blue dye was observed paralleling the measured free radical generation. Thus, superoxide and hydroxyl radicals are generated in hyperoxic pulmonary endothelial cells and this appears to be an important mechanism of cellular oxygen toxicity.
N‐acetyldopamine quinone methide/1,2‐dehydro‐N‐acetyl dopamine tautomerase
Authors: Saul Steven J. , Sugumaran Manickam
Published: July 2002

The enzyme system causing the side chain desaturation of the sclerotizing precursor, N‐acetyldopamine (NADA), was solubilized from the larval cuticle of Sarcophaga bullata and resolved into three components. The first enzyme, phenoloxidase, catalyzed conversion of NADA to NADA quinone and provided it for the second enzyme (NADA quinone isomerase), which makes the highly unstable NADA quinone methide. Quinone methide was hydrated rapidly and nonenzymatically to form N‐acetylnorepinephrine. In addition, it also served as the substrate for the last enzyme, quinone methide tautomerase, which converted it to 1,2‐dehydro‐NADA. Reconstitution of NADA side chain desaturase activity was achieved by mixing the last enzyme fraction with NADA quinone isomerase, obtained from the hemolymph of the same organism, and mushroom tyrosinase. Therefore, NADA side chain desaturation observed in insects is caused by the combined action of three enzymes rather than the action of a single specific NADA desaturase, as previously thought.
Glycolysis compared in intact, permeabilized and sonicated L‐929 cells
Authors: Jackson Susan A. , Thomson Marilyn J. , Clegg James S.
Published: July 2002

The effects of incubation time and cell density on glycolytic rate were examined in suspensions of intact, permeabilized and sonicated L‐929 cells. Sonicates exhibited strong dependence on cell density and a distinct lag in glycolytic rate, while intact cells showed no cell density dependence and linear glycolytic rates. Permeabilized cells exhibited linear glycolytic rates, but sometimes showed dependence on cell density. Rates of lactate production (nmol at 30 min/106 cells) were highest in sonicates and lowest in intact cells. These results are interpreted as support for the previously proposed hypothesis that enzymes of the glycolytic pathway are highly organized in intact L‐929 cells.
Novel pathways of oleic and cis‐vaccenic acid biosynthesis by an enzymatic double‐bond shifting reaction in higher plants
Authors: Shibahara Akira , Yamamoto Kohei , Takeoka Mariko , Kinoshita Akemi , Kajimoto Goro , Nakayama Takao , Noda Manjiro
Published: July 2002

The novel pathways of oleic acid formation from cis‐vaccenic (cis‐11‐octadecenoic) acid and of cis‐vaccenic acid formation from oleic acid by enzymatic positional isomerization have been proposed in higher plants, based on stable‐isotope experiments using[2,2‐2H2]cis‐vaccenate or [2,2‐2H2]oleate as an immediate precursor. A pulp homogenate and also pulp slices prepared from developing kaki (Diospyros kaki) fruit could catalyze these hitherto unknown isomerizations. This suggests the presence of a new type of isomerase responsible for the double‐bond shifting reaction without cis‐trans isomerization in the middle of fatty acid carbon chains.
Differential changes in lipid metabolism ofmyeloid and lymphoid cell lines induced by treatment with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)
Authors: Ponzoni Mirco , DeBell Karen E. , Huffman Thomas , Bonvini Ezio
Published: July 2002

Treatment of the myeloid cell lines, U‐937 or HL‐60, with 10 nM of the phorbol ester, 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), for 24 h increased the rate of incorporation of [3H] glycerol into total chloroform extracts. A proportionally greater labeling of the non‐polar lipid (NL) fraction compared to the polar, phospholipid (PL), fraction was observed. Chromatographic analysis showed a 6‐fold increase in the labeling of triacylglycerols (TAG), a 2‐fold increase in diacylglycerols, and no changes in monoacylglycerols. PL labeling showed a 3‐fold increase in phosphatidylcholine (PC). The effect of TPA on TAG labeling was selectively observed in myeloid cell lines. No such a change was found in the lymphoid cell line, MOLT‐3, which did respond to TPA with increased PC labeling. Incorporation of [3H]arachidonic acid (AA) into TAG by U‐937 cells was selectively increased (2.5‐fold) after treatment with TPA for 24 h. Treatment of U‐937 cells with TPA in serum‐free medium resulted in no increased labeling of TAG. These studies suggest that changes in TAG metabolism may be characteristic of myeloid differentiation and depend on the presence of serum factor(s).
Regulation of the Na+/H+ exchanger under conditions of abolished proton gradient: Isosmotic and hyperosmotic stimulation
Authors: Dascalu Avi , Nevo Zvi , Korenstein Rafi
Published: July 2002

Activation of the Na+/H+ exchanger following isosmotic and hyperosmotic stimuli was investigated in an osteoblast cell line (RCJ 1.20). The pH dependence of the transporter activity was studied under conditions of abolished proton gradient (pHi = PH*) across the membrane. The isotonic response is Na+* dependent, increases towards higher pH. values, displaying a sigmoidal dependence on pH1** (Hill coefficient ≈ 1.8) and is controlled by pH*. The greater than first order dependence of pH suggest that H+* inhibits the exchange beyond the rate expected from competition with the Na+* alone. This may be due to the existence of an external H+ regulatory site with a negative cooperative effect on the intra‐ or extracellular transport site. The hyperosmotic activation is Na+* independent, parallels the sigmoidal pH dependence of the isosmotic stimulus (Hill coefficient ≈ 2.0) and is mediated through an increase of the Vmax
without a change in the intracellular proton sensitivity.
The domain structure of transketolase from baker's yeast
Authors: Tikhomirova N.K. , Tsuprun V.L. , Kochetov G.A.
Published: July 2002

Transketolase from baker's yeast was shown by electron microscopy to have the form of a rectilinear tetrahedron and to consist of 4 structural units with a molecular mass of 30–40 kDa.
A mouse brain cDNA encodes a novel protein with the protein kinase C phosphorylation site domain common to MARCKS
Authors: Umekage Tadashi , Kato Kikuya
Published: July 2002

We have isolated a mouse brain cDNA clone encoding a protein of 200 amino acids (M
r, 20165) with partial homology with MARCKS (myristoylated alanine‐rich C‐kinase substrate). Two regions show similarity with MARCKS, one is the kinase C phosphorylation site domain which is supposed to bind calmodulin, and the other is the region near to the N‐terminus, including the consensus sequence of myristoylation. It has a similar amino acid composition to MARCKS, but the content of alanine is not as high. It is distributed throughout the mouse brain, but the pattern is not identical with that of MARCKS. Both proteins may be members of a new protein family involved in coupling the protein kinase C and calmodulin signal transduction systems.
Evidence for an NO‐rebound, mechanism for production of N2O from nitrite by the copper‐containing nitrite reductase from Achromobacter cycloclastes
Authors: Jackson M.A. , Tiedje J.M. , Averill B.A.
Published: July 2002

Reduction of NO−
2 by the Cu‐containing nitrite reductase from Achromobacter cycloclastes produces NO as the primary product initially, but as NO accumulates, NO production levels‐off and N2O production becomes significant. Reaction of the enzyme with NO−
2 in the presence of NO increases the amount of N2O product significantly, while trapping the NO product as nitrosylhemoglobin or rapid, removal of NO by sparging results in no detectable N2O production. Reaction of the enzyme with 15NO−
2 in the presence of 14NO results in rapid formation of the mixed isotope product (14N, 15N)O in ca. 45% yield. In contrast, the presence or absence of NO has no effect on N2O production by a prototypical heme cd
1‐containing nitrite reductase. These results are consistent with formation of a labile Cu+−NO+ species in the copper enzyme, which normally decomposes to NO. Production of N2O requires that the released NO must rebind to the enzyme to combine with a second NO−
2 or a species derived therefrom.
Heat shock increases turnover of 90 kDa heat shock protein phosphate groups in HeLa cells
Authors: Legagneux Vincent , Morange Michel , Bensaude Olivier
Published: July 2002

The 90 kDa heat shock protein (hsp9O) is a major phosphoprotein which associates various other cellular polypeptides such as actin, calmodulin, steroid hormone receptors and certain protein‐kinases. Little is known about the function of hsp9O in recovery from stress. In this report, we describe a dramatic increase in the rate of both phosphate uptake and dephosphorylation of hsp9O in HeLa cells submitted to acute stresses. This increased turnover of hsp9O phosphate groups might reflect a greater protein binding activity of hsp9O in stressed cells.
cDNA clones encoding leucine‐zipper proteins which interact with G‐CSF gene promoter element 1‐binding protein
Authors: Nishizawa Mikio , Nagata Shigekazu
Published: July 2002

The gene expression of granulocyte colony‐stimulating factor (G‐CSF) is induced by lipopolysaccharide (LPS). GPE1, a cis‐controlling element of the G‐CSF gene, functions as an LPS‐responsive element. GPE1‐binding protein (GPE1‐BP), a leucine‐zipper protein, did not independently activate G‐CSF gene expression. Protein blot analysis with biotinylated GPE1‐BP revealed that there were nuclear proteins that interact specifically with GPE1‐BP. Three leucine‐zipper proteins were isolated from mouse cDNA expression libraries by this method: NF‐IL6, ATF4, and a novel ATF4‐related ATFx. The interactions of these proteins with GPE1‐BP may play key roles in G‐CSF gene expression.
Purification of the 28.5 kDa cytosolic protein involved in the activation of NADPH oxidase from guinea pig neutrophils
Authors: Someya Akimasa , Yomogida Shin , Nagaoka Isao , Iwabuchi Kazuhisa , Yamashita Tatsuhisa
Published: July 2002

We tried to purify a new protein component required for the activation of NADPH oxidase from the guinea pig neutrophil cytosolic fraction which did not contain p47phox and p67phox, using HAC‐5CP, IEC‐QA and Superose 12HR columns. The NADPH oxidase‐activating activity was separated into three fractions on IEC‐QA anion‐exchange HPLC. However, when each of the fractions was purified by Superose 12HR gel filtration, the active fraction eluted at the same position, and was found to contain a common protein with a molecular weight of 28.5 kDa on SDS‐PAGE. These results suggest that the 28.5 kDa protein is a novel NADPH oxidase activating protein.
Latrotoxin‐like properties of a protein from brain
Authors: Zhukareva V.A. , Shatursky O.Ya. , Lishko V.K. , Malysheva M.K. , Terletskaya Ya.T. , Tsygankova O.M. , Grishin E.V.
Published: July 2002

In bovine brain cortex cytoplasm we have identified a soluble protein (L‐protein) of M
r ∼90 kDa interacting with polyclonal antibodies to α‐latrotoxin. The L‐protein forms potential‐dependent and cation‐selective ion channels in BLM, which are blocked by Cd2+. The fusogenic activity of the L‐protein was demonstrated on liposomes. We have arrived at the conclusion that the action mechanisms of the L‐protein and α‐latrotoxin are similar.
A DNA fragment homologous to F1‐ATPase β subunit was amplified from genomic DNA of Methanosarcina barkeri Indication of an archaebacterial F‐type ATPase
Authors: Sumi Masato , Sato Masa H. , Denda Kimitoshi , Date Takayasu , Yoshida Masasuke
Published: July 2002

A 490 bp DNA fragment was amplified from Methanosarcina barkeri genomic DNA by the polymerase chain reaction (PCR) using oligonucleotide primers designed based on conserved amino acid sequences of the F1‐ATPase β subunits. The amino acid sequence deduccd from the DNA sequence of this fragment was highly homologous to a portion of the F1‐ATPase β subunit. This indicates that this archaebacterium has a gene of F‐type ATPase in addition to a gene of V‐type ATPase.
One of the chloroplast envelope ion channels is probably related to the mitochondrial VDAC
Authors: Pottosin Igor I.
Published: July 2002

The voltage dependence of large conductance channels in the intact chloroplast envelope of Nitellopsis obtusa was examined using the patch‐clamp technique. The channel switched to the lower conducting substates with amplitudes of around 45 and 20% of that in the open state when potentials larger than 30 mV were applied. The steepness of the voltage dependence approximately corresponds to 4 elementary charges being transferred across the entire voltage drop to close the channel both at positive and negative potentials. The transition to the closed substates could also be induced by König's polyanion, a well known modulator of the mitochondrial outer membrane channel.
1 kb of the lactase‐phlorizin hydrolase promoter directs post‐weaning decline and small intestinal‐specific expression in transgenic mice
Authors: Troelsen Jesper T. , Mehlum Anja , Olsen Jørgen , Spodsberg Nikolaj , Hansen Gert H. , Prydz Hans , Norén Ove , Sjöström Hans
Published: July 2002

Adult‐type hypolactasia is a genetic condition making approximately one half of the human population intolerant to milk because of abdominal symptoms. The cause is a post‐weaning down‐regulation of the intestinal‐specific enzyme lactase‐phlorizin hydrolase (LPH) reducing the intestinal capacity to hydrolyze lactose. We here demonstrate that the stretch −17 to −994 in the pig LPH‐promoter carries cis‐elements which direct a small intestinal‐specific expression and a post‐weaning decline of a linked rabbit β‐globin gene. These data demonstrate that the post‐weaning decline of LPH is mainly due to a transcriptional down‐regulation.
2‐Thiouracil is a selective inhibitor of neuronal nitric oxide synthase antagonising tetrahydrobiopterin‐dependent enzyme activation and dimerisation
Authors: Palumbo Anna , d'Ischia Marco , Cioffi Fernando A
Published: July 2002

2‐Thiouracil (TU), an established antithyroid drug and melanoma‐seeker, was found to selectively inhibit neuronal nitric oxide synthase (nNOS) in a competitive manner (K
i=20 μM), being inactive on the other NOS isoforms. The drug apparently interfered with the substrate‐ and tetrahydrobiopterin (BH4)‐binding to the enzyme. It caused a 60% inhibition of H2O2 production in the absence of L‐arginine and BH4, and antagonised BH4‐induced dimerisation of nNOS, but did not affect cytochrome c reduction. These results open new perspectives in the understanding of the antithyroid action of TU and provide a new lead structure for the development of selective nNOS inhibitors to elucidate the interdependence of the substrate and pteridine sites and to modulate pathologically aberrant NO formation.
Biochemical and conformational characterisation of HSP‐3, a stallion seminal plasma protein of the cysteine‐rich secretory protein (CRISP) family
Authors: Magdaleno Leticia , Gasset Marı́a , Varea Julio , Schambony Alexandra M , Urbanke Claus , Raida Manfred , Töpfer-Petersen Edda , Calvete Juan J
Published: July 2002

HSP‐3 is a member of the cysteine‐rich secretory protein (CRISP) family from stallion seminal plasma. We report a large‐scale purification protocol for native HSP‐3. This protein is a non‐glycosylated polypeptide chain with a pI of 8–9 and an isotope‐averaged molecular mass of 24 987±3 Da. The molecular mass of HSP‐3, determined by equilibrium sedimentation, is 26 kDa, showing that the protein exists in solution as a monomer. The concentration of HSP‐3 in the seminal plasma of different stallions ranged from 0.3 to 1.3 mg/ml. On average, 0.9–9 million HSP‐3 molecules/cell coat the postacrosomal and mid‐piece regions of an ejaculated, washed stallion spermatozoon, suggesting a role in sperm physiology. Conformational characterisation of purified HSP‐3 was assessed by combination of circular dichroism and Fourier‐transform infrared spectroscopies and differential scanning microcalorimetry. Based on secondary structure assignment, HSP‐3 may belong to the α+β class of proteins. Thermal denaturation of HSP‐3 is irreversible and follows a non‐two state transition characterised by a T
m of 64°C, an enthalpy change of 75 kcal/mol, and a van 't Hoff enthalpy of 184 kcal/mol. Analysis of the spectroscopic and calorimetric data indicates the occurrence of aggregation of denatured HSP‐3 molecules and suggests the monomer as the cooperative unfolding unit.
Is absence of pyruvate dehydrogenase complex in mitochondria a possible explanation of significant aerobic glycolysis by normal human leukocytes?
Authors: Biswas Swati , Ray Manju , Misra Sanjoy , Dutta D.P , Ray Subhankar
Published: July 2002

The oxygen consumption of leukocyte mitochondria of both healthy donors and leukemic patients was tested by using different respiratory substrates. The results indicate that pyruvate could not be utilized by mitochondria of normal leukocytes, whereas mitochondria of leukemic leukocytes could use pyruvate as a good respiratory substrate. A search for the possible presence of pyruvate dehydrogenase complex (PDC) in leukocytes indicates that this enzyme is apparently absent in mitochondria of normal leukocytes but is quite active in mitochondria of leukemic leukocytes. The absence of PDC in normal leukocyte mitochondria can explain the phenomenon of significant aerobic glycolysis that has been observed in normal leukocytes.
Low molecular weight phosphotyrosine protein phosphatase translocation during cell stimulation with platelet‐derived growth factor
Authors: Rigacci Stefania , Bucciantini Monica , Marzocchini Riccardo , Berti Andrea
Published: July 2002

Low M
r phosphotyrosine protein phosphatase (PTP) is a cytosolic enzyme whose activity upon platelet‐derived growth factor (PDGF) and insulin receptors has been demonstrated in vivo. In our study we demonstrate that this enzyme, both naturally expressed and overexpressed in NIH/3T3 fibroblasts, translocates from the cytosol to the Triton X‐100 insoluble fraction following stimulation with PDGF. It emerges that the phosphorylation of a defined population of PDGF receptors, which is localized in this fraction and seems to be endowed with peculiar features and functions, is particularly affected by low M
r PTP overexpression.
Involvement of p21 in the PKC‐induced regulation of the G2/M cell cycle transition
Authors: Barboule Nadia , Lafon Corinne , Chadebech Philippe , Vidal Simone , Valette Annie
Published: July 2002

Activation of protein kinase C (PKC) inhibits cell cycle progression at the G1/S and G2/M transitions. We found that phorbol 12‐myristate 13‐acetate (PMA) induced upregulation of p21, not only in MCF‐7 cells arrested in the G1 phase as previously shown, but also in cells delayed in the G2 phase. This increase in p21 in cells accumulated in the G1 and G2/M phases of the cell cycle after PMA treatment was inhibited by the PKC inhibitor GF109203X. This indicates that PKC activity is required for PMA‐induced p21 upregulation and cell cycle arrest in the G1 and G2/M phases of the cell cycle. To further assess the role of p21 in the PKC‐induced G2/M cell cycle arrest independently of its G1 arrest, we used aphidicolin‐synchronised MCF‐7 cells. Our results show that, in parallel with the inhibition of cdc2 activity, PMA addition enhanced the associations between p21 and either cyclin B or cdc2. Furthermore, we found that after PMA treatment p21 was able to associate with the active Tyr‐15 dephosphorylated form of cdc2, but this complex was devoid of kinase activity indicating that p21 may play a role in inhibition of cdc2 induced by PMA. Taken together, these observations provide evidence that p21 is involved in integrating the PKC signaling pathway to the cell cycle machinery at the G2/M cell cycle checkpoint.
A stable interaction between syntaxin 1a and synaptobrevin 2 mediated by their transmembrane domains
Authors: Margittai Martin , Otto Henning , Jahn Reinhard
Published: July 2002

The proteins synaptobrevin (VAMP), SNAP‐25 and syntaxin 1 are essential for neuronal exocytosis. They assemble into a stable ternary complex which is thought to initiate membrane fusion. In vitro, the transmembrane domains of syntaxin and synaptobrevin are not required for association. Here we report a novel interaction between synaptobrevin and syntaxin that requires the presence of the transmembrane domains. When co‐reconstituted into liposomes, the proteins form a stable binary complex that cannot be disassembled by NSF and that is resistant to denaturation by SDS. Cleavage of synaptobrevin with tetanus toxin does not affect the interaction. Furthermore, the complex is formed when a truncated version of syntaxin is used that contains only 12 additional amino acid residues outside the membrane anchor. We conclude that the interaction is mediated by the transmembrane domains.
Plausible phosphoenolpyruvate binding site revealed by 2.6 Å structure of Mn2+‐bound phosphoenolpyruvate carboxylase from Escherichia coli
Authors: Matsumura Hiroyoshi , Terada Mika , Shirakata Shunsuke , Inoue Tsuyoshi , Yoshinaga Takeo , Izui Katsura , Kai Yasushi
Published: July 2002

We have determined the crystal structure of Mn2+‐bound Escherichia coli phosphoenolpyruvate carboxylase (PEPC) using X‐ray diffraction at 2.6 Å resolution, and specified the location of enzyme‐bound Mn2+, which is essential for catalytic activity. The electron density map reveals that Mn2+ is bound to the side chain oxygens of Glu‐506 and Asp‐543, and located at the top of the α/β barrel in PEPC. The coordination sphere of Mn2+ observed in E. coli PEPC is similar to that of Mn2+ found in the pyruvate kinase structure. The model study of Mn2+‐bound PEPC complexed with phosphoenolpyruvate (PEP) reveals that the side chains of Arg‐396, Arg‐581 and Arg‐713 could interact with PEP.
Neurotoxicity of prion peptide 106‐126 not confirmed
Authors: Kunz Beat , Sandmeier Erika , Christen Philipp
Published: July 2002

Prion‐related diseases are accompanied by neurodegeneration, astroglial proliferation and formation of proteinase K‐resistant aggregates of the scrapie isoform of the prion protein (PrPSc). The synthetic PrP fragment 106‐126 was reported to be neurotoxic towards cultured rat hippocampal neurons (Forloni, G., Angeretti, N., Chiesa, R., Monzani, E., Salmona, M., Bugiani, O. and Tagliavini, F. (1993) Nature 362, 543–546) and mouse cortical cells (Brown, D.R., Herms, J. and Kretzschmar, H.A. (1994) Neuroreport 5, 2057–2060). However, we found the viability of these and other neuronal cell types not to be impaired in the presence of PrP106‐126 under widely varied sets of conditions. Aged preparations of the peptide as well as synthetic deamidated and isomerized derivatives that correspond to the aging products of the peptide proved also to lack neurotoxicity. Apparently, PrP106‐126 cannot serve as a model for the interaction of PrP with neuronal cells.
Mutation of a basic sequence in the laminin α2LG3 module leads to a lack of proteolytic processing and has different effects on β1 integrin‐mediated cell adhesion and α‐dystroglycan binding
Authors: Talts Jan F , Timpl Rupert
Published: July 2002

A RRKRRQ sequence unique to the LG3 module of the laminin α2 chain was previously shown to be sensitive to endogenous proteolysis during the recombinant production of the tandem array α2LG1‐3. Mutation of RQ surrounding the cleaved peptide bond did not prevent this processing and intracellular degradation. Alanine mutagenesis of three alternate basic residues, however, was shown to prevent the cleavage in α2LG1‐3, allowing for the α2LG3 module to be obtained as a folded, globular fragment. The mutation did not change heparin and sulfatide binding or cell adhesion of α2LG1‐3 which can be mediated by α3β1 and α6β1 integrins. It did, however, cause a 10‐fold reduction in α‐dystroglycan binding. The data favor the interpretation that binding epitopes for heparin/sulfatides, β1 integrins and α‐dystroglycan occupy different parts of the α2LG1‐3 structure.
Knockout of the Sendai virus C gene eliminates the viral ability to prevent the interferon‐α/β‐mediated responses
Authors: Gotoh Bin , Takeuchi Kenji , Komatsu Takayuki , Yokoo Junko , Kimura Yoshinobu , Kurotani Atsushi , Kato Atsushi , Nagai Yoshiyuki
Published: July 2002

Sendai virus (SeV) renders cells unresponsive to interferon (IFN)‐α. To identify viral factors involved in this process, we examined whether recombinant SeVs, which could not express V protein, subsets of C proteins (C, C′, Y1 and Y2) or any of four C proteins, retained the capability of impeding IFN‐α‐mediated responses. Among these viruses, only the 4C knockout virus completely lost the ability to suppress the induction of IFN‐α‐stimulated gene products and the subsequent establishment of an anti‐viral state. These findings reveal crucial roles of the SeV C proteins in blocking IFN‐α‐mediated responses.
Hormone‐activated nuclear receptors inhibit the stimulation of the JNK and ERK signalling pathways in endothelial cells
Authors: González Marı́a Victoria , González-Sancho José Manuel , Caelles Carme , Munoz Alberto , Jiménez Benilde
Published: July 2002

Glucocorticoid hormones, retinoids, and vitamin D3 display anti‐angiogenic activity in tumor‐bearing animals. However, despite their in vivo effect on the tumor vasculature little is known about their mechanism of action. Here we show that the synthetic glucocorticoid dexamethasone (Dex) and retinoic acid (RA) inhibit the activation of c‐Jun N‐terminal kinase (JNK) and extracellular‐regulated kinase (ERK) signalling pathways by the pro‐angiogenic agents tumor necrosis factor and vascular endothelial growth factor in endothelial cells. In contrast, Dex and RA failed to inhibit the activation of the p38 mitogen‐activated protein kinase cascade. As a number of pro‐angiogenic factors activate AP‐1 transcription factor via the JNK and ERK pathways, our results suggest that the antagonism with AP‐1 may underlie at least partially the anti‐angiogenic effect of glucocorticoids and retinoids.
Purification and characterization of the single‐component nitric oxide reductase from Ralstonia eutropha H16
Authors: Cramm Rainer , Pohlmann Anne , Friedrich Bärbel
Published: July 2002

Nitric oxide (NO) reductase was purified from Ralstonia eutropha (formerly Alcaligenes eutrophus) using a two step chromatographic procedure. Unlike the common NO reductases, the enzyme consists of a single subunit of 75 kDa which contains both high‐spin and low‐spin heme b, but lacks heme c. One additional iron atom, probably a ferric non‐heme iron, was identified per enzyme molecule. Whereas reduced cytochrome c was ineffective as electron donor, NO was reduced at a specific activity of 2.3 μmol/min per mg of protein in the presence of 2‐methyl‐1,4‐naphthoquinol.
Folding and self‐assembly do not prevent ER retention and proteasomal degradation of asialoglycoprotein receptor H2a
Authors: Ayalon-Soffer Michal , Kamhi-Nesher Shiri , Lederkremer Gerardo Z
Published: July 2002

The human asialoglycoprotein receptor H2a precursor, a type II membrane protein, is cleaved to a soluble form that is secreted. Uncleaved precursor molecules are completely retained in the endoplasmic reticulum (ER) and degraded by the proteasome. To find out the causes of its fate we studied folding of H2a precursor, which was very similar to that of its alternatively spliced variant H2b which can exit to the Golgi. Proteasomal inhibition led to accumulation of folded rather than unfolded molecules. Accumulation of ER‐retained H2a did not cause an unfolded protein response. Although the receptor is a heterooligomer of the H1 and H2 subunits, single expression led to some self‐assembly. Whereas these homooligomers accumulated for H2b they were degraded for H2a. Translocation of H2a into the ER occurred efficiently. Therefore, the retention and proteasomal degradation of uncleaved membrane‐bound H2a precursor from the ER do not involve aberrant translocation or misfolding and are not prevented by self‐assembly.
α‐Actinin and spectrin structures: an unfolding family story
Authors: Viel Alain
Published: July 2002

In red blood cells, the integrity of the spectrin network is essential for normal cell shape and elasticity. To understand the molecular basis for spectrin's mechanical properties, one must determine how spectrin subunits interact with each other. The newly described crystallographic structures of two consecutive homologous repeats of human α‐actinin, a member of the spectrin superfamily, shed new light on α‐actinin interchain binding properties. Here I present evidence that interchain binding at the tail end of the spectrin molecule is likely to occur via a mechanism similar to that observed for α‐actinin.
Dominant‐negative alleles of 14‐3‐3 proteins cause defects in actin organization and vesicle targeting in the yeast Saccharomyces cerevisiae
Authors: Roth Dagmar , Birkenfeld Jörg , Betz Heinrich
Published: July 2002

14‐3‐3 Proteins are thought to function as adapters in signaling complexes [1,2], thereby participating in cellular processes including vesicle trafficking and exocytosis [3,4]. To delineate further the function of 14‐3‐3 proteins during vesicle trafficking, we generated dominant‐negative alleles of the two 14‐3‐3 homologues, Bmh1p and Bmh2p, in budding yeast and analyzed their phenotype in respect to exocytosis. Cells overexpressing the carboxy‐terminal region of Bmh2p failed to polarize vesicular transport although bulk exocytosis remained unaffected and showed a disrupted actin cytoskeleton. Our data suggest that 14‐3‐3 proteins may act primarily on the actin cytoskeleton to regulate vesicle targeting.
Unitary exocytotic and endocytotic events in guard‐cell protoplasts during osmotically driven volume changes
Authors: Homann Ulrike , Thiel Gerhard
Published: July 2002

Osmotically driven swelling and shrinking of guard‐cell protoplasts (GCPs) requires adjustment of surface area which is achieved by addition and removal of plasma membrane material. To investigate the mechanism for adaptation of surface area we have used patch‐clamp capacitance measurements. The recorded membrane capacitance (C
m) trace of swelling and shrinking GCPs occasionally revealed discrete upward and downward deflecting capacitance steps, respectively, with a median value of about 2 fF. The observed capacitance steps resulted from the fusion and fission of single vesicles with a diameter of around 300 nm. We conclude that exo‐ and endocytosis of these vesicles accommodate for osmotically driven surface area changes in GCPs.
Sphingosine‐1‐phosphate modulates growth and adhesion of ovarian cancer cells
Authors: Hong Guiying , Baudhuin Linnea M. , Xu Yan
Published: July 2002

Sphingosine‐1‐phosphate (S1P) is a bioactive lipid molecule. It stimulates the growth of some cells, but inhibits the growth of others. In this study, we describe the detection of sub‐μM to μM concentrations of S1P in the ascitic fluids of patients with ovarian cancer. In ovarian cancer cells cultured in vitro, S1P exhibited a dual effect on growth and/or survival. S1P (10 μM) induced cell death when cells were in suspension but stimulated cell growth when cells were attached. The calcium‐dependent induction of cell death by S1P is apparently associated with its inhibitory effect on cell attachment and cell adhesion. S1P (10–30 μM) also induced calcium‐dependent cell‐cell aggregation.
Voltage‐dependent interaction of the peptaibol antibiotic zervamicin II with phospholipid vesicles
Authors: Kropacheva T.N. , Raap J.
Published: July 2002

The effect of a transmembrane potential on ion channel formation by zervamicin II (ZER‐II) was studied in a vesicular model system. The dissipation of diffusion potential caused by addition of ZER‐II to small phosphatidylcholine vesicles was monitored using fluorescent (Safranine T) and optical (Oxonol YI) probes. Cis‐positive potentials facilitated channel formation, while at cis‐negative potentials, ion fluxes were inhibited. A potential‐independent behavior of ZER‐II was observed at high peptide concentrations, most likely due to its membrane modifying property.
The inhibitory effect of glucose on phosphoenolpyruvate carboxykinase gene expression in cultured hepatocytes is transcriptional and requires glucose metabolism
Authors: Cournarie Fabienne , Azzout-Marniche Dalila , Foretz Marc , Guichard Colette , Ferre Pascal , Foufelle Fabienne
Published: July 2002

Phosphoenolpyruvate carboxykinase (PEPCK) is the rate‐limiting enzyme of gluconeogenesis in the liver. PEPCK gene expression is controlled at the transcriptional level and is mainly regulated by hormones that are involved in glucose homeostasis. In this study, we have investigated the role of glucose on PEPCK gene expression in cultured hepatocytes. We demonstrate that glucose counteracts the stimulatory effect of glucocorticoids and cAMP on PEPCK expression. Glucose must be metabolized through glucokinase to have its inhibitory effect. The effect of glucose is mainly transcriptional and the region responsible for glucose inhibition is localized in the first 490 bp of the promoter.
The PAN module: the N‐terminal domains of plasminogen and hepatocyte growth factor are homologous with the apple domains of the prekallikrein family and with a novel domain found in numerous nematode proteins
Authors: Tordai Hedvig , Bányai László , Patthy László
Published: July 2002

Based on homology search and structure prediction methods we show that (1) the N‐terminal N domains of members of the plasminogen/hepatocyte growth factor family, (2) the apple domains of the plasma prekallikrein/coagulation factor XI family, and (3) domains of various nematode proteins belong to the same module superfamily, hereafter referred to as the PAN module. The patterns of conserved residues correspond to secondary structural elements of the known three‐dimensional structure of hepatocyte growth factor N domain, therefore we predict a similar fold for all members of this superfamily. Based on available functional informations on apple domains and N domains, it is clear that PAN modules have significant functional versatility, they fulfill diverse biological functions by mediating protein‐protein or protein‐carbohydrate interactions.
Spinach hexokinase I is located in the outer envelope membrane of plastids
Authors: Wiese Anika , Gröner Ferdi , Sonnewald Uwe , Deppner Heike , Lerchl Jens , Hebbeker Ulrike , Flügge Ulf-Ingo , Weber Andreas
Published: July 2002

The subcellular localization of hexokinase activities in plant cells has been a matter of debate for a long time. We have isolated a hexokinase cDNA fragment from glucose‐fed spinach leaves using a differential display reverse transcription‐PCR approach. The corresponding cDNA was expressed in Escherichia coli and an antiserum, raised against the recombinant protein, was used in subcellular localization studies. The spinach hexokinase could be localized primarily to the outer envelope membrane of chloroplasts where it is inserted via its N‐terminal membrane anchor. We suggest that the chloroplast envelope hexokinase is involved in the energization of glucose export from plastids rather than in the sugar‐sensing pathway of the plant cell.
Transcriptional activation of mouse cytosolic chaperonin CCT subunit genes by heat shock factors HSF1 and HSF2
Authors: Kubota Hiroshi , Matsumoto Shuji , Yokota Shin-ichi , Yanagi Hideki , Yura Takashi
Published: July 2002

The chaperonin containing TCP‐1 (CCT) is a eukaryotic molecular chaperone consisting of eight subunit species and assists in the folding of cytosolic proteins. We show here that all eight mouse CCT subunit genes contain sequences called heat shock elements for binding heat shock transcription factors (HSFs) by electrophoretic mobility shift assays and that these genes are transcriptionally activated by HSFs in reporter gene assays using HeLa cells transiently overexpressing HSFs. These results suggest that HSF1 and/or HSF2 play a role in Cct gene expression.
Interaction with GroEL destabilises non‐amphiphilic secondary structure in a peptide
Authors: Preuss Monika , Miller Andrew D
Published: July 2002

The Escherichia coli molecular chaperone GroEL can functionally interact with non‐native forms of many proteins. An inherent property of non‐native proteins is the exposure of hydrophobic residues and the presence of secondary structure elements. Whether GroEL unfolds or stabilises these structural elements in protein substrates as a result of binding has been the subject of extended debate in the literature. Based on our studies of model peptides of pre‐formed helical structure, we conclude that the final state of a GroEL‐bound substrate is dependent on the conformational flexibility of the substrate protein and the distribution of hydrophobic residues, with optimal association when these are able to present a cluster of hydrophobic residues in the binding interface.
The mechanism of the alkaline phosphatase reaction: insights from NMR, crystallography and site‐specific mutagenesis
Authors: Holtz Kathleen M , Kantrowitz Evan R
Published: July 2002

The proposed double in‐line displacement mechanism of Escherichia coli alkaline phosphatase (AP) involving two‐metal ion catalysis is based on NMR spectroscopic and X‐ray crystallographic studies. This mechanism is further supported by the X‐ray crystal structures of the covalent phospho‐enzyme intermediate of the H331Q mutant AP and of the transition state complex between the wild‐type enzyme and vanadate, a transition state analog. Kinetic and structural studies on several genetically engineered versions of AP illustrate the overall importance of the active site's metal geometry, hydrogen bonding network and electrostatic potential in the catalytic mechanism.
Antisense suppression of proline degradation improves tolerance to freezing and salinity in Arabidopsis thaliana
Authors: Nanjo Tokihiko , Kobayashi Masatomo , Yoshiba Yoshu , Kakubari Yoshitaka , Yamaguchi-Shinozaki Kazuko , Shinozaki Kazuo
Published: July 2002

Synthesis, degradation, and transport of proline (Pro) are thought to cooperatively control its endogenous levels in higher plants in response to environmental conditions. To evaluate the function of Pro degradation in the regulation of the levels of Pro and to elucidate roles of Pro in stress tolerance, we generated antisense transgenic Arabidopsis plants with an AtProDH cDNA encoding proline dehydrogenase (ProDH), which catalyzes Pro degradation. Several transgenic lines accumulated Pro at higher levels than wild‐type plants, providing evidence for a key role of ProDH in Pro degradation in Arabidopsis. These antisense transgenics were more tolerant to freezing and high salinity than wild‐type plants, showing a positive correlation between Pro accumulation and stress tolerance in plants.
Role of sphingosine kinase in Ca2+ signalling by epidermal growth factor receptor
Authors: Meyer zu Heringdorf Dagmar , Lass Holger , Kuchar Igor , Alemany Regina , Guo Yuanjian , Schmidt Martina , Jakobs Karl H
Published: July 2002

Contribution of sphingosine kinase (SPK)‐catalyzed production of sphingosine‐1‐phosphate (SPP), in comparison to phospholipase C (PLC), to Ca2+ signalling by epidermal growth factor (EGF) was studied in two HEK‐293 cell clones (HEK2 and HEK3), expressing functional EGF receptors and exhibiting release of stored Ca2+ by intracellular SPP. In HEK3 cells, EGF increased [Ca2+]i and stimulated both, SPK and PLC. [Ca2+]i increase, but not PLC stimulation, was strongly reduced by SPK inhibition. In HEK2 cells, EGF similarly stimulated PLC, but did not increase [Ca2+]i or stimulate SPK, suggesting that intracellular SPP production plays a major role for Ca2+ signalling by EGF in HEK‐293 cells.
Structure of influenza haemagglutinin at neutral and at fusogenic pH by electron cryo‐microscopy
Authors: Böttcher Christoph , Ludwig Kai , Herrmann Andreas , van Heel Marin , Stark Holger
Published: July 2002

The three‐dimensional structures of the complete haemagglutinin (HA) of influenza virus A/Japan/305/57 (H2N2) in its native (neutral pH) and membrane fusion‐competent (low pH) form by electron cryo‐microscopy at a resolution of 10 Å and 14 Å, respectively, have been determined. In the fusion‐competent form the subunits remain closely associated preserving typical overall features of the trimeric ectodomain at neutral pH. Rearrangements of the tertiary structure in the distal and the stem parts are associated with the formation of a central cavity through the entire ectodomain. We suggest that the cavity is essential for relocation of the so‐called fusion sequence of HA towards the target membrane.
Amino acid sequence of a putative transposase protein of the medaka fish transposable element Tol2 deduced from mRNA nucleotide sequences
Authors: Koga Akihiko , Suzuki Miho , Maruyama Yuki , Tsutsumi Makiko , Hori Hiroshi
Published: July 2002

Tol2 is a terminal‐inverted repeat transposable element of the medaka fish Oryzias latipes. It is one of a few elements of this class so far demonstrated to be active in vertebrates, thus providing a unique tool for establishing a gene tagging system. For the purpose of identifying its transposase, we analyzed the structures of mRNAs originating from the Tol2 element. The results indicated that transcription of Tol2 is initiated at several sites, the four open reading frames in Tol2 roughly corresponding to exons, and that two main forms of mRNAs, covering exons 1–4 and exons 2–4, are present in medaka fish cells. One or both of these mRNAs are likely to encode a transposase, the amino acid sequence of which was deduced.