bluuebunny/crossref_metadata_2025 huggingface dataset:
Determination of the sequence coding for the β subunit of the human high‐affinity IgE receptor
Authors: Maekawa Kazuhiko , Imagawa Noriko , Tanaka Yoshiaki , Harada Shigenori
Published: July 2002

The cDNA encoding the β subunit of the human high‐affinity IgE receptor was cloned by a combination or various polymerase chain reactions (PCR). A major portion of the β cDNA was amplified using primers homologous within the sequences of rat and mouse. The 3′ unknown sequence was preferentially amplified using the RNA template‐specific PCR and the improved two‐step PCR. The 5′ unknown sequence was specifically amplified by our newly developed PCR walking. Random heptanucleotides tagged with a unique sequence at the 5′ end were used as the walking primer. Finally, the entire coding region was amplified and sequenced. The two extracellular loops of the human β subunit were the least homologous to those of rat and mouse.
Detection of a new mutant α‐1‐antichymotrypsin in patients with occlusive‐cerebrovascular disease
Authors: Tsuda Michio , Sei Yukari , Yamamura Masaichi , Yamamoto Masahiro , Shinohara Yukito
Published: July 2002

A new mutant α‐1‐antichymotrypsin (variant ACT) was found by direct sequencing and PCR‐single strand conformation polymorphism (PCR‐SSCP). This variant ACT was a point mutation of exon V of ACT, with the substitution of Met by Val. Four out of six individuals with this variant ACT had occlusive‐cerebrovascular disease, leading to one hypothesis that there might be an association between this mutation and occlusive‐cerebrovascular disease.
The envelope glycoprotein of Ebola virus contains an immunosuppressive‐like domain similar to oncogenic retroviruses
Authors: Volchkov Viktor E. , Blinov Vladimir M. , Netesov Sergey V.
Published: July 2002

Genomic RNA of a Zaire strain of Ebola virus was cloned, and cDNA inserts specific for the glycoprotein gene were isolated and sequenced. The determined sequence has only one open reading frame encoding 318 amino acids and is part of ORF‐4 on the plus RNA strand. The putative transcriptional stop site (3′ AAUUCUUUUU 5′) and the transcriptional start site (3′ AACUACUUCUAAUU.. 5′) were identified. Computer‐assisted comparison of the amino acid sequence of the C‐terminal part of protein encoded by ORF‐4 of Ebola virus with sequence of the proteins present in the SWISSPROT and EMBL banks revealed significant homology with the immunosuppressive domain of the p15E envelope proteins of various oncogenic retroviruses. The possible role of such a homology is discussed.
Aluminum inhibits neurofilament protein degradation by multiple cytoskeleton‐associated proteases
Authors: Shea Thomas B. , Balikian Philip , Beermann Mary Lou
Published: July 2002

The environmental neurotoxin aluminum exerts several distinct biochemical effects on neurofilament proteins, including subunit aggregation, disruption or the normal segregation of phosphorylated subunits within axons leading to abnormal perikaryal accumulation, and inhibition of in vitro degradation by the calcium‐dependent neutral protease, calpain. In the present study, we demonstrate that exposure of mouse CNS cytoskeletal preparations to aluminum chloride inhibits the degradation of neurofilament proteins by both calcium‐dependent and ‐independent proteases that co‐purify with cytoskeletons. Aluminum inhibited both calcium‐dependent and calcium‐independent proteolysis of the high and middle molecular weight neurofilament subunits, but inhibited only calcium‐dependent, and not calcium‐independent proteolysis of the low molecular weight neurofilament subunit. These findings demonstrate that aluminum interferes with multiple aspects or neurofilament protein metabolism.
A selective inhibitor of cyclic AMP‐dependent protein kinase, N‐[2‐bromocinnamyl(amino)ethyl]‐5‐isoquinolinesulfonamide (H‐89), inhibits phosphatidylcholine biosynthesis in HeLa cells
Authors: Geilen Christoph C. , Wieprecht Marcus , Wieder Thomas , Reutter Werner
Published: July 2002

In this study, we report that the potent and selective inhibitor of cyclic AMP‐dependent protein kinase, N‐[2‐bromocinnamyl(amino)ethyl]‐5‐isoquinolinesulfonamide (H‐89) interferes with the incorporation of choline into phosphatidylcholine in HeLa cells. Treatment of cells with 10 μM H‐89 for 1 h decreases the phosphatidylcholine biosynthesis by 50%. This inhibition is prevented by simultaneous addition of 10 μM forskolin, while the choline uptake itself is not affected by H‐89.
Endotoxin transduces Ca2+ signaling via platelet‐activating factor receptor
Authors: Nakamura Motonao , Honda Zen-ichiro , Waga Iwao , Matsumoto Takashi , Noma Masana , Shimizu Takao
Published: July 2002

Lipopolysaccharide (LPS) is a pathogenic substance causing severe multiple organ failures and high mortality, Although several LPS binding proteins have been identified, the molecular mechanism underlying the LPS signaling pathway still remains obscure. We have found that the LPS‐induced Ca2+ increase in platelets and platelet aggregation is blocked by selective platelet‐activating factor (PAF) receptor antagonists, thus suggesting a cross‐talk between LPS and the PAF receptor. Next, we confirmed this hypothesis using the cloned PAF receptors [(1991) Nature 349, 342–346; (1991) J. Biol. Chem. 266, 20400–20405] expressed in Xenopus occytes and Chinese hamster ovary (CHO) cells. In both systems, cells responded to LPS only when PAF receptors were expressed, and specific PAF binding was successfully displaced and reversibly dissociated by LPS. PAF receptor activation by LPS may represent a novel important pathway in the pathogenesis of circulatory collapse and systemic thrombosis caused by endotoxin.
Structure of the small ribosomal subunit RNA of the pulmonate snail, Limicolaria kambeul, and phylogenetic analysis of the Metazoa
Authors: Winnepennickx Birgitta , Backeljau Thierry , van de Peer Yves , De Wachter Rupert
Published: July 2002

The complete nucleotide sequence of the small ribosomal subunit RNA of the gastropod, Limicolaria kambeul, was determined and used to infer a secondary structure model. In order to clarify the phylogenetic position of the Mollusca among the Metazoa, an evolutionary tree was constructed by neighbor‐joining, starting from an alignment of small ribosomal subunit RNA sequences. The Mollusca appear to be a monophyletic group, related to Arthropoda and Chordata in an unresolved trichotomy.
The organisation of the mouse secretogranin II gene
Authors: Schimmell Angelika , Bräunling Otto , Rüther Ulrich , Huttner Wieland B. , Gerdes Hans-Hermann
Published: July 2002

We have characterized the gene which encodes mouse secretogranin II (previously also referred to as chromogranin C), a tyrosine‐sulfated secretory protein belonging to the granin (chromogranin/secretogranin) family which is found in secretory granules of most endocrine cells and neurons. The secretogranin II gene was found to contain 2 exons. In contrast to chromogranin A and chromogranin B, the two previously characterized granin genes, the entire secretogranin II protein is encoded by a single exon, exon 2, with exon 1 containing only a 5′‐untranslated sequence. Consistent with previous data on the expression of secretogranin II, the putative promoter region was found to contain a cAMP‐responsive element and a potential AP‐1 binding site.
Release of the FAD domain from cellobiose oxidase by proteases from cellulolytic cultures of Phanerochaete chrysosporium
Authors: Habu Naoto , Samejima Masahiro , Dean Jeffrey F.D. , Eriksson Karl-Erik L.
Published: July 2002

Evidence has previously suggested that cellobiose:quinone oxidoreductase (CBQ) in cellulolytic cultures of Phanerochaete chrysosporium might be produced from cellobiose oxidase (CBO) by proteolytic cleavage. This study demonstrates that the ratio of CBO activity to (CBO + CBQ) activity declines with decreasing culture pH, while protease activity increases. Furthermore, we demonstrate that endogenous P. chrysosporium proteases can only cleave CBO when the enzyme is bound to cellulose. This is the first demonstration that the proteases produced in cellulolytic cultures of P. chrysosporium can release the FAD domain from CBO.
The crystal structure of triacylglycerol lipase from Pseudomonas glumae reveals a partially redundant catalytic aspartate
Authors: Noble M.E.M. , Cleasby A. , Johnson L.N. , Egmond M.R. , Frenken L.G.J.
Published: July 2002

The family of lipases (triacylglycerol‐acyl‐hydrolases, EC 3.1.1.3) constitutes an interesting class of enzymes because of their ability to interact with lipid‐water interfaces, their wide range of substrate specificities, and their potential industrial applications [1,2]. Here we report the first crystal structure of a bacterial lipase, from Pseudomonas glumae. The structure is formed from three domains, the largest of which contains a subset of the α/β hydrolase fold and a calcium site. Asp263, the acidic residue in the catalytic triad, has previously been mutated into an alanine with only a modest reduction in activity [3].
Interaction of homologues of Hsp70 and Cpn60 with ferredoxin‐NADP+ reductase upon its import into chloroplasts
Authors: Tsugeki Ryuji , Nishimura Mikio
Published: July 2002

A homologue of the 70‐kDa heat‐shock protein (Hsp70) was purified from pumpkin chloroplasts. The molecular mass of the purified protein was approximately 75 kDa and its N‐terminal amino acid sequence was very similar to those of homologues of Hsp70 from bacterial cells and from the mitochondrial matrix and stroma of pea chloroplasts. The purified homologue of Hsp70 was found in the stroma of chloroplasts. To investigate the role(s) of the homologue of Hsp70 in the chloroplast stroma, we examined the possibility that the homologue of Hsp70 might interact with newly imported proteins to assist in their maturation (for example, in their folding and assembly). Ferredoxin NADP+ reductase (FNR) imported into chloroplasts in vitro could be immunoprecipitated with antisera raised against the homologue of Hsp70 from pumpkin chloroplasts and against GroEL from Escherichia coli, which is a bacterial homologue of chaperonin 60 (Cpn60), in an ATP‐dependent manner, an indication that newly imported FNR interacts physically with homologues of Hsp70 and Cpn60 in chloroplasts. Time‐course analysis of the import of FNR showed that imported FNR interacts transiently with the homologue of Hsp70 and that the association of FNR with the homologue of Hsp70 precedes that with the homologue of Cpn60. These results suggest that homologues of Hsp70 and Cpn60 in chloroplasts might sequentially assist in the maturation of newly imported FNR in an ATP‐dependent manner.
The genes aroA and trnQ are located upstream of psbO in the chromosome of Synechocystis 6803
Authors: Mayes Steve R. , Chiesa Marta Dalla , Zhang Zhihong , Barber James
Published: July 2002

We have identified the existence of two genes, trnQ and aroA, located upstream of the psbO gene in Synechocystis sp. PCC 6803. The trnQ gene encodes a glutamine‐specific transfer RNA (tRNAGln) and the sequence given is the first reported for any cyanobacterium. The gene seems to exist as a single copy since its deletion results in nonviable mutation. The aroA gene encodes for 5‐enolpyruvylshikimate 3‐phosphate synthase and its discovery in the genome of Synechocystis 6803 is the first genetic evidence for the existence of the shikimate biosynthetic pathway in cyanobacteria. Interestingly, the partial sequence shares close homologies with the sequences of aroA from Gram‐positive bacteria.
Synthetic peptides derived from the sequence around the plasmin cleavage site in vitronectin
Authors: Gechtman Zeev , Sharma Raman , Kreizman Tamar , Fridkin Mati , Shaltiel Shmuel
Published: July 2002

A series of 8 peptides derived from the amino acid sequence accommodating the plasmin cleavage site in vitronectin were synthesized and used to map its binding site for the type I plasminogen activator inhibitor (PAI‐1). This mapping assigned the inhibitor binding site to the K348‐R370 region with high affinity recognition elements within the K348‐R357 sequence. These results account for our previous finding that cleavage of the R361 ‐S362 bond by plasmin significantly reduces the affinity between PAI‐1 and vitronectin, since it splits the PAI‐1 binding site in two. Furthermore, in the case of the two‐chain form of vitronectin, this cleavage detaches the S362‐R379 peptide which provides some of the affinity elements for the binding of PAI‐1.
Evidence for a role for protein kinase C in the stimulation of protein synthesis by insulin in swiss 3T3 fibroblasts
Authors: Welsh Gavin I. , Proud Christopher G.
Published: July 2002

We have examined the role of protein kinase C (PKC) in the stimulation of protein synthesis by insulin using two complementary approaches. In the first, fibroblasts were pretreated with phorbol esters to down‐regulate PKC. In these cells, the effects of insulin and of phorbol esters on protein synthesis were completely abolished, although serum still elicited an effect approaching that seen in control cells. Secondly, we used newly developed inhibitors of PKC which, again, blocked the effects of insulin and phorbol esters without greatly reducing the response to serum. Thus PKC apparently plays an important role in the stimulation of translation by insulin.
A product of phosphatidylinositol‐3 kinase is elevated in dividing HT29 colonic epithelial cells
Authors: Dobos Gustav J. , Wu Xiao Rong , Traynor-Kaplan Alexis
Published: July 2002

Phosphatidylinositol 3 kinase (PI‐3 kinase) activity has been linked to cell proliferation and growth regulation. Therefore, we studied changes in phosphoinositide metabolism during the cell cycle of HT‐29 cells, a colonic epithelial cell line. HT29 cells were treated with the microtubule disrupter, nocodozole, separated into mitotic and quiescent populations and their phospholipid composition was analyzed. Radiolabelled phospholipids from cells labelled with 32PO4 or [3H]myoinositol were analyzed by TLC and/or deacylated and analyzed by HPLC. In all cases, levels of phosphatidyl‐inositol 3 phosphate from mitotic phase cells were double that in resting populations. Therefore, levels of a product of PI‐3 kinase are elevated and may play a role in cell division.
NAD(P)H‐flavin oxidoreductase from the bioluminescent bacterium, Vibrio fischeri ATCC 7744, is a flavoprotein
Authors: Inouye Satoshi
Published: July 2002

The NAD(P)H‐flavin oxidoreductase gene from the bioluminescent bacterium, Vibrio fischeri ATCC 7744, was expressed in Escherichia coli, and the enzyme purified using Cibacron Blue 3G‐A affinity column chromatography from crude extracts in a single step. The purified enzyme had a typical flavoprotein absorption spectrum and flavin mononucleotide (FMN) was identified as a prosthetic group, non‐covalently bound in a molar ratio of 1:1. The enzyme catalyzed the electron transfer from NADH via FMNH2 to various other electron acceptors. Reduced flavin produced by flavin reductase participated non‐enzymatically in the following reactions: H2O2‐forming NADH oxidase‐like, oxygen‐insenstive nitroreductase‐like, diaphorase (quinone reductase)‐like and bacterial luciferase reactions.
Ganglioside antigen of DU‐PAN‐2 in a human pancreatic cancer
Authors: Hamanaka Yuichiro , Hamanaka Sumiko , Shinagawa Yuji , Suzuki Takashi , Inagaki Fuyuhiko , Suzuki Minoru , Suzuki Akemi
Published: July 2002

DU‐PAN‐2 reactive gangliosides were isolated from the tumor of a patient with pancreatic cancer (duct cell carcinoma, moderately differentiated adenocarcinoma), having a negative Lewis blood phenotype, and were analyzed by means of TLC‐immunostaining, enzyme‐linked immunosorbent assay (ELISA), permethylation study, 1H NMR spectroscopy and fast atom bombardment mass spectrometry. The structures of the gangliosides were found to be NeuAcα2‐3Ga1β1‐3G1cNAcβ1‐3Ga1β1‐4Glcβ1‐1′Cer, containing normal and hydroxy fatty acids. By TLC‐immunostaining and ELISA with chemically synthesized gangliosides, DU‐PAN‐2 was demonstrated to react strongly with IV3αNeuAc‐Lc4Cer, weakly with IV3αNeuAc‐nLc4Cer, and moderately with IV6αNeuAc‐Lc4Cer and IV6αNeuAc‐nLc4Cer. Thus it was concluded that the DU‐PAN‐2 reactive ganglioside in the tumor is IV3αNeuAc‐Lc4Cer and that DU‐PAN‐2 has a rather broad specificity.
PVP‐containing solutions for analysis of divalent cation‐dependent NMDA responses in Xenopus oocytes
Authors: Raditsch Martin , Witzemann Veit
Published: July 2002

The electrophysiological analysis of Ca2+‐conducting ion channels in Xenopus oocytes is difficult due to secondary intracellular effects induced by Ca2+. In the presence of polyvinylpyrrolidone (PVP) membrane currents can be recorded in nominally divalent cation‐free solutions. The Ca2+‐permeable recombinant NMDA receptors of the NR1/NR2A subtype were used as assay system and the results show that PVP has no effect on NMDA receptor‐induced currents. Ca2+ and Ba2+ depress NMDA‐induced currents at submillimolar concentrations probably by interfering with the Na+/K+ flux. This block is fully reversible as also observed for Mg2+ but shows in contrast no pronounced voltage dependence. PVP‐containing solutions may be useful for the analysis of divalent cation‐dependent ion channels.
Distinct cadherin—catenin complexes in Ca2+ dependent cell—cell adhesion
Authors: Butz Stefan , Kemler Rolf
Published: July 2002

Catenins are peripheral cytoplasmic proteins originally identified in association with the mouse epithelial cell adhesion molecule E‐cadherin. Molecular cloning and primary structure analysis demonstrated that α‐catenin is homologous to vinculin and that β‐catenin is homologous to human plakoglobin and the Drosophila gene product armadillo. With the use of peptide‐specific anti plakoglobin antibodies we confirm here that plakoglobin is a component of the cadherin‐catenin complex and that it is most likely identical to γ‐catenin. We show that plakoglobin binds directly to E‐cadherin. We consolidate the biochemical evidence for the existence of two distinct and separable E‐cadherin‐catenin complexes in the same cell. One complex is composed of E‐cadherin, α‐and β‐catenin, the other of E‐cadherin, α‐catenin and plakoglobin. A similar distinct association with catenins is also found for other cadherins. Comparison of different cell lines revealed that the relative amounts of the two complexes vary depending on cell types.
A pre‐existing protease is a common effector of thymocyte apoptosis mediated by diverse stimuli
Authors: Fearnhead Howard O. , Rivett A.Jennifer , Dinsdale David , Cohen Gerald M.
Published: July 2002

Data from a number of model systems support a role for proteolysis in apoptotic cell death. Using immature rat thymocytes, we demonstrate that the inhibitors chloromethylketone (TLCK) and chloromethylketone (TPCK) have very different effects on apoptosis. TLCK inhibits apoptosis induced by diverse stimuli at an early stage prior to both DNA fragmentation and cytoplasmic changes. We show that the TLCK‐sensitive target is pre‐existing and not synthesized in response to apoptotic stimuli. The contrasting effects of TLCK and TPCK support the hypothesis that the TLCK target is a trypsin‐like protease which is a common effector of thymocyte apoptosis.
On the two forms of bacteriorhodopsin
Authors: Komrakov Andrey Yu. , Kaulen Andrey D.
Published: July 2002

In our previous work [(1993) FEBS Lett. 313, 248‐250; (1993) Biochem. Int. 30,461‐469] M‐intermediate formation of wild‐type bacteriorhodopsin was shown to involve two components differing in time constants (τ1 = 60–70 μs and τ2 = 220–250 μs), which were suggested to reflect two independent pathways of M‐intermediate formation. The contribution of the fast M was 4‐times higher than the slow one. Our present research on M‐intermediate formation in the D115N bacteriorhodopsin mutant revealed the same components but at a contribution ratio of 1:1. Upon lowering the pH, the slow phase of M‐formation vanished at a pK of 6.2, and in the pH region 3.0–5.5 only the M‐intermediate with a rise time of 60 μs was present. A 5–6 h incubation of D115N bacteriorhodopsin at pH 10.6 resulted in the irreversible transformation of 50% of the protein into a form with a difference absorbance maximum at 460 nm. This form was stable at pH 7.5 and had no photocycle, including M‐intermediate formation. The remaining bacteriorhodopsin contained 100% fast M‐intermediate. The disappearance of the 250‐μs phase concomitant with bR460 formation indicates that at neutral pH bacteriorhodopsin exists as two spectroscopically indistinguishable forms.
Effects of l‐carnitine on the pyruvate dehydrogenase complex and carnitine palmitoyl transferase activities in muscle of endurance athletes
Authors: Arenas Joaquín , Huertas Rosa , Campos Yolanda , Díaz A.Enrique , Villalón José María , Vilas Esther
Published: July 2002

The effects of l‐carnitine on the pyruvate dehydrogenase (PDH) complex and carnitine palmitoyl transferase (CPT) were studied in muscle of 16 long‐distance runners (LDR). These subjects received placebo or l‐carnitine (2 g orally) during a 4‐week period of training. Athletes receiving l‐carnitine showed a dramatic increase (P < 0.001) in the PDH complex activities. By contrast, the levels of CPT, both 1 and 2, were unchanged. No significant changes were observed after placebo administration. We previously reported [Huertas R. et al., Biochem. Biophys. Res. Commun. 188 (1992) 102‐107] that l‐carnitine induces an increase in the activities of complexes I, III and IV of the respiratory chain in muscle of LDR. Taken together, our data suggest that the improvement in (maximal oxygen consumption) V
O2max observed in LDR after l‐carnitine administration is based on these biochemical findings.
The atomic structure of Carnation Mottle Virus capsid protein
Authors: Morgunova E.Yu. , Dauter Z. , Fry E. , Stuart D.I. , Stel'mashchuk V.Ya. , Mikhailov A.M. , Wilson K.S. , Vainshtein B.K.
Published: July 2002

The structure of the Carnation Mottle Virus (CMtV) capsid protein has been determinated at 3.2 Å resolution by the method of molecular replacement. Three‐dimensional data were collected from a small number of crystals (sp.g.I23, a = 382.6 Å) using the synchrotron radiation with an image plate as detector. The coordinates of Tomato Bushy Stunt Virus (TBSV) were used as a searching model. Refinement of the coordinates of 7,479 non‐hydrogen atoms performed by the program XPLOR, has led to an R‐factor of 18.3%. It was found that the amino acid chain fold of capsid protein is very similar to that in other icosahedral viruses. However, there are some differences in the contact regions between protein subunits and also the lack of the β‐annulus around the 3‐fold icosahedral axes. The structural and biochemical results lead us to consider an alternative assembly pathway.
Effects of temperature shifts on the activities of Neurospora crassa glycogen synthase, glycogen phosphorylase and trehalose‐6‐phosphate synthase
Authors: Noventa-Jordão Maria A. , de Lourdes Maria , Polizeli T.M. , Beatriz  , Bonini M. , Jorge João A. , Terenzi Héctor F.
Published: July 2002

Conidiospore germlings of Neurospora crassa submitted to a heat shock at 45°C accumulate trehalose and degrade glycogen. The opposite occurs upon reincubation at a physiologic temperature (30°C). These observations suggest a temperature‐dependent mechanism for the preferential synthesis of one or the other sugar reserve. Here we show that concomitant with these shifts of temperature, occurred reversible changes in the activities of glycogen synthase and phosphorylase. Glycogen synthase was inactivated at 45°C while phosphorylase was activated. The reverse was true when the cells were shifted back to 30°C. Addition of cycloheximide did not prevent the reversible enzymatic changes, which remained stable after gel filtration. Apparently, the effects of temperature shifts occurred at the level of reversible covalent enzymatic modifications. Trehalose‐6‐phosphate synthase properties were also affected by temperature. For instance, the enzyme was less sensitive to in vitro inhibition by inorganic phosphate at 50°C than at 30°C. Fructose‐6‐phosphate partially relieved the inhibitory effect of phosphate at 30°C but not at 50°C. These effects of the assay temperature, inorganic phosphate, and fructose‐6‐phosphate, on trehalose‐6‐phosphate synthase activity, were more evident for crude extracts obtained from heat‐shocked cells. Altogether, these results may contribute to explain the preferential accumulation of trehalose 45°C, or that of glycogen at 30°C.
Heuristic scheduling of capital constrained projects
Authors: Smith‐Daniels Dwight E. , Padman Rema , Smith‐Daniels Vicki L.
Published: July 2002

The movement to product and process development projects that involve joint ventures among strategic partners, as well as the increasing prevalence of projects within organizations has led to increased implementation of project scheduling methods. It is frequently the case that a capital constraint is placed on a project, thus limiting the number and value of activities that can be scheduled to occur simultaneously. However, the quantity of capital available to schedule activities can increase as additional cash is received as progress payments for completed activities. Since the project manager'so objective is to maximize project Net Present Value (NPV), it is important for the manager to develop a schedule that balances the early receipt of progress payments (which improve NPV and increase the capital balance available), with the delay of particularly large expenditures. Due to the intractability of optimal methods, the use of heuristic methods is required to solve problems of practical size. This paper presents the first test of heuristic methods for solving this problem. We use information from a relaxed optimization‐guided model that employs information from the unconstrained NPV‐optimal problem in heuristic procedure for solving the capital constrained problem. An experimental design is employed to test the heuristics that includes multiple factor levels for a number of project characteristics, including capital utilization, frequency of progress payments, and project network structure. The results indicate very good relative performance for the optimization‐guided procedures as compared to two benchmark heuristics.
Cell type specific localization of sphingomyelin biosynthesis
Authors: Sadeghlar Farsaneh , Sandhoff Konrad , van Echten-Deckert Gerhild
Published: July 2002

We have studied the incorporation of [14C]serine and of [3H]sphingosine into sphingomyelin in the presence or absence of brefeldin A (BFA) in three different cell types. Administration of BFA (1 μg/ml) to fibroblasts for 24 h increased the incorporation of label into sphingomyelin 1.5–3 fold compared with untreated controls. In contrast, BFA strongly decreased sphingomyelin biosynthesis (4–5 fold) in cerebellar neurons as well as in neuroblastoma cells. The effect of BFA on glycosphingolipid formation, however, was similar in all three cell types studied: an increased labeling of the precursor glycolipids GlcCer, LacCer, GM3 and GD3 was paralleled by a decreased formation of complex gangliosides, GM1, GD1a, GT1b and GQ1b. Our data therefore suggest that in neuronal cells sphingomyelin synthesis, like the formation of complex gangliosides, is localized primarily distal to the BFA block, in a post‐Golgi compartment, most probably the trans‐Golgi network, whereas in fibroblasts sphingomyelin biosynthesis is mainly localized prior to the BFA block, in the Golgi apparatus, as has been shown for LacCer, GlcCer, GM3 and GD3 synthases.
A putative binding protein for lipophilic substances related to butterfly oviposition
Authors: Tsuchihara Kazuko , Ueno Kohei , Yamanaka Akira , Isono Kunio , Endo Katsuhiko , Nishida Ritsuo , Yoshihara Kazuo , Tokunaga Fumio
Published: July 2002

A unique protein of 23 kDa (Jf23) was found in the tarsus of the female swallowtail butterfly, Atrophaneura alcinous. Jf23 has 38% identity with a bilin‐binding protein, which was found in the cabbage butterfly, Pieris brassicae, and which has two consensus sequences in common with the members of the lipocalin family, suggesting that it is a binding protein for lipophilic ligands. Western blot analysis showed that Jf23 was expressed only in the female, and not in the male. Electrophysiological response of the female tarsi was stimulated by methanolic extract of their host plant, Dutchman's pipe (Aristolochia debilis). The stimulated response was depressed by the presence of Jf23 antiserum. These results suggest that Jf23 is one of the chemosensory signaling proteins, which plays one or more roles in female butterfly oviposition.
Hypochlorite modified LDL are a stronger agonist for platelets than copper oxidized LDL
Authors: Volf Ivo , Roth Astrid , Cooper Julian , Moeslinger Thomas , Koller Elisabeth
Published: July 2002

Experimental low density lipoprotein (LDL) oxidation is usually performed using trace copper, although the in vivo relevance of this method has been called into question. Such LDL augment adenosine 5′‐diphosphate (ADP) induced platelet aggregation, presumably by the action of lipid derived compounds. In striking contrast, we find that LDL oxidized to a comparable extent by hypochlorite, an in vivo occurring oxidant, reveal themselves to be potent promoters of platelet aggregation. Interestingly, hypochlorite modified LDL seem to mediate their influence on human platelets by means of the modified apolipoprotein B‐100 (apoB) moiety. Also, the finding that hypochlorite modified albumin is able to trigger platelet aggregation suggests an essential role for hypochlorite modified protein(s) in the process of platelet activation.
Mitochondria, aging and longevity – a new perspective
Authors: Salvioli Stefano , Bonafè Massimiliano , Capri Miriam , Monti Daniela , Franceschi Claudio
Published: July 2002

A new perspective is emerging indicating that mitochondria play a critical role in aging not only because they are the major source and the most proximal target of reactive oxygen species, but also because they regulate stress response and apoptosis. Recent literature indicates that, in response to stress, a variety of molecules translocate to and localise in mitochondria. These molecules are likely to interact with each other, in order to mediate mitochondria/nucleus cross‐talk and to regulate apoptosis. We surmise that an integration of signals in multimolecular complexes occurs at mitochondrial level. These phenomena can be of critical importance for human aging and longevity.
The scaffolding protein CASK mediates the interaction between rabphilin3a and β‐neurexins
Authors: Zhang Yan , Luan Zhidong , Liu Aihua , Hu Gengxi
Published: July 2002

CASK, a member of the membrane‐associated guanylate kinase (MAGUK) superfamily, binds to the carboxyl‐terminus of β‐neurexins on the intracellular side of the presynaptic membrane. The guanylate kinase‐like (GUK) domains of MAGUKs lack kinase activities, but might be important for mediating specific protein–protein interaction. By a yeast two‐hybrid approach, we identified an interaction between the GUK domain of CASK and the C2B domain of rabphilin3a, a presynaptic protein involved in synaptic vesicle exocytosis. The interaction was confirmed by in vitro GST pull‐down and co‐immunoprecipitation assays. It was proposed that presynaptic vesicles might be guided to the vicinity of points of exocytosis defined by β‐neurexins via the interaction between rabphilin3a–CASK–β‐neurexins.
An interplay between the TOM complex and porin isoforms in the yeast Saccharomyces cerevisiae mitochondria
Authors: Antos Nina , Budzińska Malgorzata , Kmita Hanna
Published: July 2002

The outer mitochondrial membrane of Saccharomyces cerevisiae contains two isoforms of mitochondrial porin, known also as the voltage‐dependent anion channel. The isoform termed here porin1 displays channel‐forming activity enabling metabolite transport whereas the second one, termed here porin2, does not form a channel and its function is still not clear. We have shown recently that in the absence of porin1, the channel within the protein import machinery (the TOM complex) is essential for metabolite transport across the outer membrane [Kmita and Budzińska, Biochim. Biophys. Acta 1509 (2000) 6044–6050]. Here, we report that the TOM complex channel may also serve as a supplementary pathway for metabolites in the presence of porin1 when the permeability of the latter is limited and the role of the TOM complex seems to increase when porin2 is depleted.
Chloroplast TatC plays a direct role in thylakoid ΔpH‐dependent protein transport
Authors: Mori Hiroki , Summer Elizabeth J. , Cline Kenneth
Published: July 2002

The thylakoid ΔpH‐dependent pathway transports folded proteins. Identified components include Hcf106 and Tha4. Orthologs of these proteins plus a membrane protein called TatC are essential for the homologous bacterial Tat system. Here we report identification of a chloroplast TatC (cpTatC). cpTatC is an integral thylakoid membrane protein as determined by in vitro chloroplast import and immunoblotting. Antibody to cpTatC specifically inhibited the thylakoid ΔpH‐dependent pathway in vitro. cpTatC is present in about the same quantity as estimated translocation sites, whereas Hcf106 and Tha4 are present in 5–8‐fold excess. These results are relevant to mechanistic models for this system.
Influence of the conformational flexibility on the kinetics and dimerisation process of two Candida rugosa lipase isoenzymes
Authors: Pernas Marı́a A. , López Cristina , Rúa M.Luisa , Hermoso Juan
Published: July 2002

We have investigated the interfacial activation process of two isoenzymes from Candida rugosa (Lip1 and Lip3) using triacetin as substrate. Kinetics were coupled to inhibition experiments in order to analyse the transition between the open and closed conformers. This process was slow, particularly for Lip1, in the absence of an interface provided by the substrate or a detergent. Dimers of Lip3 were also purified and their catalytic action was closer to that of a typical esterase. In spite of the high sequence homology between Lip1 and Lip3, small changes enhance hydrophobicity in the binding pocket of Lip3 and increase the flexibility of its flap. We postulated that these factors account for the higher tendency of Lip3 to dimerise fixing its open conformation.
Cholesterol effects on the phospholipid condensation and packing in the bilayer: a molecular simulation study
Authors: Róg Tomasz , Pasenkiewicz-Gierula Marta
Published: July 2002

A 15‐ns molecular dynamics simulation of the fully hydrated liquid–crystalline dimyristoylphosphatidylcholine–cholesterol (DMPC–Chol) bilayer containing ∼22 mol% Chol was carried out. The generated trajectory was analysed to investigate the mechanism of the Chol condensing effect on DMPC hydrocarbon chains and the influence of Chol on the chain packing in the membrane. Chol was found to induce stronger van der Waals interactions among the chains, whereas its interactions with the chains were weak. In the DMPC–Chol bilayer, as in the DMPC bilayer, DMPC chains were regularly packed around a chosen chain but around a Chol molecule they were not. DMPC γ chains made closer contacts with Chol than the β chains.
Structural study of novel antimicrobial peptides, nigrocins, isolated from Rana nigromaculata
Authors: Park Sangho , Park Sang-Ho , Ahn Hee-Chul , Kim Sunkyu , Kim Sunny S. , Lee Byeong Jae , Lee Bong-Jin
Published: July 2002

Novel cationic antimicrobial peptides, named nigrocin 1 and 2, were isolated from the skin of Rana nigromaculata and their amino acid sequences were determined. These peptides manifested a broad spectrum of antimicrobial activity against various microorganisms with different specificity. By primary structural analysis, it was revealed that nigrocin 1 has high sequence homology with brevinin 2 but nigrocin 2 has low sequence homology with any other known antimicrobial peptides. To investigate the structure–activity relationship of nigrocin 2, which has a unique primary structure, circular dichroism (CD) and homonuclear nuclear magnetic resonance spectroscopy (NMR) studies were performed. CD investigation revealed that nigrocin 2 adopts mainly an α‐helical structure in trifluoroethanol (TFE)/H2O solution, sodium dodecyl sulfate (SDS) micelles, and dodecylphosphocholine micelles. The solution structures of nigrocin 2 in TFE/H2O (1:1, v/v) solution and in SDS micelles were determined by homonuclear NMR. Nigrocin 2 consists of a typical amphipathic α‐helix spanning residues 3–18 in both 50% TFE solution and SDS micelles. From the structural comparison of nigrocin 2 with other known antimicrobial peptides, nigrocin 2 could be classified into the family of antimicrobial peptides containing a single linear amphipathic α‐helix that potentially disrupts membrane integrity, which would result in cell death.
Cystic fibrosis, lung infections, and a human tracheal antimicrobial peptide (hTAP)
Authors: Ko Young Hee , Delannoy Michael , Pedersen Peter L
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Post‐translational modification of heterologously expressed Streptomyces type II polyketide synthase acyl carrier proteins
Authors: Cox Russell J , Hitchman Timothy S , Byrom Kate J , Findlow I.Stuart C , Tanner Julian A , Crosby John , Simpson Thomas J
Published: July 2002

Expression in Escherichia coli of Streptomyces acyl carrier proteins (ACPs) associated with polyketide biosynthesis using the pT7‐7 expression system of Tabor and Richardson led to the production predominantly of inactive apo‐proteins lacking the 4′‐phosphopantetheinyl prosthetic group essential for polyketide synthase activity. Modification of growth conditions led to an increase of production of active holo‐protein for the actinorhodin (act) ACP, but this technique was ineffective for oxytetracycline (otc) and griseusin (gris) ACPs. Labelling experiments revealed that a low level of otc ACP expressed prior to induction was produced mainly as active holo‐protein, while post‐induction 15N‐labelled protein was almost exclusively in the apo‐ACP form. Limiting endogenous holo‐acyl carrier protein synthase (ACPS) concentration was implicated as responsible for low apo‐ACP to holo‐ACP conversion, rather than limiting substrate (coenzyme A) and cofactor (Mg2+) concentrations. Co‐expression of act and gris ACPs with ACPS in E. coli led to high levels of production of active holo‐ACPs and ACPS. We have also made the significant observation that ACPS is able to transfer acylated CoA moieties to act apo‐ACP.
Cloning and recombinant expression of rat and human kynureninase
Authors: Toma Salvatore , Nakamura Masayuki , Toné Shigenobu , Okuno Etsuo , Kido Ryo , Breton Jerome , Avanzi Nilla , Cozzi Liviana , Speciale Carmela , Mostardini Marina , Gatti Silvia , Benatti Luca
Published: July 2002

Kynureninase [E.C.3.7.1.3.] is one of the enzymes involved in the biosynthesis of NAD cofactors from tryptophan through the kynurenine pathway. By tryptic and CNBr digestion of purified rat liver kynureninase, we obtained about 28% of the amino acid sequence of the enzyme. The rat kynureninase cDNA, isolated by means of reverse‐transcribed polymerase chain reaction and hybridization screening, codes for a polypeptide of 464 amino acids. Northern blot analysis revealed the synthesis of a 2.0 kb rat kynureninase mRNA. A cDNA encoding human liver kynureninase was also isolated. The deduced amino acid sequence is 85% identical to that of the rat protein. COS‐1 cells were transfected with both cDNAs. The K
m values of the rat enzyme, for l‐kynurenine and dl‐3‐hydroxykynurenine, were 440±20 μM and 32±5 μM and of the human enzyme 440±20 μM and 49±6 μM, respectively. Interestingly, COS‐1 cells transfected with the cDNA coding for rat kynureninase also display cysteine‐conjugate β‐lyase activity.
Molecular cloning of a novel phosphorylation‐dependent inhibitory protein of protein phosphatase‐1 (CPI17) in smooth muscle: its specific localization in smooth muscle1
Authors: Eto Masumi , Senba Shingo , Morita Fumi , Yazawa Michio
Published: July 2002

The cDNA encoding a phosphorylation‐dependent inhibitory protein of protein phosphatase‐1 (PP1) was isolated from a porcine aorta library. The coding region represented the complete amino acid sequence of this protein comprised of a novel 147‐residue polypeptide, which we termed CPI17, a 17‐kDa PKC‐potentiated inhibitory protein of PP1. As well as the native CPI17 from porcine aorta, the recombinant protein completely suppressed the PP1 activity (IC50=0.18 nM) by the stoichiometric thiophosphorylation. The CPI17 mRNA is expressed in smooth muscle tissues such as aorta and bladder, whereas little expression was observed in heart, skeletal muscle, and non‐muscle tissues. These results suggest a specific regulatory mechanism of the PP1 activity through CPI17 in smooth muscle.
Specificity of different isoforms of protein phosphatase‐2A and protein phosphatase‐2C studied using site‐directed mutagenesis of HMG‐CoA reductase
Authors: Pang Ching Yick , Kobayashi Takayasu , Tamura Shinri , Grahame Hardie D
Published: July 2002

We have expressed the catalytic domain of Chinese hamster HMG‐CoA reductase, and 13 point mutations involving the region around the single phosphorylation site for AMP‐activated protein kinase. After phosphorylation, these were used to test the specificity of isoforms of protein phosphatase‐2A [bovine PP2AC (catalytic subunit) and PP2A1 (ABC heterotrimer)] and protein phosphatase‐2C (human α; mouse α, β1, β2, β3, β4, β5). PP2A1 had >50‐fold higher activity for HMG‐CoA reductase variants than PP2AC, but their relative selectivity for different variants was similar. Although the specificities of PP2A and PP2C were distinct, no dramatic differences in selectivity were observed between different PP2C isoforms.
Substrate binding to the inactive mutants of Streptomyces sp. N174 chitosanase: indirect evaluation from the thermal unfolding experiments
Authors: Honda Yuji , Fukamizo Tamo , Boucher Isabelle , Brzezinski Ryszard
Published: July 2002

Oligosaccharide binding to chitosanase from Streptomyces sp. N174 was indirectly evaluated from thermal unfolding experiments of the protein. Thermal unfolding curves were obtained by fluorescence spectroscopy in the presence of d‐glucosamine oligosaccharides ((GlcN)
n
, n=3,4,5, and 6) using the inactive mutant chitosanase in which the catalytic residue, Glu22, is mutated to glutamine (E22Q), aspartic acid (E22D), or alanine (E22A). The midpoint temperature of the unfolding transition (T
m) of E22Q was found to be 44.4°C at pH 7.0. However, the T
m increased upon the addition of (GlcN)
n
by 1.3°C (n=3), 2.5°C (n=4), 5.2°C (n=5), or 7.6°C (n=6). No appreciable change in T
m was observed when (GlcNAc)6 was added to E22Q. The effect of (GlcN)
n
on the thermal stability was examined using the other protein, RNase T1, but the oligosaccharide did not affect T
m of the protein. Thus, we concluded that the stabilization effect of (GlcN)
n
on the chitosanase results from specific binding of the oligosaccharides to the substrate binding cleft. When E22D or E22A was used instead of E22Q, the increases in T
m induced by (GlcN)6 binding were 2.7°C for E22D and 4.2°C for E22A. In E22D or E22A, interaction with (GlcN)6 seems to be partly disrupted by a conformational distortion in the catalytic cleft.
Phytoalexin production elicited by exogenously applied jasmonic acid in rice leaves (Oryza sativa L.) is under the control of cytokinins and ascorbic acid
Authors: Tamogami Shigeru , Rakwal Randeep , Kodama Osamu
Published: July 2002

Jasmonic acid (JA) has been shown to be a signaling compound which elicits the production of secondary metabolites including phytoalexins in plants. It has been shown that the phytoalexin production is elicited by exogenously applied JA in rice leaves. We now show that this phytoalexin production by exogenously applied JA is significantly counteracted by cytokinins, kinetin and zeatin. Kinetin and zeatin also inhibit the induction of naringenin‐7‐O‐methyltransferase (a key enzyme in rice phytoalexin production) by JA. A natural free radical scavenger, ascorbic acid (AsA) shows both counteractive and enhancing effects on JA‐inducible phytoalexin production, depending on its concentration. This effect of AsA suggests that active oxygen species (AOS) may play important roles in phytoalexin production by JA in rice leaves.
Membrane association of FtsY, the E. coli SRP receptor
Authors: de Leeuw Erik , Poland Dennis , Mol Olaf , Sinning Irmgard , ten Hagen-Jongman Corinne M , Oudega Bauke , Luirink Joen
Published: July 2002

FtsY, the Escherichia coli homologue of the eukaryotic SRP receptor (SRα), is located both in the cytoplasm and in the inner membrane of E. coli. Similar to SRα, FtsY consists of two major domains: a strongly acidic N‐terminal domain (A) and a C‐terminal GTP binding domain (NG) of which the crystal structure has recently been determined. The domains were expressed both in vivo and in vitro to examine their subcellular localization. The results suggest that both domains associate with the membrane but that the nature of the association differs.
Nuclear translocation of the Y‐box binding protein by ultraviolet irradiation
Authors: Koike Koji , Uchiumi Takeshi , Ohga Takefumi , Toh Satoshi , Wada Morimasa , Kohno Kimitoshi , Kuwano Michihiko
Published: July 2002

The Y‐box binding protein, YB‐1, is a member of a DNA binding protein family with a structurally and functionally conserved cold shock domain. Using Western blotting and immunohistochemical methods, larger amounts of YB‐1 were detected in the cytosol, particularly at the perinuclear region, than in the nucleus of human cancer cells. UV irradiation increased accumulation of YB‐1 in the nucleus at 20 min and thereafter. This translocation of YB‐1 into the nucleus by UV irradiation was blocked by the protein kinase inhibitor H‐7, but not HA‐1004. Both green fluorescent protein (GFP)‐YB‐1 and GFP‐YB‐1C with the C‐terminus (248–317) of YB‐1 were located mainly in the cytosol, but GFP‐YB‐1ΔC with a deletion at the C‐terminus of YB‐1 was located in the nucleus. YB‐1 is translocated into the nucleus by UV irradiation, possibly through a protein kinase C‐mediated signal transduction pathway, and the C‐terminal region of YB‐1 might be important for cytoplasmic retention of YB‐1.
The p38‐MAPK inhibitor, SB203580, inhibits cardiac stress‐activated protein kinases/c‐Jun N‐terminal kinases (SAPKs/JNKs)
Authors: Clerk Angela , Sugden Peter H
Published: July 2002

SB203580 is a recognised inhibitor of p38‐MAPKs. Here, we investigated the effects of SB203580 on cardiac SAPKs/JNKs. The IC50 for inhibition of p38‐MAPK stimulation of MAPKAPK2 was approximately 0.07 μM, whereas that for total SAPK/JNK activity was 3–10 μM. SB203580 did not inhibit immunoprecipitated JNK1 isoforms. Three peaks of SAPK/JNK activity were separated by anion exchange chromatography, eluting in the isocratic wash (44 kDa), and at 0.08 M (46 and 52 kDa) and 0.15 M NaCl (54 kDa). SB203580 (10 μM) completely inhibited the 0.15 M NaCl activity and partially inhibited the 0.08 M NaCl activity. Since JNK1 antibodies immunoprecipitate the 46 kDa activity, this indicates that SB203580 selectively inhibits 52 and 54 kDa SAPKs/JNKs.
A cysteine‐11 to serine mutant of Gα12 impairs activation through the thrombin receptor
Authors: Ponimaskin Evgeni , Harteneck Christian , Schultz Günter , Schmidt Michael F.G.
Published: July 2002

We have recently reported that Gα12 is acylated with palmitic acid [Veit et al., FEBS Lett. 339 (1994) 160–164]. Here we identify cysteine 11 as the sole palmitoylation site and assess the function of Gα12 palmitoylation after expression of wild type and acylation‐deficient mutant in insect cells. Our experimental approach yielded the following results. (1) Palmitoylation of Gα12 has no influence on the subunit interactions. (2) Palmitoylation promotes membrane binding of Gα12 when this protein is expressed alone. Membrane attachment of the heterotrimer occurs independent of the presence of fatty acids in Gα12. (3) Assays for agonist‐stimulated binding of [35S]GTPγS after expression of the human thrombin receptor (PAR1) along with Gα12 and the βγ subunits revealed a 70% inhibition with the palmitoyl‐deficient mutant.