bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of pteridine derivatives on intracellular calcium concentration in human monocytic cells
Authors: Wöll E. , Weiss G. , Fuchs D. , Lang F. , Wachter H.
Published: July 2002

Pteridines are heterocyclic compounds which are synthesized and released by human monocytes/macrophages following stimulation by interferonγ. Their concentration in various body fluids proved to be indicative for the stimulation of the cellular immune system, and determination of pteridines has become an important diagnostic tool. We show that pteridine derivatives, namely neopterin (N), 7,8‐dihydroneopterin (NH2), and 5,6,7,8‐tetrahydrobiopterin (BH4) increase intracellular calcium (Cai) in human monocytic cells. Significant increases of Cai, are observed at 10 nmol/l NH2, at 100 nmol/l BH4 and at 1 mol/l N, i.e. at concentrations encountered in vivo. At a concentration of 1 μmol/l, Cai is increased (from a control value of 145 ± 7 nmol/l) to 464 ± 62 nmol/l (NH2), 340 ± 41 nmol/l (BH4) and 344 ± 46 nmol/l (N), respectively. The increase of Cai depends on the presence of extracellular calcium and is likely to be due to activation of a calcium channel. We show that the absence of extracellular calcium or the addition of lanthanum ions to the extracellular fluid fully reverses the pteridine‐induced increase of Cai. According to these observations, pteridines may mimick the effects of other inflammatory mediators on monocytic cells and seem to be involved in the crosstalk of immunocompetent cells.
The structure of apo‐calmodulin
Authors: Finn Bryan E. , Drakenberg Torbjörn , Forsén Sture
Published: July 2002

The structure of the carboxy‐terminal domain of bovine calmodulin, TR2C, in the calcium‐free form was investigated using two‐dimensional 1H NMR. Sequential resonance assignments were made using standard methods. Using information from medium and long range contacts revealed by nuclear Overhauser enhancement, the secondary structure and global fold were determined. The apo protein possesses essentially the same secondary structure as that in the calcium activated form of intact calmodulin. However, the secondary structural elements are rearranged so that the hydrophobic binding pocket is closed in the apo‐form.
Multiple genes for G protein‐coupled receptors and their expression in lingual epithelia
Authors: Abe Keiko , Kusakabe Yuko , Tanemura Kentaro , Emori Yasufumi , Arai Soichi
Published: July 2002

Using the polymerase chain reaction (PCR), we identified a gene family including more than 60 members which encoded similar G protein‐coupled seven‐transmembrane receptors. Sequence analyses of six representatives out of the 60 PCR clones showed that they had significant structural similarity to olfactory and optic receptors. Their expression is restricted in the surface of lingual epithelia.
Binding of retinoids to uteroglobin
Authors: de Haro M.S.López , Martinez M.Pérez , García C. , Nieto A.
Published: July 2002

Uteroglobin, a progesterone‐binding secretory protein, was shown to bind retinoic acid and retinol in a non‐saturable manner, at least up to concentrations of retinoids of 20 μM. Binding is increased about 10‐fold by previous reduction of uteroglobin with 10 mM dithothreitol and it is not affected by previous saturation of the progesterone binding site, suggesting different binding sites for the steroid and the retinoids. The results are discussed in relation to a possible physiological role for this protein.
Immunoreactivity of chimeric proteins carrying the HIV‐1 epitope IGPGRAF Correlation between predicted conformation and antigenicity
Authors: Tisminetzky Sergio G. , Scodeller Eduardo A. , Evangelisti Paolo. , Chen Yaunding , Schiappacassi Monica , Porro Fabiola , Bizik Frantisek , Zacchi Tullio , Lunazzi Giancarlo , Miertus Stanislav , Baralle Francisco E.
Published: July 2002

Sera from HIV‐1 infected individuals were examined for their reactivity to the principal neutralizing domain, IGPGRAF sequence, of the V3‐loop of HIV‐1. Four hybrid proteins carrying this sequence inserted in four different outer loops of a protein that makes up the capsid of an insect virus were used as antigen in a Western blot assay for this survey. All the four antigens showed different activity: sera that recognise all antigens to sera that reacted with only one of them. Competition experiments indicated that the antibodies recognised these proteins with different affinity. Molecular modelling of the hybrid proteins predicted that the inserted sequence adopted different conformations in each position. Comparison of predicted most stable conformations for IGPGRAF indicated that there is a close relationship between conformational similarity to a V3‐loop reference structure and the degree of reactivity with sera.
A specific carotenoid is required for reconstitution of the Rubrivivaxgelatinosus B875 light harvesting complex from its subunit form B820
Authors: Jirsakova Vladimira , Reiss-Husson Françoise
Published: July 2002

The core light‐harvesting complex B875 from Rubrivivax gelatinosus has been reconstituted from its subunit form B820 with hydroxyspheroidene, the carotenoid which is bound to native B875 antenna. Other carotenoids which are chemically similar to hydroxyspheroidene (spheroidene, spheroidenone, neurosporene and spirilloxanthin) gave only low levels of partial reconstitution. Absorption and circular dichroism spectra of the hydroxyspheroidene‐containing, reconstituted B875 were identical with those of original B875 antenna isolated directly from chromatophores, indicating that the two hydroxyspheroidene molecules bind to their native sites within the (αβ)Bchl2, subunit during the reconstitution process. These observations point to a structural role for this carotenoid in determining the architecture of Rv. gelatinosus B875 antenna.
Fusion complex formation protects synaptobrevin against proteolysis by tetanus toxin light chain
Authors: Pellegrini Lorenzo L. , O'Connor Vincent , Betz Heinrich
Published: July 2002

The clostridial neurotoxin, tetanus toxin, is a Zn2+‐dependent protease which inhibits neurotransmitter exocytosis by selective cleavage of the synaptic vesicle protein, synaptobrevin. Synaptobrevin is thought to serve as a receptor for two neuronal plasma membrane proteins, syntaxin and SNAP‐25, which in the presence of non‐hydrolyzable ATP analogs form a 20 S fusion complex with the soluble fusion proteins NSF and α‐SNAP. Here we show that synaptobrevin, when in this 20 S complex, or its 7 S precursor, is protected against proteolysis by the enzymatically active tetanus toxin light chain. Our data define distinct pools of synaptobrevin, which provide markers of different steps of vesicle/plasma membrane interaction.
Enhancement of hydrogen peroxide‐induced luminol‐dependent chemiluminescence by neopterin depends on the presence of iron chelator complexes
Authors: Murr Christian , Fuchs Dietmar , Gössler Walter , Hausen Arno , Reibnegger Gilbert , Werner Ernst R. , Werner-Felmayer Gabriele , Esterbauer Hermann , Wachter Helmut
Published: July 2002

We have previously shown that neopterin, 6‐d‐erythro‐trihydroxypropyl‐pteridine, synthesized by human monocytes/macrophages upon stimulation by interferon‐γ, enhances toxicity of reactive oxygen at neutral or slightly alkaline pH (7.5), but not at acidic pH (below 6.5). In the present study, we explored in more detail the necessary requirements for neopterin to modulate the effects of hydrogen peroxide in a luminol‐dependent chemiluminescence assay. We demonstrate that neopterin enhances hydrogen peroxide effects only in the presence of iron chelator complexes like iron‐(III)‐ or iron‐(II)‐EDTA or iron‐(III)‐DTPA. Thus, iron chelator complexes together with neopterin may play an important role in macrophage‐mediated effector mechanisms.
A novel plant glutathione peroxidase‐like protein provides tolerance to oxygen radicals generated by paraquat in Escherichia coli
Authors: Holland Doron , Faltin Zehava , Perl Avi , Ben-Hayyim Gozal , Eshdat Yuval
Published: July 2002

Citrus salt‐stress associated protein (Cit‐SAP) reveals significant sequence homology to mammalian glutathione peroxidase (GP). In an attempt to assign biological function to this protein, transformed E. coli cells expressing Cit‐SAP were examined for their ability to tolerate free radicals formed by paraquat, an O−
2 radical forming agent. In the presence of paraquat, the survival rate of the transformed bacteria expressing Cit‐SAP was much higher as compared to the wild‐type bacteria. The results support the assumption that Cit‐SAP is a plant GP‐like protein which participate in the enzymatic system aimed at scavenging oxygen free‐radicals in plants.
Succinyl phosphonate inhibits α‐ketoglutarate oxidative decarboxylation, catalyzed by α‐ketoglutarate dehydrogenase complexes from E. coli and pigeon breast muscle
Authors: Biryukov A.I. , Bunik V.I. , Zhukov Yu.N. , Khurs E.N. , Khomutov R.M.
Published: July 2002

Effects of a set of α‐ketoglutarate phosphoanalogues on the activity of α‐ketoglutarate dehydrogenase (EC 1.2.4.2) complexes from E. coli and pigeon breast muscle, as well as on α‐ketoglutarate dehydrogenase isolated from the pigeon breast muscle, have been studied. α‐Ketoglutarate phosphoanalogues (succinyl phosphonate and its monomethyl ester) were found to be effective inhibitors of α‐ketoglutarate oxidative decarboxylation, catalyzed by both muscle and bacterial α‐ketoglutarate dehydrogenase complexes, as well as muscle α‐ketoglutarate dehydrogenase. The ability of glutamate phosphoanalogues to inhibit α‐ketoglutarate oxidative decarboxylation has been shown in E. coli extract and a model system.
The myrosinase‐glucosinolate interaction mechanism studied using some synthetic competitive inhibitors
Authors: Iori Renato , Rollin Patrick , Streicher Harald , Thiem Joachim , Palmieri Sandro
Published: July 2002

Using synthetic deoxy‐glucotropaeolins (6d‐GTL, 4d‐GTL, 3d‐GTL, 2d‐GTL) as substrates, myrosinase activity was studied in comparison to that determined on native glucotropaeolin (GTL) isolated from ripe Lepidium sativum seeds. When the deoxy substrates were used, in addition to an overall strong reaction rate decline, a significant decrease in the reaction rate was observed in going from 6d‐ to 2d‐GTL. This finding allows us to propose a mechanism of catalysis which appears to be similar in many respects to that established for β‐glucosidases. Finally, 2d‐GTL was shown to be the first strong competitive inhibitor of myrosinase ever reported.
Genomes with distinct function composition
Authors: Tamames Javier , Ouzounis Christos , Sander Chris , Valencia Alfonso
Published: July 2002

The functional composition of organisms can be analysed for the first time with the appearance of complete or sizeable parts of various genomes. We have reduced the problem of protein function classification to a simple scheme with three classes of protein function: energy‐, information‐ and communication‐associated proteins. Finer classification schemes can be easily mapped to the above three classes. To DAal with the vast amount of information, a system for automatic function classification using database annotations has been DAveloped. The system is able to classify correctly about 80% of the query sequences with annotations. Using this system, we can analyse samples from the genomes of the most represented species in sequence databases and compare their genomic composition. The similarities and differences for different taxonomic groups are strikingly intuitive. Viruses have the highest proportion of proteins involved in the control and expression of genetic information. Bacteria have the highest proportion of their genes DAdicated to the production of proteins associated with small molecule transformations and transport. Animals have a very large proportion of proteins associated with intra‐ and intercellular communication and other regulatory processes. In general, the proportion of communication‐related proteins increases during evolution, indicating trends that led to the emergence of the eukaryotic cell and later the transition from unicellular to multicellular organisms.
Laparoscopic para‐aortic lymph node sampling in the staging of invasive cervical carcinoma: including a comparative study of 21 laparotomy cases
Authors: Su T.H. , Wang K.G. , Yang Y.C. , Hong B.K. , Huang S.H.
Published: July 2002

Objectives: To assess the efficacy and risks of laparoscopic para‐aortic lymph node sampling compared with standard laparotomy in staging cervical carcinoma. Methods: From August 1993 through July 1994, 38 patients with biopsyproven invasive cervical carcinoma (24 early and 14 advanced invasive cancers) were entered into the study. This was a prospective study of laparoscopic para‐aortic lymphadenectomy in invasive cervical carcinoma, with patients serving as their own controls. Video laparoscopic lymph node sampling was performed. In patients with early invasive cancer, the nodes were sent for frozen section and, if negative, laparotomy was performed to look for any residual nodes. Radical hysterectomy was performed immediately if residual nodes were negative. Patients with either nodal metastasis on frozen section or with advanced cancer underwent para‐aortic lymphadenectomy only. The operative technique was also evaluated. Results: Laparoscopy required an average of 77 min (S.D. 40), with an average blood loss of 116 ml (S.D. 321). The average number of nodes was 15 (S.D. 7). At subsequent laparotomy the average number of residual nodes found was 0.4 (S.D. 0.9) and none showed metastasis. One vena cava laceration and one ureteral injury required immediate repair, and two patients were too obese to undergo laparoscopy. Conclusions: Laparoscopic para‐aortic lymph node sampling is a less invasive, reliable method for staging invasive cervical carcinoma and can substitute for traditional open procedures. The incidence of risks with this method appears to be low.
The development of cash flow weight procedures for maximizing the net present value of a project
Authors: Baroum Sami M. , Patterson James H.
Published: July 2002

We describe in this paper the development of heuristic procedures for obtaining improved solutions to the maximize ion of net present value in a network problem. Optimization techniques have not been successful in solving this limited resource problem for large projects. As a result, rule‐of‐thumb heuristic approaches have historically been used to improve the financial returns from project activity. The heuristic procedures we describe assign weights to activities based upon the cumulative cash flows for the activity and all of its logical successors. Subsequent application of multi‐pass shifting algorithms search locally for improved activity assignments, resulting in increased project net present value over the initial solution obtained. Results reported demonstrate the efficacy of this approach over more traditional methods for solving this problem. We also contrast the cash flow weighting heuristics developed with other implementations of cash flow weighting procedures. Despite their inherent simplicity, the procedures described quite often produce improved results over other approaches when maximum net present value solutions are desired.
Termination of IL‐6‐induced STAT activation is independent of receptor internalization but requires de novo protein synthesis
Authors: Thiel Stefan , Sommer Ulrike , Kortylewski Marcin , Haan Claude , Behrmann Iris , Heinrich Peter C , Graeve Lutz
Published: July 2002

In this paper we have studied whether the down‐modulation of the STAT signal requires the endocytosis of the receptor complex. Our results suggest that the desensitization of the IL‐6 signal is not due to internalization of the receptor complex but requires de novo protein synthesis.
Mit1/Lb9andCopg2, new members of mouse imprinted genes closely linked toPeg1/Mest
Authors: Lee Young Jae , Park Chang Won , Hahn Yoonsoo , Park Jeongmin , Lee Jieun , Yun Ji Hye , Hyun Byunghwa , Chung Jae Hoon
Published: July 2002

Two mouse genes,Mit1/Lb9andCopg2, linked toPeg1/Meston mouse chromosome 6, were identified to be imprinted maternally and paternally, respectively.Mit1/Lb9encoding untranslated transcripts resides within the intron 20 ofCopg2. The gene is maternally imprinted in adult mouse brain, partially imprinted in other tissues.Copg2consists of 24 exons within the >40 kb genomic region, being expressed ubiquitously in mouse tissues with a partial imprinting pattern in embryos, neonates, and adult brain in contrast to maternally imprinted humanCOPG2. In addition, we identified an antisense transcript ofCopg2,Copg2AS, which overlaps 3′‐UTRs ofCopg2andPeg1/Mest. TheCopg2AStranscript is maternally imprinted in embryos, neonates, and adult tissues.
In vivo measurements of control coefficients for hexokinase and glucose‐6‐phosphate dehydrogenase in Xenopus laevis oocytes
Authors: Ureta Tito , Fernández Waldy Y. , Centelles Josep J. , Cascante Marta
Published: July 2002

Hexokinase and glucose‐6‐phosphate dehydrogenase activities were increased in Xenopus laevis oocytes by microinjection of commercial pure enzymes. The effect of increased fractional activities on glycogen synthesis or on the production of 14CO2 (the oxidative portion of the pentose phosphate pathway) was investigated by microinjection of [1‐14C]glucose and measurements of the radioactivity in glycogen and CO2. Control coefficients calculated from the data show that hexokinase plays an important role in the control of glycogen synthesis (control coefficient=0.7) but its influence on the control of the pentose phosphate pathway is almost nil (control coefficient=−0.01). Glucose‐6‐phosphate dehydrogenase injections did not affect the production of 14CO2 by the pentose phosphate pathway, indicating that other factors control the operation of this pathway. In addition, an almost null control of this enzyme on glycogen synthesis flux was observed.
Production of polyomavirus structural protein VP1 in yeast cells and its interaction with cell structures
Authors: Palková Z , Adamec T , Liebl D , Štokrová J , Forstová J
Published: July 2002

The gene for mouse polyomavirus major structural protein VP1 was expressed in Saccharomyces cerevisiae from the inducible GAL7 promoter. VP1 pseudocapsids were purified from cell lysates. Their subpopulation contained fragments of host DNA, which, in contrast to those of VP1 pseudocapsids produced in insect cells, did not assemble with cellular histones into pseudonucleocores. VP1 pseudocapsids accumulated in the yeast cell nuclei. A strong interaction of VP1 with tubulin fibres of the mitotic spindle was observed. The fibres of spindles were larger in diameter, apparently due to tight VP1 binding. Substantial growth inhibition of yeast cells producing VP1 was observed.
Colocalization of leukotriene C synthase and microsomal glutathione S‐transferase elucidated by indirect immunofluorescence analysis
Authors: Surapureddi Sailesh , Svartz Jesper , Magnusson Karl-Eric , Hammarström Sven , Söderström Mats
Published: July 2002

We have previously shown that the two membrane bound enzymes leukotriene C synthase and microsomal glutathione S‐transferase interact in vitro and in vivo. Rat basophilic leukemia cells and murine mastocytoma cells, two well‐known sources of leukotriene C synthase, both expressed microsomal glutathione S‐transferase as determined by Western blot analyses. Several human tissues were found to contain both leukotriene C synthase and microsomal glutathione S‐transferase mRNA. These data suggest that the interaction may be physiologically important. To study this further, expression vectors encoding the two enzymes were cotransfected into mammalian cells and the subcellular localization of the enzymes was determined by indirect immunofluorescence using confocal laser scanning microscopy. The results showed that leukotriene C synthase and microsomal glutathione S‐transferase were both localized on the nuclear envelope and adjacent parts of the endoplasmic reticulum. Image overlay demonstrated virtually identical localization. We also observed that coexpression substantially reduced the catalytic activity of each enzyme suggesting that a mechanism involving protein–protein interaction may contribute to the regulation of LTC4 production.
Genomic structure and promoter analysis of the rat Kir7.1 potassium channel gene (Kcnj13)
Authors: Döring Frank , Karschin Andreas
Published: July 2002

In the brain inwardly rectifying potassium channel Kir7.1 subunits are predominantly expressed in the choroid plexus and meninges. To investigate this tissue‐specific expression pattern, we characterized the genomic organization and the 5′ proximal promoter of the rat Kir7.1 gene (Kcnj13). Starting from the major transcriptional initiation site, three exons in Kcnj13 give rise to the dominant ∼1.45 kb transcript in brain. Adjacent to the transcriptional start the minimal promoter which, uncommon for ion channels, contains a TATA‐ and CAAT‐box is controlled by AP‐1 factors and accounts for high gene expression levels. Luciferase reporter gene responses driven by the first 2.1 kb of the 5′ flanking region were similarly high in epithelial FRTL‐5 and neuronal N2A cells, suggesting that neuron‐specific repressor elements are located remote from the non‐selective minimal promoter.
Phosphorylated, but not native, tau protein assembles following reaction with the lipid peroxidation product, 4‐hydroxy‐2‐nonenal
Authors: Pérez M. , Cuadros R. , Smith M.A. , Perry G. , Avila J.
Published: July 2002

A correlation between hyperphosphorylation of tau protein and its aberrant assembly into paired helical filaments has lead to suggestions that phosphorylation controls assembly, but lacked a mechanistic basic. In this work, we have found that phosphorylated, but not native, tau protein is able to form polymers after the reaction with 4‐hydroxy‐2‐nonenal, a highly toxic product of lipid peroxidation. Phosphorylation of tau by both proline or non‐proline directed kinases, was able to assemble it into polymers.
Immunocytological localization of two plant fatty acid desaturases in the endoplasmic reticulum
Authors: Dyer John M. , Mullen Robert T.
Published: July 2002

The subcellular location of two integral membrane‐bound fatty acid desaturases (Fads), Fad2 and Fad3, was elucidated by immunofluorescence microscopic analyses of tobacco suspension cells transiently transformed with different epitope‐tagged versions of the enzymes. Both myc‐ or hemagglutinin‐tagged Fad2 and Fad3 localized to the same region of the endoplasmic reticulum (ER), as evidenced by their co‐localization with the ER lumenal protein calreticulin. Results from differential permeabilization experiments revealed that the N‐termini of both epitope‐tagged Fad2 and Fad3 were exposed on the cytosolic side of ER membranes. These data define the subcellular location and topological orientation of plant desaturases in ER membranes.
Alcohol dehydrogenase in human tissues: localisation of transcripts coding for five classes of the enzyme
Authors: Estonius Mats , Svensson Stefan , Höög Jan-Olov
Published: July 2002

Tissue distribution of the five identified classes of human alcohol dehydrogenase was studied by assessment of mRNA levels in 23 adult and four fetal tissues. Alcohol dehydrogenase of class I was found in most tissues, brain and placenta excluded, but expression levels among tissues differed widely. The distribution pattern of class III transcripts was consistent with those of housekeeping enzymes while, in contrast, class IV transcripts were found only in stomach. Transcripts of multiple length were detected for most classes and were due to different gene products arising through the use of different poly A signals or transcription from different gene loci. Both class II and class V showed a pattern of liver‐enriched expression. However, low mRNA levels were detected also in stomach, pancreas and small intestine for class II, and in fetal kidney and small intestine for class V. Significantly higher levels of class V transcripts were present in fetal liver when compared with levels in adult liver, which suggests that human class V is a predominantly fetal alcohol dehydrogenase.
Analysis of the promoter region of the murine gephyrin gene
Authors: Ramming Markus , Betz Heinrich , Kirsch Joachim
Published: July 2002

© 1997 Federation of European Biochemical Societies.
HIV‐1 protein Vpr causes gross mitochondrial dysfunction in the yeast Saccharomyces cerevisiae
Authors: Macreadie Ian G , Thorburn David R , Kirby Denise M , Castelli Laura A , de Rozario Nicole L , Azad Ahmed A
Published: July 2002

The biological effects of the HIV‐1 accessory protein, Vpr, have been studied in yeast expression systems. In our previous study [1], employing the pCUP1‐vpr copper‐inducible expression cassette, Vpr was shown to cause growth arrest and structural defects. In this study yeast constitutively expressing vpr, through elevated copy number and/or elevated transcription levels, displayed no growth arrest in fermentative growth conditions while Vpr was produced at much lower levels than in the inducible expression system. However, such cells were respiratory deficient and unable to utilise ethanol or glycerol as the sole carbon source. They exhibited gross mitochondrial dysfunction displayed in the loss of respiratory chain complex I, II, III, IV and citrate synthase activities. The effects on mitochondria required a C‐terminal domain of Vpr that contains a conserved amino acid sequence motif HFRIGCRHSRIG. These results suggest that the widely observed phenomenon of ‘Vpr‐induced growth arrest’ in human cells could be due to mitochondrial dysfunction.
Poly(ethylene glycol)‐lipid conjugates inhibit phospholipase C‐induced lipid hydrolysis, liposome aggregation and fusion through independent mechanisms
Authors: Basáñez Gorka , Goñi Félix M , Alonso Alicia
Published: July 2002

Poly(ethylene glycol)‐phosphatidylethanolamine (PEG‐PE) conjugates have been introduced in liposomal compositions. The resulting large unilamellar vesicles were subjected to the action of phospholipase C. Enzyme‐promoted vesicle aggregation and fusion were assayed in liposomes containing various proportions of PEG‐PE. At PEG‐PE concentrations above 1 mol% the rate of phospholipid hydrolysis decreases, perhaps because the PEG moiety hinders the enzyme from reaching the membrane surface. At concentrations above 0.1 mol% vesicle aggregation occurs at a slower rate, presumably because of the repulsive barrier properties or surface‐grafted PEG. Lipid mixing decreases in parallel with vesicle aggregation. Finally, liposomal fusion rates measured as mixing of vesicle aqueous contents are decreased at or even below 0.1 mol%. The latter inhibition is due, apart from the reduced rates of lipid hydrolysis, vesicle aggregation and lipid mixing, to a PEG‐PE‐based stabilization of the lipid bilayer structure. Thus the observed low rates of contents mixing arise from three combined and independent inhibitory effects of PEG‐PE.
1,2‐Diacylglycerol hydroperoxides induce the generation and release of superoxide anion from human polymorphonuclear leukocytes
Authors: Yamamoto Yorihiro , Kambayashi Yasuhiro , Ito Takashi , Keiichi Watanabe  , Nakano Minoru
Published: July 2002

We examined the effect of 1‐palmitoyl‐2‐linoleoylglycerol (PLG), PLG hydroxide (PLG‐OH), and PLG hydroperoxide (PLG‐OOH) on the release of superoxide anion from human PMNs monitored by the chemiluminescence generated by the superoxide anion‐sensitive reagent, 2‐methyl‐6‐(p‐methoxyphenyl)‐3,7‐dihydroimidazo[1,2‐a]pyrazin‐3‐one (MCLA). PLG‐OOH at low micromolar concentrations stimulated human PMNs whereas PLG and PLG‐OH did not. 1,3‐Dilinoleoylglycerol hydroperoxide, 1‐palmitoyl‐2‐linoleoylphosphatidylcholine hydroperoxide, and linoleic acid hydroperoxide were much less efficient in stimulating human PMNs than PLG‐OOH. The PKC inhibitors, chelerythrine chloride and staurosporine, inhibited the stimulation of PMNs. Possible pathophysiological role of 1,2‐diacylglycerol hydroperoxides is discussed.
Release of opioid peptides, gluten exorphins by the action of pancreatic elastase
Authors: Fukudome Shin-ichi , Jinsmaa Yunden , Matsukawa Taiji , Sasaki Ryuzo , Yoshikawa Masaaki
Published: July 2002

The release of opioid peptides, gluten exorphins A, which have been isolated from the pepsin–thermolysin digest of wheat gluten, with gastrointestinal proteases was examined. High levels of gluten exorphin A5 (Gly–Tyr–Tyr–Pro–Thr) immunoreactive materials were detected in the pepsin–pancreatic elastase digest by a competitive ELISA. From this digest, gluten exorphin A5, B5 and B4 were isolated. This means that these peptides are released in the gastrointestinal tracts after ingestion of wheat gluten. The yield of gluten exorphin A5 in the pepsin–elastase digest was larger than that in the pepsin–thermolysin digest. The gluten exorphin A5 sequence is found 15 times in the primary structure of the high molecular weight glutenin. The region from which gluten exorphin A5 was released by the action of pancreatic elastase was identified using synthetic fragment peptides.
Co‐refolding denatured‐reduced hen egg white lysozyme with acidic and basic proteins
Authors: Trivedi V.D , Raman B , Rao Ch.Mohan , Ramakrishna T
Published: July 2002

Refolding of denatured‐reduced lysozyme and the effect of co‐refolding it with other proteins such as RNase A, bovine serum albumin, histone, myelin basic protein, alcohol dehydrogenase and DNase I on the renaturation yield and the aggregation of lysozyme have been studied. Basic proteins consistently increase the renaturation yield of the basic protein lysozyme (10–20% more than in their absence) with little or no aggregation. On the other hand, co‐refolding of lysozyme with acidic proteins leads to aggregation and a significant decrease in renaturation yields. Our results show that hetero‐interchain interactions (non‐specific interactions) occur when the basic protein lysozyme is refolded together with acidic proteins such as bovine serum albumin, alcohol dehydrogenase or DNase I. Our results also suggest that the net charge on proteins plays a significant role in such non‐specific aggregation. These results should prove useful in understanding the hetero‐interchain interactions between folding polypeptide chains.
Maturation and degradation of β‐galactosidase in the post‐Golgi compartment are regulated by cathepsin B and a non‐cysteine protease
Authors: Okamura-Oho Yuko , Zhang Sunqu , Callahan John W , Murata Mitsuo , Oshima Akihiro , Suzuki Yoshiyuki
Published: July 2002

Lysosomal β‐galactosidase precursor is processed to a mature form and associated with protective protein in lysosomes. In this study we used two cysteine protease proinhibitors, E64‐d for cathepsins B, S, H, and L, and CA074Me for cathepsin B. They are converted intracellularly to active forms, E‐64c and CA074, respectively. Both active compounds inhibited maturation of the exogenous β‐galactosidase precursor, but E‐64c did not inhibit further degradation to an inactive 50‐kDa product. We concluded that cathepsin B participated exclusively in maturation of β‐galactosidase, and a non‐cysteine protease was involved in further degradation and inactivation of the enzyme molecule.
Antibacterial activity of a novel 26‐kDa serine protease in the yellow body of Sarcophaga peregrina (flesh fly) pupae
Authors: Tsuji Yumiko , Nakajima Yuki , Homma Ko-ichi , Natori Shunji
Published: July 2002

We have reported a novel serine protease produced by Sarcophaga peregrina (Nakajima et al., J. Biol. Chem. 272 (1997) 23805–23810). This 26‐kDa protease showed antibacterial activity against several bacteria. This activity was an intrinsic characteristic of the enzyme protein and not directly related to its protease activity, because treating the 26‐kDa protease with diisopropyl fluorophosphate had no appreciable effect on its antibacterial activity. Unlike bovine trypsin, the 26‐kDa protease interacted with acidic phospholipids, suggesting that its antibacterial activity is attributable to interaction with bacterial membranes.
Effect of P‐chirality of oligo(deoxyribonucleoside phosphorothioate)s on the activity of terminal deoxyribonucleotidyl transferase
Authors: Koziołkiewicz Maria , Maciaszek Anna , Stec Wojciech J , Semizarov Dmitry , Victorova Lyubov , Krayevsky Alexander
Published: July 2002

Phosphorothioate analogues of oligonucleotides (PS‐oligos) of predetermined chirality at the phosphorus atom at each internucleotide linkage have been used as primers for terminal deoxyribonucleotidyl transferase (TdT, EC 2.7.7.31). The enzyme catalyzes efficient elongation of PS primers in which all phosphorothioate internucleotide linkages are uniformly of the [RP] configuration, while the presence of the linkage(s) of the [SP] configuration significantly decreases or completely inhibits the primer extension. Our results indicate that for the elongation of phosphorothioate oligomers the most important is the internucleotide bond located between the second and the third nucleoside from the 3′‐end. The presence of [SP] linkage at this position strongly reduces the enzyme activity while the [RP] bond allows for effective elongation of the primer. The activity of the enzyme is also influenced by base composition and sequence of phosphorothioate primer as well as the dNTP used for elongation process.
Identification of arginine‐700 as the residue that binds the C‐5 carboxyl group of 2‐oxoglutarate in human lysyl hydroxylase 1
Authors: Passoja Kaisa , Myllyharju Johanna , Pirskanen Asta , Kivirikko Kari I
Published: July 2002

Lysyl hydroxylase catalyzes the formation of hydroxylysine in collagens by a reaction that involves oxidative decarboxylation of 2‐oxoglutarate. Its binding site can be divided into two main subsites: subsite I consists of a positively charged side‐chain which binds the C‐5 carboxyl group, while subsite II consists of two coordination sites of the enzyme‐bound Fe2+ and is chelated by the C‐1‐C‐2 moiety. In order to identify subsite I, we converted Arg‐697, Arg‐700 and Ser‐705 individually to alanine and Arg‐700 also to lysine, and expressed the mutant enzymes in insect cells. Arg‐700‐Ala inactivated lysyl hydroxylase completely, whereas Arg‐697‐Ala and Ser‐723‐Ala had only a relatively minor effect. Arg‐700‐Lys produced 93% inactivation under standard assay conditions, the main effect being a 10‐fold increase in the K
m for 2‐oxoglutarate, whereas the V
max was unchanged. Arg‐700 thus provides the positively charged residue that binds the C‐5 carboxyl group of 2‐oxoglutarate, whereas Ser‐705 appears to be of no functional significance in this binding.
Regulation of cardiac L‐type Ca2+ channel by coexpression of Gαs in Xenopus oocytes
Authors: Blumenstein Yakov , Ivanina Tatiana , Shistik Elena , Bossi Elena , Peres Antonio , Dascal Nathan
Published: July 2002

Activation of Gαs via β‐adrenergic receptors enhances the activity of cardiac voltage‐dependent Ca2+ channels of the L‐type, mainly via protein kinase A (PKA)‐dependent phosphorylation. Contribution of a PKA‐independent effect of Gαs has been proposed but remains controversial. We demonstrate that, in Xenopus oocytes, antisense knockdown of endogenous Gαs reduced, whereas coexpression of Gαs enhanced, currents via expressed cardiac L‐type channels, independently of the presence of the auxiliary subunits α2/δ or β2A. Coexpression of Gαs did not increase the amount of α1C protein in whole oocytes or in the plasma membrane (measured immunochemically). Activation of coexpressed β2 adrenergic receptors did not cause a detectable enhancement of channel activity; rather, a small cAMP‐dependent decrease was observed. We conclude that coexpression of Gαs, but not its acute activation via β‐adrenergic receptors, enhances the activity of the cardiac L‐type Ca2+ channel via a PKA‐independent effect on the α1C subunit.
1α,25‐Dihydroxy‐3‐epi‐vitamin D3: In vivo metabolite of 1α,25‐dihydroxyvitamin D3 in rats
Authors: Sekimoto H , Siu-Caldera M.-L , Weiskopf A , Vouros P , Muralidharan K.R , Okamura W.H , Uskokovic M.R , Reddy G.S
Published: July 2002

We recently identified 1α,25‐dihydroxy‐3‐epi‐vitamin D3 as a major in vitro metabolite of 1α,25‐dihydroxyvitamin D3, produced in primary cultures of neonatal human keratinocytes. We now report the isolation of 1α,25‐dihydroxy‐3‐epi‐vitamin D3 from the serum of rats treated with pharmacological doses of 1α,25‐dihydroxyvitamin D3. 1α,25‐dihydroxy‐3‐epi‐vitamin D3 was identified through its co‐migration with synthetic 1α,25‐dihydroxy‐3‐epi‐vitamin D3 on both straight and reverse phase high performance liquid chromatography systems and by mass spectrometry. Along with 1α,25‐dihydroxy‐3‐epi‐vitamin D3, other previously known metabolites, namely, 1α,24(R),25‐trihydroxyvitamin D3, 1α,25‐dihydroxy‐24‐oxo‐vitamin D3 and 1α,25‐dihydroxyvitamin D3‐26,23‐lactone, were also identified. Thus, our study for the first time provides direct evidence to indicate that 1α,25‐dihydroxy‐3‐epi‐vitamin D3 is an in vivo metabolite of 1α,25‐dihydroxyvitamin D3 in rats.
Identification of a Ca2+/calmodulin‐binding domain within the carboxyl‐terminus of the angiotensin II (AT1A) receptor
Authors: Thomas Walter G. , Pipolo Luisa , Qian Hongwei
Published: July 2002

To identify regulators of the type 1A angiotensin II receptor (AT1A), we investigated the interaction of cellular proteins with a fusion protein containing the rat AT1A receptor carboxyl‐terminus. An ∼20 kDa cytoplasmic protein interacted with the fusion protein in a Ca2+‐dependent manner and was identified as calmodulin. A control peptide with high affinity for Ca2+/calmodulin and a peptide corresponding to a membrane proximal portion of the AT1A receptor carboxyl‐terminus with analogy to known calmodulin‐binding sequences were synthesised and tested for calmodulin‐binding. Using in vitro binding assays combined with gel shift analysis, we demonstrated the formation of complexes between calmodulin and both peptides, which were Ca2+‐dependent and of 1:1 stoichiometry. Affinity gels produced from these peptides also purified calmodulin from cell extracts. These results suggest a novel feedback regulation of the AT1A receptor by Ca2+/calmodulin and identify the membrane proximal region of the carboxyl‐terminus as a focal point for interactions important for AT1A receptor function.
Human neutrophil elastase regulates the expression and secretion of elafin (elastase‐specific inhibitor) in type II alveolar epithelial cells
Authors: Reid P.T , Marsden M.E , Cunningham G.A , Haslett C , Sallenave J.-M
Published: July 2002

Elafin is a low molecular weight antiproteinase believed to be important in the regulation of elastase mediated tissue damage. The expression of elafin is known to be regulated by proinflammatory cytokines such as interleukin‐1β and tumour necrosis factor but little was known regarding the effect of human neutrophil elastase (HNE). Employing a chloramphenicol acetyltransferase reporter construct of the human elafin gene, reverse transcription PCR from total cellular RNA and ELISA techniques, we have examined the effect of human neutrophil elastase on the transcription and secretion of human elafin in the pulmonary epithelial A549 cell line. Stimulation with HNE at concentrations of 10−10 and 10−11 M resulted in a significant upregulation of elafin promoter activity. Similarly, transcription of the endogenous human elafin gene was upregulated with HNE concentrations ranging from 10−10 to 10−12 M. In addition, we demonstrate that stimulation with HNE at concentrations ranging from 10−9 and 10−12 M resulted in a significant reduction in the secreted elafin protein as measured in the cell supernatant. These results provide further evidence for a role of elafin in the regulation of HNE driven proteolysis of the extracellular matrix.
Evidence that the gene YLR070c of Saccharomyces cerevisiae encodes a xylitol dehydrogenase
Authors: Richard Peter , Toivari Mervi H , Penttilä Merja
Published: July 2002

The open reading frame YLR070c of Saccharomyces cerevisiae has high sequence similarity to S. cerevisiae sorbitol dehydrogenase and to xylitol dehydrogenase of Pichia stipitis. Overexpression of this open reading frame in S. cerevisiae resulted in xylitol dehydrogenase activity. The enzyme is specific for NADH. The following Michaelis constants were estimated: D‐xylulose, 1.1 mM; NADH, 240 μM (at pH 7.0); xylitol, 25 mM; NAD, 100 μM (at pH 9.0). Xylitol dehydrogenase activity with the same kinetic properties can also be induced by xylose in wild type S. cerevisiae cells.
CA‐MMP: a matrix metalloproteinase with a novel cysteine array, but without the classic cysteine switch
Authors: Pei Duanqing
Published: July 2002

A matrix metalloproteinase (MMP)‐like gene was identified in mouse to contain a conserved MMP catalytic domain and an RRRR motif. It lacks a classic cysteine switch, but it possesses two novel motifs: a cysteine array (Cys‐X6‐Cys‐X8‐Cys‐X10‐Cys‐X3‐Cys‐X2‐Cys), and a novel Ig‐fold. It is named CA‐MMP after the distinct cysteine array motif, and little is known about its biochemical function. In an attempt to characterize CA‐MMP activity, the full‐length sequence was expressed in mammalian cells and its product found to be cell‐associated without detectable secretion. In light of this unusual finding, a chimera combining the catalytic domain of CA‐MMP with the prodomain of stromelysin‐3 was constructed to express a fully active enzyme in mammalian cells. Purified CA‐MMP catalytic domain expresses proteolytic activity against protein substrates in an MMP inhibitor sensitive fashion. Taken together, it is concluded that CA‐MMP is an MMP with distinct structure, biochemical properties and evolutionary history that may define a new subclass of the MMP superfamily.
Identification of melanin concentrating hormone (MCH) as the natural ligand for the orphan somatostatin‐like receptor 1 (SLC‐1)
Authors: Bächner Dietmar , Kreienkamp Hans-Jürgen , Weise Christoph , Buck Friedrich , Richter Dietmar
Published: July 2002

To identify possible ligands of the orphan somatostatin‐like receptor 1 (SLC‐1), rat brain extracts were analyzed by using the functional expression system of Xenopus oocytes injected with cRNAs encoding SLC‐1 and G protein‐gated inwardly rectifying potassium channels (GIRK). A strong inward current was observed with crude rat brain extracts which upon further purification by cation exchange chromatography and high performance liquid chromatography (HPLC) yielded two peptides with a high agonist activity. Mass spectrometry and partial peptide sequencing revealed that one peptide is identical with the neuropeptide melanin concentrating hormone (MCH), the other represents a truncated version of MCH lacking the three N‐terminal amino acid residues. Xenopus oocytes expressing the MCH receptor responded to nM concentrations of synthetic MCH not only by the activation of GIRK‐mediated currents but also by the induction of Ca2+ dependent chloride currents mediated by phospholipase C. This indicates that the MCH receptor can couple either to the Gi‐ or Gq‐mediated signal transduction pathway, suggesting that MCH may serve for a number of distinct brain functions including food uptake behavior.
The puzzle of RNA recombination
Authors: Chetverin Alexander B
Published: July 2002

For more than three decades, RNA recombination remained a puzzle and has only begun to be solved in the last few years. The available data provide evidence for a variety of RNA recombination mechanisms. Non‐homologous recombination seems to be the most common for RNA. Recent experiments in both the in vitro and the in vivo systems indicate that this type of recombination may result from various transesterification reactions which are either performed by RNA molecules themselves or are promoted by some proteins. The high frequency of homologous recombination manifested by some RNA viruses can be easier explained by a replicative template switch.
Protection against peroxynitrite by cocoa polyphenol oligomers
Authors: Arteel Gavin E , Sies Helmut
Published: July 2002

Flavonoids, natural plant constituents, protect against peroxynitrite and can thereby play a role in defense against this mediator of inflammation. Procyanidin oligomers of different size (monomer through nonamer), isolated from the seeds of Theobroma cacao, were examined for their ability to protect against peroxynitrite‐dependent oxidation of dihydrorhodamine 123 and nitration of tyrosine. By molarity, oligomers were more effective than the monomeric epicatechin; the tetramer was particularly efficient at protecting against oxidation and nitration reactions. These results suggest that epicatechin oligomers found in cocoa powder and chocolate may be a potent dietary source for defense against peroxynitrite.
Emergency contraception in clinical practice: global perspectives
Authors: Bártfai Gy
Published: July 2002

Access to reliable contraception is often unavailable. Unsafe abortion yearly causes death for thousands and disabling illness for millions worldwide. Insufficient information, negligence, inappropriate contraception, poverty and poor education contribute to these serious sequelae of unintended pregnancy. Identification of those at risk, the provision of appropriate information and access to emergency contraception (EC), and male involvement are emphasized. Improved knowledge, better attitudes, enhanced practice of EC, and determined providers might meet the requirements of the next century.
Challenges of managing the development of breakthrough products in Japan
Authors: Song X.Michael , Parry Mark E
Published: July 2002

We propose a contingency model and hypothesize that increases in product innovativeness weaken the influence of product synergies and development proficiencies on product performance. We incorporate these hypotheses in a path model linking measures of product synergy, development proficiency, product competitive advantage, and product performance. We test the model using data from Japan on 412 high‐innovativeness projects and 375 low‐innovativeness projects. We find that product innovativeness weakens three key relationships that determine new product success: (i) the impact of technical synergy on technical proficiency, (ii) the impact of technical proficiency on product competitive advantage, and (iii) the impact of product competitive advantage on new product success.
Effect of a long‐term nerve growth factor treatment on body weight, blood pressure,and serum corticosterone in rats
Authors: Taglialatela Giulio , Foreman P.Jay , Perez‐Polo J.Regino
Published: July 2002

Nerve growth factor is a well‐characterized neurotrophin essential for the development and maintenance of certain central and peripheral neurons. As many neurons affected by aging depend for their survival on a constant supply of neurotrophins, nerve growth factor has been proposed as a possible treatment to prevent aging‐associated neurodegeneration. There is evidence that nerve growth factor also plays a role in the immune system and modulates certain aspects of endocrine function. Here we have determined the effects of prolonged peripheral (intraperitoneal) treatment with nerve growth factor on body weight, blood pressure, and serum corticosterone levels in the rat. Our data indicate that intraperitoneally‐injected nerve growth factor can affect body weight gain in rats. This effect may not be mediated by nerve growth factor‐induced increases in serum corticosterone levels, as exogenous administration of corticosterone did not result in a similar body weight loss. These results show that, as previously reported for intracerebroventricular treatment with nerve growth factor, intraperitoneally‐injected nerve growth factor also reduces body weight gain in rats. The data also suggest that exogenous delivery of nerve growth factor as part of therapeutic regimens is likely to have several effects.