bluuebunny/crossref_metadata_2025 huggingface dataset:
Heterologous desensitization of bombesin‐ and vasopressin‐stimulated phospholipase D activity in Swiss 3T3 fibroblasts
Authors: Briscoe Celia P. , Wakelam Michael J.O.
Published: April 2003

Bombesin‐ and vasopressin‐stimulated phospholipase D (PLD) activities are rapidly desensitized in 3T3 cells, in addition both agonists are subject to heterologous desensitization. Binding studies showed that homologous desensitization was partly a result of loss of cell surface receptors, whilst heterologous desensitization was independent of receptor changes. Pretreatment with either agonist reduced subsequent GTPγS‐stimulated PLD activity by 50% whereas a pretreatment with GTPγS did not attenuate the response, suggesting that the G‐protein or downstream effector systems were affected by receptor activation resulting in desensitization. The desensitization of receptor‐stimulated PLD activation provides support for the phospholipase functioning in a key signalling pathway.
Molecular cloning and characterisation of the gene encoding the murine D4 dopamine receptor
Authors: Simone Fishburn C. , Carmon Shari , Fuchs Sara
Published: April 2003

The murine D4 dopamine receptor was isolated from a murine genomic DNA library. The receptor's entire coding region was contained within a 6 kb EcoRI genomic fragment, indicating that the murine D4 receptor gene is significantly smaller than the corresponding D2 and D3 receptor genes, the coding regions of which each stretch over 30 kb. The murine D4 receptor gene has three introns and four exons, in common with the rat and human D4 receptor genes. RT‐PCR on mRNA from different brain regions shows that the D4 receptor mRNA is expressed in various areas of the brain, with some differences from the rat and human receptor homologues.
Expression in Escherichia coli, purification and functional activity of recombinant human chaperonin 10
Authors: Legname Giuseppe , Fossati Gianluca , Gromo Gianni , Monzini Nicoletta , Marcucci Fabrizio , Modena Daniela
Published: April 2003

We have recently reported the cloning of a cDNA coding for a stress inducible human chaperonin 10. The protein was shown to possess 100% identity with the bovine homologue and a single amino acid replacement (glycine to serine at position 52) compared to rat chaperonin 10. Here we report the heterologous expression of human chaperonin 10 in Escherichia coli, its purification and its functional characterization. The recombinant protein was purified to homogeneity as judged by different analytical techniques, and mass spectrometry analysis showed a MW of 10,801 Da in agreement with the predicted sequence. This molecular weight accounts for a protein which is not modified post‐translationally. In fact, natural rat chaperonin 10 has been shown to be acetylated at the N‐terminus, a feature suggested to be important for targeting and functional activity. Here we show that recombinant human chaperonin 10 is fully active in assisting the chaperonin 60 GroEL in the refolding of denatured yeast enolase, thereby showing that, at least in the present system, post‐translational acetylation is not necessary for its activity.
Expression and characterization of a B cell growth promoting polypeptide derived from the 12 kDa B cell growth factor gene (BCGF 1)
Authors: Kovanen Panu E. , Virtaneva Kimmo , Harju Leena , Kantor Carmela , Gahmberg Carl G. , Timonen Tuomo
Published: April 2003

The expression and partial purification of recombinant 12 kDa B cell growth factor are reported. The polypeptide was derived from the genomic sequence of the gene (BCGF 1) which is here shown to be a single copy gene that localizes to human chromosome 16. When expressed as a glutathione S‐transferase fusion protein in E. coli, the protein appears as a 38 kDa polypeptide in Western blot analysis using a peptide antibody. The purified fusion protein stimulates the proliferation of activated human B cells in a dose‐dependent manner, and the active site resides within the 104 carboxy‐terminal amino acids. The availability of biologically active recombinant 12 kDa B cell growth factor will enable its evaluation in B cell growth regulation, and provides a new means of in vitro culturing of human B lymphocytes.
Diaminoalkanes with an odd number of carbon atoms induce compaction of a single double‐stranded DNA chain
Authors: Yoshikawa Yuko , Yoshikawa Kenichi
Published: April 2003

We have performed direct observations on the conformational change of a single double‐stranded T4 DNA molecule induced by diaminoalkanes, NH3
+(CH2)nNH3
+ (n = 1 ∼ 6), by use of fluorescence microscopy. It was found that diamines with three and five methylene groups show a significant effect on the compaction of individual single DNA molecules. On the other hand, diamines with two, four and six methylene groups are not effective in the compaction of DNA. Such a drastic difference in the action of diamines suggests that not only the number of charge but also the length between the amino groups in the diamines plays an essential role in their interactions with DNA.
Purification of the hepatic glycogen‐associated form of protein phosphatase‐1 by microcystin‐Sepharose affinity chromatography
Authors: Moorhead Greg , MacKintosh Carol , Morrice Nick , Cohen Philip
Published: April 2003

The form of protein phosphatase‐1 associated with hepatic glycogen (PP1G) was purified to near homogeneity from rat liver by affinity chromatography on microcystin‐Sepharose and gel‐filtration. The enzyme is a heterodimer consisting of the catalytic subunit of PP1 (the α and β isoforms) complexed to a 33 kDa glycogen‐binding (GL) subunit. The GL subunit binds phosphorylase a with high affinity, and is responsible for the enhanced dephosphorylation of glycogen synthase by PP1G and its allosteric inhibition by phosphorylase a.
The arrestin superfamily: cone arrestins are a fourth family
Authors: Craft Cheryl M. , Whitmore Donald H.
Published: April 2003

Arrestins constitute a superfamily of regulatory proteins that down‐regulate phosphorylated G‐protein membrane receptors, including rod and cone photoreceptors and adrenergic receptors. The potential role of arrestin in color visual processes led us to identify a cDNA encoding a cone‐like arrestin in Xenopus laevis, the principle amphibian biological model system. Alignment of 18 deduced amino acid sequences of all known arrestins from both invertebrate and vertebrate species reveals five arrestin families. Further analysis identifies 7 variable and 4 conservative arrestin structural motifs that may identify potential functional domains. The adaptive evolutionary relationship of Xenopus cone arrestin to the arrestin gene tree suggests high intrafamily homology and early gene duplication events.
The Sec61 complex is essential for the insertion of proteins into the membrane of the endoplasmic reticulum
Authors: Oliver Jason , Jungnickel Berit , Görlich Dirk , Rapoport Tom , High Stephen
Published: April 2003

Cross‐linking studies have implicated Sec61α as the principal component adjacent to newly synthesised membrane proteins during insertion into the endoplasmic reticulum. Using proteoliposomes which have been reconstituted from purified components of the endoplasmic reticulum [Görlich, D and Rapoport, T.A., Cell 75 (1993) 615–630] we have found that the Sec61 complex, consisting of three subunits, is essential for the insertion of single‐spanning membrane proteins. This is true for signal‐anchor proteins of both orientations, and for proteins with a cleavable signal sequence. These results support the view that Sec61α is a major component of the ER translocation site and promotes both the insertion of membrane proteins and the translocation of secretory proteins.
Properties of N‐terminus truncated and C‐terminus mutated muscle acylphosphatases
Authors: Taddei Niccolò , Modesti Alessandra , Bucciantini Monica , Stefani Massimo , Magherini Francesca , Vecchi Manuela , Raugei Giovanni , Ramponi Giampietro
Published: April 2003

Enzymatic activity and structure of N‐terminus truncated and C‐terminus substituted muscle acylphosphatase mutants were investigated by kinetic studies under different conditions and 1H NMR spectroscopy, respectively. The N‐terminus truncated mutant lacked the first six residues (Δ6), whereas arginine 97 and tyrosine 98 were replaced by glutamine giving two C‐terminus substituted mutants (R97Q and Y98Q, respectively). All acylphosphatase forms were obtained by modifications of a synthetic gene coding for the human muscle enzyme which was expressed in E. coli. The Δ6 deletion mutant elicited a reduced specific activity and a native‐like structure. The kinetic and structural properties of R97Q and Y98Q mutants indicate a possible role of Arg‐97 in the stabilisation of the active site correct conformation, most likely via back‐bone and side chain interactions with Arg‐23, the residue involved in phosphate binding by the enzyme. This study also suggests a possible involvement of Tyr‐98 in the stabilisation of the acylphosphatase overall structure.
Enhanced susceptibility of cholesteryl sulfate‐enriched low density lipoproteins to copper‐mediated oxidation
Authors: Blache D. , Rodriguez C. , Davignon J.
Published: April 2003

Cholesteryl sulfate (CS) is a minor component of cell membranes, also present in lipoproteins, and its exact function is unknown. Since oxidation of low density lipoproteins (LDL) is thought to be an important determinant of atherogenesis, we investigated the influence of CS enrichment on copper‐mediated oxidation of LDL. CS was found to act as a pro‐oxidant, as measured by lipid oxidation parameters. The results also suggest that these effects were dependent on the sulfate group since pure cholesterol or cholesteryl acetate did not promote Cu2+‐mediated oxidation. Our findings imply that CS may affect the oxidizability and hence the potential atherogenicity of LDL.
Liposome destabilization induced by the HIV‐1 fusion peptide Effect of a single amino acid substitution
Authors: Pereira Francisca B. , Goñi Félix M. , Nieva JoséL.
Published: April 2003

The 23‐residue synthetic peptide representing the N‐terminus of HIV‐1 gp41 is known to induce either leakage or fusion of lipid vesicles depending on the experimental conditions. In this paper we report that a polar amino acid substitution V → E at position 2, known to block gp41 activity in vivo, makes the peptide unable to destabilize and/or fuse membranes. Moreover this variant, unlike the parent peptide, is never found in the membrane‐associated β conformation.
A minimum catalytic unit of F1‐ATPase shows non‐cooperative ATPase activity inherent in a single catalytic site with a Km 70 μM
Authors: Saika Koji , Yoshida Masasuke
Published: April 2003

F1‐ATPase has three interacting catalytic sites and shows complicated kinetics. Here, we report reconstitution of a complex, most likely composed of one α subunit and one β subunit, with a single catalytic site from thermophilic Bacillus PS3 F1‐ATPase on the solid surface. The complex has an ATPase activity which obeys a simple non‐cooperative kinetics with a K
m(ATP) of 70 μM and a V
max of 0.1 unit/mg. Different from F1‐ATPase, the complex is not inactivated by 7‐chrolo‐4‐nitrobenzofrazan. Thus, the inherent activity attributable to a single catalytic site unaffected by other catalytic sites of F1‐ATPase is characterized.
Plasma 8‐epi‐PGF2α levels are elevated in individuals with non‐insulin dependent diabetes mellitus
Authors: Gopaul N.K. , Änggård E.E. , Mallet A.I. , Betteridge D.J. , Wolff S.P. , Nourooz-Zadeh J.
Published: April 2003

This study reports plasma levels of a specific nonenzymatic peroxidation product of arachidonic acid, esterified 8‐epi‐PGF2α, from healthy‐ and NIDDM individuals as an index of oxidative stress in vivo. Plasma 8‐epi‐PGF2α was isolated by solid‐phase extraction on a C,8 followed by an NH2 cartridge and analyzed by GC‐MS/NICI as PFB‐ester/TMS‐ether derivative. We found that the average concentration of esterified 8‐epi‐PGF2α among NIDDM subjects (0.93 ± 0.07 nM, n = 39) was higher (P < 0.0001, Mann‐Whitney test) than in healthy individuals (0.28 ± 0.04 nM, n = 15). These data indicate that NIDDM is associated with increased plasma lipid peroxidation.
Induction of the antioxidant stress proteins heme oxygenase‐1 and MSP23 by stress agents and oxidised LDL in cultured vascular smooth muscle cells
Authors: Siow Richard C.M. , Ishii Tetsuro , Sato Hideyo , Taketani Shigeru , Leake David S. , Sweiry Jacob H. , Pearson Jeremy D. , Bannai Shiro , Mann Giovanni E.
Published: April 2003

Enhanced expression of the antioxidant stress proteins heme oxygenase‐1 (HO‐1) and macrophage stress protein (MSP23) by oxidative stress agents and oxidatively modified low density lipoproteins (LDL) was investigated in cultured porcine aortic smooth muscle cells. Treatment of smooth muscle cells with glucose oxidase, CdCl2 or diethylmaleate resulted in a time‐dependent (6–48 h) induction of HO‐1 and MSP23 expression. Exposure of cells to 100 μg protein/ml highly oxidised LDL increased the expression of HO‐1 and MSP23 within 24 h, and the induction was dependent on the degree of LDL oxidation. The induction of HO‐1 and MSP23 may thus play an important cytoprotective role against oxidative stress in atherogenesis.
Asn‐265 of frog kainate binding protein is a functional glycosylation site: implications for the transmembrane topology of glutamate receptors
Authors: Galen Wo Z. , Christine Bian Z. , Oswald Robert E.
Published: April 2003

Kainate binding proteins (KBPs) from frog and goldfish brain are glycosylated, integral membrane proteins. These KBPs are homologous (35–40%) to the C‐terminal half of AMPA and kainate receptors which have been shown to form glutamategated ion channels. We report here that the frog KBP has three functional N‐glycosylation sites. Of particular interest, Asn‐265, a residue located between two putative membrane spanning regions of the frog KBP, is a functional N‐glycosylation site. A mutation of Ser‐267 to Gly renders this site non‐functional as shown using an in vitro translation system and by transient expression in human embryonic kidney (HEK 293) cells. The mutant receptor protein (S267G), when expressed in HEK cells, binds kainate with high affinity (K
d = 16 nM). These results further support a topology with three transmembrane segments for KBPs and, by sequence homology, for glutamate‐gated ion channels.
Ras binding to a C‐terminal region of GAP
Authors: Molloy David P. , Owen Darerca , Grand Roger J.A.
Published: April 2003

Using fluorescence spectroscopy we have identified a binding region for Ras on the GTPase activating protein (GAP) lying within residues 715–753. A synthetic peptide Y922, corresponding to residues 716–753 of GAP binds to wild type Ras showing 3.3‐fold higher affinity for the GTP‐ over the GDP‐bound forms of Ras. Binding is stabilised by Mg2+, although Y922 does not stimulate the GTPase activity of Ras. Peptide binding to the Y32A and Y40F Ras mutants showed equal affinity for both GDP‐ and GTP‐bound forms, with binding to Y32A · GDP abolished in the absence of Mg2+. These results suggest that Y922 mimics the in vivo interactions shown by the intact p120GAP protein and provide the first direct demonstration of Ras interaction with GAP in the region 715–753.
Cell‐specific effects of RAS oncogene and protein kinase C agonist TPA on P‐glycoprotein function
Authors: Stromskaya T.P. , Grigorian I.A. , Ossovskaya V.S. , Rybalkina E.Y. , Chumakov P.M. , Kopnin B.P.
Published: April 2003

We compared the influence of exogenous N‐ras oncogene and treatment with PKC agonist 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) on P‐glycoprotein (Pgp) function in various human, rat and dog cell lines. Two approaches were used: (a) flow cytometry analysis of Rhodamine 123 (Rh123) exclusion; and (b) sensitivity to cytotoxic action of colchicine. We have found that in Rat1 fibroblasts, rat IAR2 epithelial cells and rat McA RH 7777 (hepatoma), ras activates Pgp function, while in MDCK (dog kidney), K562 (human chronic myelogenous leukaemia) and LIM1215 (human colon carcinoma) cells it either has no effect or even acts in opposite direction. TPA‐induced Pgp function shows dissimilar pattern of cell specificity. It is assumed that PKC and ras oncogene regulate mdr1 gene expression through at least partially distinct signalling pathways.
Evidence for SecA‐ and ΔpH‐independent insertion of D1 into thylakoids
Authors: van Wijk Klaas Jan , Knott Tracy G. , Robinson Colin
Published: April 2003

Many nuclear‐encoded proteins are targeted into chloroplast thylakoids by an azide sensitive Sec‐related mechanism or by a ΔpH‐driven mechanism. In this report, the requirements for the integration of chloroplast‐encoded thylakoid proteins have been analysed in pulse‐labeled intact chloroplasts. We show that the integration of the photosystem II reaction centre protein, D1, continues in the absence of a ΔpH and in the presence of azide. A range of other proteins are similarly targeted to thylakoids in the presence of azide, suggesting that the SecA‐related mechanism is not widely used for the targeting of chloroplast‐encoded proteins.
Phenylarsine oxide inhibits tyrosine phosphorylation of phospholipase Cγ2 in human platelets and phospholipase Cγ1 in NIH‐3T3 fibroblasts
Authors: Yanaga Fumi , Asselin Judith , Schieven Gary L. , Watson Steve P.
Published: April 2003

The sulphydryl reagent phenylarsine oxide (PAO) (1 μM) inhibited completely formation of inositol phosphates in human platelets induced by collagen or by cross‐linking of the platelet low affinity Fc receptor, FcγRIIA, but did not alter the response to the G protein receptor agonist thrombin. PAO also inhibited completely tyrosine phosphorylation of PLCγ2 in collagen and FcγRIIA‐stimulated cells, although tyrosine phosphorylation of other proteins including the tyrosine kinase syk was relatively unaffected. PAO (1 μM) also inhibited completely tyrosine phosphorylation of PLCγ1 induced by platelet derived growth factor (PDGF) in NIH‐3T3 fibroblasts but only partially reduced phosphorylation of the PDGF receptor. These results provide further evidence that collagen and FcγRIIA cross‐linking activate platelets through a pathway distinct from that used by thrombin and suggest that PAO may be a selective inhibitor of PLCγ relative to PLC β isozymes.
1H NMR T1 relaxation rate study on substrate orientation of fluoromethylanilines in the active sites of microsomal and purified cytochromes P450 1A1 and 2B1
Authors: Koerts Janneke , Rietjens Ivonne M.C.M. , Boersma Marelle G. , Vervoort Jacques
Published: April 2003

The present study describes 1H NMR T
1 relaxation rate studies on fluoromethylanilines bound to the active sites of microsomal and purified cytochromes P450 1A1 and 2B1. From the data obtained, insights into the average orientation of the substrates with respect to the paramagnetic Fe3+ centre in the cytochromes P450 could be derived. Particular attention was paid to a possible extra relaxation pathway for methyl protons compared to the aromatic protons, due to the rotational motion of the CH3 around the σ‐C‐CH3 bond. However, this effect appeared to be minimal and to result in at most a few percent underestimation of the actual distance of the methyl protons to the Fe3+ centre. Furthermore, the data obtained demonstrate that all aromatic protons are at about the same average distance from the paramagnetic centre. The results also demonstrate that the fluoromethylanilines are bound to the active sites of cytochromes P450 1A1 and 2B1 in a similar way. A time‐averaged orientation of the substrate with the Fe3+ above the aromatic ring, with the π‐orbitals of the aromatic ring and those of the porphyrin rings in a parallel position, providing possibilities for energetically favourable π−π interaction defines the orientation which best fits the results of the present study. Possibilities for a flip‐flop rotation around an axis in the plane of the aromatic ring can be included in this picture, as such rotations would still result in a similar average distance of all aromatic protons to the Fe3+ paramagnetic centre. The results obtained also indicate that possible differences in metabolite patterns resulting from conversion of the fluoromethylanilines by different cytochromes P450, especially P450 1A1 and 2B1, are unlikely to be caused by a specific orientation of the substrate imposed by the substrate binding site of the enzyme.
The three‐dimensional structure of thymidine kinase from Herpes simplex virus type 1
Authors: Wild Klemens , Bohner Thomas , Aubry André , Folkers Gerd , Schulz Georg E.
Published: April 2003

Recombinant thymidine kinase from Herpes simplex virus type 1 (ATP:thymidine 5′‐phosphotransferase; EC 2.7.1.21), an enzyme of therapeutic importance, was purified and crystallized in an N‐terminally truncated but still fully active form. The three‐dimensional structure was solved by X‐ray diffraction analysis at 3.0 Å resolution using isomorphous replacement. The chain fold is presented together with the bound substrates thymidine and ATP. Three chain segments at the surface could not be located. The chain fold, the location of the substrates and presumbly also the catalytic mechanism resemble the well‐known adenylate kinases.
Lipopolysaccharide treatment in vivo induces tissue expression of GTP cyclohydrolase I mRNA
Authors: Hattori Yoshiyuki , Oka Muneo , Kasai Kikuo , Nakanishi Nobuo , Shimoda Shin-Ichi
Published: April 2003

A significant induction of GTP cyclohydrolase I (GTPCH) mRNA was observed in lung, heart and kidney of rats treated with lipopolysaccharide (LPS; 10 mg/kg i.v.). GTPCH mRNA levels in liver were high even in untreated rats, and remained elevated after LPS treatment. Parallel induction of nitric oxide synthase (NOS) mRNA was observed in these tissues of LPS‐treated rats. Our results demonstrate induction of GTPCH mRNA after LPS treatment in vivo and provide molecular evidence for the increased GTPCH activity which may up‐regulate NOS activity in vivo.
Cooperative interactions in the tandem of oligonucleotide derivatives arranged at complementary target. Quantitative estimates and contribution of the target secondary structure
Authors: Fedorova Olga S. , Adeenah-Zadah Abdussalam , Knorre Dmitri G.
Published: April 2003

The intraduplex reaction of the alkylating reagent CIRCH2NHpd(TTCCCA) (X, CIR is p‐(N‐2‐chloroethyl‐N‐methylaminophenyl) residue) with the target 26‐mer d(TTGCCTTGAATGGGAAGAGGGTCATT) (P) in the presence of effectors was studied. The effectors used were Phn‐L‐pd(TTCAAGGC)p‐L‐Phn (E1) and Phn‐L‐pd(TGACCCTC)p‐L‐Phn (E2), where Phn is N‐(2‐hydroxyethyl)‐phenazinium residue and L is NHCH2CH2NH spacer. The dependence of the alkylation extent of the target on the reagent concentration was treated using the equation derived earlier for the two‐component system (reagent+target) to calculate association constants of X with P, PE1, PE2 and PE1E2. The latters were found to be K
xe1 = 6.75 · 105 M−1, K
xe2 = 4.15 · 104 M− andK
xe12 = 5.87 · 106 106 M−1 as compared with the affinity of X to P K
x = 2.16 · 104 M−1 in the absence of effectors. Taking into account the internal structure of the target, co‐operativity parameters describing interactions in the tandem E1 · X · E2 arranged at the target were calculated as α
1 = 16, α
2 = 10 and α
12 = 139 for the duplexes PXE1, PXE2 and PXE1E2.
Ribosomal protein L22 from Thermus thermophilus: sequencing overexpression and crystallisation
Authors: Davydova Natalia L. , Gryaznova Olga I. , Mashchenko Oksana V. , Vysotskaya Valentina S. , Jonsson Bengt-Harald , Al-Karadaghi Salam , Liljas Anders , Garber Maria B.
Published: April 2003

The gene for the ribosomal protein L22 from Thermus thermophilus has been sequenced and overexpressed in Escherichia coli. A multiple sequence alignment was carried out for all proteins of the L22 family reported so far. The recombinant protein was purified and crystallized. The crystals belong to the space group P212121 with cell parameters of .
Coordination of a histidine residue of the protein‐component S to the cobalt atom in coenzyme B12‐dependent glutamate mutase from Clostridium cochlearium
Authors: Zelder Oskar , Beatrix Birgitta , Kroll Friedrich , Buckel Wolfgang
Published: April 2003

Electron paramagnetic resonance (EPR) spectroscopy of glutamate mutase from Clostridium cochlearium was performed in order to test the idea, that a histidine residue of component S replaces the dimethylbenzimidazole ligand of the Co‐atom during binding of coenzyme B12 to the enzyme. The shapes and the superhyperfine splitting of the g
z‐lines of the Co(II) EPR spectra were used as indicators of the interaction of the axial base nitrogen with the Co‐atom. A mixture of completely 15N‐labelled component S, unlabelled component E, coenzyme B12 and glutamate gave slightly sharper g
z‐lines than that with unlabelled component S. A more dramatic change was observed in the Co(II) spectrum of the inactivated enzyme containing tightly bound cob(II)alamin, in which unlabelled component S caused a threefold superhyperfine‐splitting of the g
z‐line, whereas the 15N‐labelled protein only caused a twofold splitting, as expected for a direct interaction of a nitrogen of the enzyme with the Co‐atom. By using a sample of 15N‐labelled component S, in which only the histidines were 14N‐labelled, the EPR spectra showed no difference to those with unlabelled component S. The experiments indeed demonstrate a replacement of the dimethylbenzimidazole ligand in coenzyme B12 by a histidine when bound to glutamate mutase. The most likely candidate is H16, which is conserved among the carbon skeleton rearranging mutases and methionine synthase.
Nitric oxide and oxygen radicals: a question of balance
Authors: Darley-Usmar Victor , Wiseman Helen , Halliwell Barry
Published: April 2003

The production of superoxide and nitric oxide individually has been associated with the development of several diseases but only recently has it been realised that interactions between them may also be important in disease pathology. The central hypothesis which is emerging is that the balance between nitric oxide and superoxide generation is a critical determinant in the aetiology of many human diseases including atherosclerosis, neurodegenerative disease, ischaemia‐reperfusion and cancer. These ideas are discussed in this short overview and placed in the context of the current and future status of therapies which could modulate the balance between nitric oxide and superoxide.
Effects of vasoactive substances and cAMP related compounds on adrenomedullin production in cultured vascular smooth muscle cells
Authors: Sugo Shin , Minamino Naoto , Shoji Hiroki , Kangawa Kenji , Matsuo Hisayuki
Published: April 2003

To elucidate the regulation mechanism of adrenomedullin (AM) production in blood vessels, we examined the effects of 30 substances on AM production in cultured rat vascular smooth muscle cells (VSMCs). Forskolin and 8‐bromo‐cAMP suppressed production and gene transcription of AM. Since VSMC expresses AM receptors coupled with adenylate cyclase, AM production may be regulated by intracellular cAMP concentration. Thrombin, vasoactive intestinal polypeptide and interferon‐gg also inhibited AM production, while angiotensin II, endothelin‐1, bradykinin, substance P, adrenaline, phorbol ester and fetal calf serum stimulated AM production in VSMC. These results suggest that AM production is regulated by a variety of substances, indicating complex systems regulating AM production.
Expression of Aequorea green fluorescent protein in plant cells
Authors: Wei Hu  , Chi-Lien Cheng
Published: April 2003

The coding region of the green fluorescent protein (GFP) from Aequorea victoria has been fused to the cauliflower mosaic virus 35S promoter and introduced into maize leaf protoplasts. Transient expression of GFP was observed. In addition, the coding region of GFP was fused to an Arabidopsis heat shock promoter and co‐transformed with another construct in which GFP has been replaced with chloramphenicol acetyltransferase (CAT). The heat‐induced expression of GFP in maize protoplasts parallels that of CAT. While GFP was expressed in both darkgrown and green maize leaf protoplasts, no green fluorescence was observed in similarly transformed Arabidopsis protoplasts.
The first untranslated exon of the human tenascin‐C gene plays a regulatory role in gene transcription
Authors: Gherzi Roberto , Ponassi Marco , Gaggero Barbara , Zardi Luciano
Published: April 2003

The transcription of the human tenacin‐C (TN‐C) gene is directed by a single promoter. Here we demonstrate, in transiently transfected cells, that two distinct regions of the untranslated 179 bp‐long exon 1 play antagonistic roles in transcriptional regulation: bases from 1 to 20 strongly increase the transcription of the reporter gene CAT directed by the human TN‐C gene promoter, while bases from 79 to 179 significantly reduce this activation.
Native and/or asialo‐Tamm‐Horsfall glycoproteins Sd(a+) are important receptors for Triticum vulgaris (wheat germ) agglutinin and for three toxic lectins (abrin‐a, ricin and mistletoe toxic lectin‐I)
Authors: Wu Albert M. , Watkins Winifred M. , Chen Chie-Pein , Song Shuh-Chyung , Chow Lu-Ping , Lin Jung-Yaw
Published: April 2003

The binding properties of human Tamm‐Horsfall Sd(a+) urinary glycoprotein (THGP) and asialo‐THGP with Triticum vulgaris agglutinin(WGA) and three toxic lectins (abrin‐a, ricin, and Mistletoe toxic lectin‐I) were investigated by quantitative precipitin and precipitin inhibition assays. Both glycoproteins reacted strongly with abrin‐a, precipitating over 80% of the lectin nitrogen tested. THGP also bound well to mistletoe toxic lectin‐I and precipitated 86% of this lectin added, while the precipitability of its asialo product decreased by 28%. The native glycoprotein completely precipitated the WGA added, but its reactivity was reduced dramatically after desialylation. On the contrary, the poor reactivity of THGP with ricin increased substantially after removal of sialic acid and completely precipitated the lectin added. The glycoprotein‐lectin interactions were inhibited by one or several of the following haptens, p‐NO2‐phenylαGalNAc, p‐NO2‐phenylβGalNAc, Galβ1→4GlcNAc, Galβ1→4Glc, GlcNAcβ1→4GlcNAc, and/or GlcNAc. From the above results, it is concluded that native and/or asialo Tamm‐Horsfall glycoproteins serve as important receptors for these three toxic lectins and for WGA.
Oxidative DNA damage induced by simultaneous generation of nitric oxide and superoxide
Authors: Inoue Sumiko , Kawanishi Shosuke
Published: April 2003

Incubation of calf thymus DNA with 3‐morpholinosydnonimine (SIN‐1), which simultaneously generates nitric oxide (NO) and superoxide (O2
−), induced a significant increase of 8‐hydroxydeoxyguanosine (8‐OH‐dG). Peroxynitrite also increased 8‐OH‐dG in calf thymus DNA. Addition of free hydroxyl radical (•OH) scavengers inhibited the increase of 8‐OH‐dG by SIN‐1 or peroxynitrite. Incubation of 32P‐labeled DNA fragment with SIN‐1 or peroxynitrite caused DNA cleavage at every nucleotide with a little dominance at guanine residues. The results suggest that NO reacts with O2
− to form peroxynitrite and the peroxynitrite induces oxidative DNA damage through an active intermediate of which reactivity is similar to •OH.
Genomic structure and alternative splicing of the murine bhk/ctk/ntk gene
Authors: Ershler M.A. , Samokhvalov I.M. , Belyavsky A.V. , Visser J.W.M.
Published: April 2003

Recently, we and others have cloned cDNAs encoding a second member of the Csk family of inhibitory protein kinases, which we termed Bhk [M.A. Ershler et al. (1994) Dokl. Akad. Nauk. 339, 679–683]. In the present study, two new distinct types of bhk mRNA were found in addition to the third form described previously. Analysis of the bhk genomic structure established that three exons participate in the alternative splicing of bhk mRNA.
Temperature‐induced rapid increase in cytoplasmic free Ca2+ in pathogenic Leishmania donovani promastigotes
Authors: Sarkar Dhiman , Bhaduri Amar
Published: April 2003

We demonstrate that the cytoplasmic free Ca2+ concentration in three virulent Leishmania donovani promastigote strains is maintained at 20–30 nM level whereas the avirulent promastigotes maintain [Ca2+]i at 80–100 nM. Rapid exposure of virulent promastigotes to higher physiological temperature increases [Ca2+]i many‐fold. Use of CaCl2, EGTA and Mn2+ confirmed that both release of Ca2+ from internal pool(s) and influx from outside milieu are responsible for this increase in [Ca2+]i. Application of oligomycin, CN−, and nigericin indicated the non‐mitochondrial pHi sensitive pool as the possible intracellular origin for internal Ca2+ release. Exposure of these cells to acidic environment had no influence on [Ca 2+]i. In contrast, the avirulent promastigotes or freshly isolated amastigotes had shown no effect of heat‐shock or pH shock on [Ca 2+]i. of these cells. Our results indicate that the upward shift in temperature may play a role in signal transduction events in morphogenetic transformation of L. donovani promastigotes that involves mobilization of Ca2+ in cytoplasm.
In vitro glycation of glomerular basement membrane alters its permeability: a possible mechanism in diabetic complications
Authors: Cochrane Sheila M. , Robinson Garth B.
Published: April 2003

The permeability of glomerular basement membrane (GBM) was assessed in vitro by the filtration of solutions of proteins across films formed from isolated pig GBM. Incubation of the films with fructose or glucose increased their permeability to water and serum albumin. The effect of fructose was similar to that previously noted for films crosslinked with glutaraldehyde. The metal chelator DTPA abolished the effects of glycation; EDTA was partially effective in this respect. Transition metal catalysed formation of glycoxidation induced crosslinks may explain the increased permeability of glycated GBM.
Stimulation of synaptosomal free radical production by fatty acids: Relation to esterification and to degree of unsaturation
Authors: Bondy S.C. , Marwah S.
Published: April 2003

The ability of three fatty acids and their respective ethyl esters, to promote generation of reactive oxygen species (ROS), was compared in a preparation of rat brain synaptosomes. Arachidonic but not palmitic or linoleic acids promoted ROS generation. Ethyl esterification of each fatty acid significantly enhanced ROS production and also levels of lipid peroxidation. Pro‐oxidant activity was enhanced by fatty acids, proportionally to their degree of unsaturation. Since ethanol consumption is known to lead to esterification of membrane lipids, this transformation may in part account for the ROS‐promoting potential of alcohol.
Molecular cloning of a human polypeptide related to yeast sds22, a regulator of protein phosphatase‐1
Authors: Renouf Sylvie , Beullens Monique , Wera Stefaan , Van Eynde Aleyde , Sikela James , Stalmans Willy , Bollen Mathieu
Published: April 2003

sds22 is a regulatory polypeptide of protein phosphatase‐1 that is required for the completion of mitosis in both fission and budding yeast. We report here the cDNA cloning of a human polypeptide that is 46% identical to yeast sds22. The human homolog of sds22 consists of 360 residues, has a calculated molecular mass of 41.6 kDa and shows a tandem array of 11 leucinerich repeat structures of 22 residues. Northern analysis revealed a major transcript of 1.39 kb in all 8 investigated human tissues. sds22 was detected by western analysis in both the cytoplasm and the nucleus of rat liver cells as a polypeptide of 44 kDa.
Ligands of purinergic receptors stimulate electrogenic H+‐transport of neutrophils
Authors: Suszták Katalin , Káldi Krisztina , Kapus András , Ligeti Erzsébet
Published: April 2003

The possible role of ATP, acting as a ligand on cell surface receptor was investigated in the activation of the electrogenic H+‐transporting pathway of porcine neutrophil granulocytes. (1) ATP brought about 2.1‐fold increase in the rate of H+‐efflux. (2) The order of potency of different nucleotides suggests, that ATP acts on P2 type purinoceptor. (3) The effect of the nucleotides was prevented by inhibition of phospholipase A2. (4) Inhibition of the metabolism of arachidonic acid (AA) via the cyclooxygenase pathway had no effect, whereas inhibition of the lipoxygenase pathway significantly enhanced H+‐release. This is the first report about activation of the H+‐transporter by physiological stimulator acting on the cell surface.
Occurrence and postmortem generation of anandamide and other long‐chain N‐acylethanolamines in mammalian brain
Authors: Schmid Patricia C. , Krebsbach Randy J. , Perry Stephen R. , Dettmer Timothy M. , Maasson Jeffrey L. , Schmid Harald H.O.
Published: April 2003

Long‐chain N‐acylethanolamines (NAEs) were assayed in pig, sheep and cow brain by gas chromatography/mass spectrometry of their tert.‐butyldimethylsilyl derivatives in the presence of deuterium‐labeled internal standards. Immediately after death, total NAEs ranged from about 2.7 μg/g wet weight (sheep, cow) to 6.5 μg/g wet weight (pig) and consisted almost exclusively (99%) of saturated and monounsaturated species. Anandamide (20:4n‐6 NAE) comprised about 1% of total NAE in pig and cow brain, but was absent in freshly extracted sheep brain. When pig brain was analysed after 0.5, 1, 3, 4 and 23 h at ambient temperature, NAE levels were found to increase substantially over the entire time period with 20:4n‐6 NAE formation exhibiting a time lag compared to that of saturated and monounsaturated NAEs.
The peripheral cannabinoid receptor: adenylate cyclase inhibition and G protein coupling
Authors: Bayewitch Michael , Avidor-Reiss Tomer , Levy Rivka , Barg Jacob , Mechoulam Raphael , Vogel Zvi
Published: April 2003

Two cannabinoid receptors, designated neuronal (or CB1) and peripheral (or CB2), have recently been cloned. Activation of CB1 receptors leads to inhibition of adenylate cyclase and N‐type voltage‐dependent Ca2+ channels. Here we show, using a CB2 transfected Chinese hamster ovary cell line, that this receptor binds a variety of tricyclic cannabinoid ligands as well as the endogenous ligand anandamide. Activation of the CB2 receptor by various tricyclic cannabinoids inhibits adenylate cyclase activity and this inhibition is pertussis toxin sensitive indicating that this receptor is coupled to the Gi/G0 GTP‐binding proteins. Interestingly, contrary to results with CB1, anandamide did not inhibit the CB2 coupled adenylate cyclase activity and δ
9‐tetrahydrocannabinol had only marginal effects. These results characterize the CB2 receptor as a functional and distinctive member of the cannabinoid receptor family.
The fastest‐actin‐based motor protein from the green algae, Chara, and its distinct mode of interaction with actin
Authors: Higashi-Fujime Sugie , Ishikawa Ryoki , Iwasawa Hidehiro , Kagami Osamu , Kurimoto Eiji , Kohama Kazuhiro , Hozumi Tetsu
Published: April 2003

The endoplasmic streaming in Characean cells is an actin‐dependent movement. The motor protein responsible for the streaming was partially purified and characterized. It was soluble at low ionic strength, an ATPase of a molecular mass of 225 kDa and activated more than 100 times by muscle F‐actin. Surprisingly, in an in vitro motility assay, the motor protein moved muscle F‐actin at 60 μm/s, which is similar to the velocity of streaming in a living cell and 10 times faster than muscle myosin. Proteolytic cleavage of actin impaired movement crucially on muscle myosin, but did not affect movement at all on the Chara motor protein, suggesting that the Chara motor protein would interact with actin via a set of sites different from those of muscle myosin.
Sequence‐specific DNA binding activity of rat cdc37‐related gene product
Authors: Ozaki Toshinori , Sakiyama Shigeru
Published: April 2003

The rat cdc37‐related gene product (RCdc37), which is possibly involved in the regulation of cell cycle progression, contains a putative basic/leucine zipper (bZIP) domain in its N‐terminal portion. In this study, we have identified a rat genomic sequence which can interact with RCdc37 using an in vitro binding assay. The specificity of this interaction was confirmed by gel retardation experiments. These results raise a possibility that RCdc37 might play an important role in the control of cell cycle progression via a sequence‐specific DNA binding mechanism.
Transcriptional repression by methylation: cooperativity between a CpG cluster in the promoter and remote CpG‐rich regions
Authors: Hug Martin , Silke John , Georgiev Oleg , Rusconi Sandro , Schaffner Water , Matsuo Koichi
Published: April 2003

Cytosine methylation of binding sites for transcription factors is a straightforward mechanism to prevent transcription, while data on an indirect mechanism, by methylation outside of the factor binding sites, are still scarce. We have studied the latter effect using a model promoter construct. For this, a 69 bp G + C rich DNA segment with a cluster of 14 CpG sites was inserted between upstream lexA sites and the TATA box. Transcription was measured in transient transfection assays with lexA‐VP16 as an activating factor. When the entire plasmid was methylated at all CpGs before transfection, transcription was blocked (to 3% residual activity), whereas transcription was only mildly inhibited (to 60%) by methylation of a control plasmid that lacked the 69 bp CpG cluster. However, the effect could not simply be attributed to methylation of the CpG cluster: neither a methylated CpG cluster in an otherwise methylation‐free reporter gene plasmid, nor the methylated plasmid with an unmethylated CpG cluster, inhibited transcription considerably (69% and 44% remaining activity, respectively). The data presented here suggest that a minimal length of methylated DNA in the promoter is required for repression, and imply that concomitant methylation of CpGs in the promoter region and in remote sequences can cooperatively block transcription, without the need to methylate any binding sites for transcription factors. We also note that the cooperation for a negative effect described here bears an analogy to transcriptional activation, where a promoter often cooperates with a remote enhancer.
Topology of the secondary structure elements of ribosomal protein L7/L12 from E. coli in solution
Authors: Bocharov E.V. , Gudkov A.T. , Arseniev A.S.
Published: April 2003

Topology of the secondary structure elements of ribosomal protein L7/L12 has been studied. The sequential assignment was obtained for main and side chain resonances. This allows the overall secondary structure to be described. The results of high resolution NMR studies show that dimer of the ribosomal protein L7/L12 from Escherichia coli has a parallel (head‐to‐head) orientation of subunits, and N‐terminal domain (NTD, residues Ser1‐Ser33) has no contracts with the C‐terminal domain (CTD, residues Lys51‐Lys120). The NMR data for CTD are in line with crystallographic structure. The flexible interdomain (hinge) region (residues Ala34‐Glu50) has an unordered structure, the Pro44 forming both cis and trans peptide bonds. Due to the conformational exchange the intensities of the peaks from the NTD are low. The conformation of the NTD, which is responsible for the formation of the L7/L12 dimer, is α‐helical hairpin. The NTD dimer forms an antiparallel four‐α‐helix bundle.
On the mechanism by which 6‐ketocholestanol protects mitochondria against uncoupling‐induced Ca2+ efflux
Authors: Chávez Edmundo , Moreno-Sánchez Rafael , Zazueta Cecilia , Cuéllar Adela , Ramírez Jorge , Reyes-Vivas Horacio , Bravo Concepción , Rodríguez-Enríquez Sara
Published: April 2003

This work shows that 6‐ketocholestanol (kCh) inhibits the effect of carbonyl cyanide‐m‐chlorophenyl hydrazone (CCP) on mitochondrial Ca2+ efflux. Such an effect proved to be caused by diminution of membrane fluidity, therefore, it is affected by the incubation temperature. Furthermore, kCh reversed CCP‐induced Ca2+ efflux depending on the accumulation of phosphate. It is also shown that kCh enhances the effect of carboxyatractyloside on membrane permeability transition.
Molecular chaperones are present in the thylakoid lumen of pea chloroplasts
Authors: Schlicher Thomas , Soll Jürgen
Published: April 2003

A soluble protein fraction was obtained from pea chloroplast thylakoids, which represents highly enriched lumenal components. Using antisera against chaperonin 60 (cpn60), chaperonin 10 (cpn10) and the heat shock cognate protein of 70 kDa (hsc70) we are able to demonstrate, that the thylakoid lumen contains a separate set of molecular chaperones, which is distinct from the stromal one. In contrast to the α and β subunits of cpn60 present in the stroma the lumen contains only one cpn60 isoform of distinct isoelectric point. Furthermore the lumenal cpn10 is of ‘normal’ size and not like its stromal counterpart of a double‐domain tandem architecture. The immunoreactive hsc70 isoforms in the lumen seem also to be different from the stromal ones. Thus, chloroplasts seem to contain the largest number of molecular chaperone isoforms present in one organelle.
Altered voltage sensitivity of mutant OmpC porin channels
Authors: Bishop N.D. , Lea E.J.A. , Mobasheri H. , Spiro S.
Published: April 2003

Single OmpC porin channels have been reconstituted in planar bilayer membranes. Wild‐type OmpC forms trimers which are largely insensitive to voltages below 250 mV. A single‐point mutation of the ompC gene has been prepared resulting in replacement of Trp56 by Cys in the eyelet region of the channel wall in a highly conserved segment of the polypeptide. The monomeric channels of which the trimer is composed have smaller conductivity in 1 M NaCl (400 ± 20 pS, mean and S.E.M.,n= 30) and increased voltage sensitivity by comparison with the wild‐type under similar conditions, whereas other (Dex) mutants form larger channels and display different behaviour. Further, by treatment in SDS solutions at different temperatures, the W56C mutant has been shown to be less stable than either the wild‐type or the Dex mutants.
The H+/ATP coupling ratio of the ATP synthase from thiol‐modulated chloroplasts and two cyanobacterial strains is four
Authors: Van Walraven Hendrika S. , Strotmann Heinrich , Schwarz Oliver , Rumberg Bernd
Published: April 2003

In this paper the authors emphasise that the proton translocating ATP synthase from thiol‐modulated chloroplasts and two cyanobacterial strains has a coupling ratio of 4 protons per ATP synthesised or hydrolysed. This ratio is determined by several thermodynamic studies at equilibrium between phosphate potential (ΔG
p) and proton gradient (), and is confirmed by measurement of proton flux during ATP hydrolysis. Ratios lower than 4 H+/ATP that have been published in the past have predominantly been determined with the oxidised chloroplast enzyme. Errors in these measurements will be discussed.
Evaluation of the metal ion requirement of the human deoxyhypusine hydroxylase from HeLa cells using a novel enzyme assay
Authors: Csonga Robert , Ettmayer Peter , Auer Manfred , Eckerskorn Christoph , Eder Jörg , Klier Hannelore
Published: April 2003

Hypusine synthesis in the eukaryotic initiation factor 5A is a unique two‐step posttranslational modification. After deoxyhypusine is generated by the deoxyhypusine synthase, the deoxyhypusine hydroxylase (EC 1.14.99.29) catalyzes the formation of mature hypusine. A rapid assay for monitoring the deoxyhypusine hydroxylase activity was established, employing the oxidative cleavage of the hypusyl residue and subsequent extraction of the generated aldehydes. As metal ion chelators have been reported to inhibit the deoxyhypusine hydroxylase, the mechanism of this inhibition and the effect of transition metal ions on the enzyme activity were investigated. A ferric ion appears to be essential for enzymatic activity, the inhibition of which is entirely attributed to the metal ion bunding capacity of the chelators.