bluuebunny/crossref_metadata_2025 huggingface dataset:
Microheterogeneity of parsley plastocyanin
Authors: Dimitrov Mitko I. , Donchev Anthony A. , Egorov Tsezi A.
Published: July 2002

A procedure for isolation of two iso‐plastocyanins from parsley has been described here. Three consecutive Chromatographic steps on DE‐52‐Whatman cellulose were applied for isolation of two total plastocyanin (PC) fractions, oxidized [PC(II)] and reduced [PC(I)]. By chromatofocusing of PC(II) on Polybuffer exchanger 74 two different plastocyanins, designated as plastocyanin a (PCa) and plastocyanin b (PCb), were obtained. The isoelectric points (pI) of PCa and PCb at 10°C are 4.16 and 4.14, respectively. The complete amino acid sequences of PCa and PCb were determined. The two iso‐proteins consist of 97 amino acid residues and differ only at sequence position 53, where Glu in PCa is replaced by Asp in PCb.
Studies on the synthesis and secretion of transthyretin by the human hepatoma cell line Hep G2
Authors: Blaner William S. , Bonifácio Maria Joao , Feldman Hal D. , Piantedosi Roseann , Saraiva Maria Joao Mascarenhas
Published: July 2002

Transthyretin (TTR) is a circulatory protein which plays an important role in the transport of both thyroid hormone and retinol. Hep G2 cells, a human hepatoma‐denved cell line. have been used extensively in studies of protein secretion by liver cells. The original description of this cell line indicated that this line, unlike primary hepatocytes, does not secrete TTR. We now report studies which reexamine the ability of Hep G2 cells to synthesize and secrete TTR. For this purpose, total RNA was isolated from Hep G2 cells grown on both uncoated and collagen‐coated plastic plates and was examined for TTR expression by Northern blot analysis. TTR mRNA was found to be present m nearly equal amounts in Hep G2 cells cultured in either condition. When Hep G2 cells were cultured in [35S]jmethionine‐containing medium, the cells were found both to synthesize and to secrete immunoprecipitable [35S]SJTTR. Hep G2 cells were found, by sensitive and specific radioimmunoassay, to contain 142 ± 91 ng TTR/106 cells and to secrete TTR into the medium at a nearly constant rate for at least 24 h after medium change. Our data demonstrate that Hep G2 cells do synthesize and secrete TTR and suggest that this cell line might be useful for studies of the secretion of TTR.
Formation of free sphingosine and ceramide from exogenous ganglioside GM1 by cerebellar granule cells in culture
Authors: Riboni Laura , Bassi Rosaria , Sonnino Sandro , Tettamanti Guido
Published: July 2002

Cerebellar granule cells differentiated in culture were incubated with ganglioside [3H‐Sph]GM1 in order to have it inserted into the plasma membrane and metabolized. Among the formed metabolites radioactive sphingosine and ceramide were identified. [3H]Ceramide started to be measurable after 10 min of incubation (pulse), and [3H]sphingosine after 15 min. Their concentrations increased with pulse time, and after a 1‐hour pulse, with chase time. After a 1‐hour pulse with 2 × 10−6 M [3H‐Sph]GM1 followed by a 4‐hour chase, the amount of [3H]sphingosine and [3H]ceramide formed were 0.04 and 0.4 pmol/106 cells, respectively. Particularly the ability to produce sphingosine was higher in differentiated than in undifferentiated cells. It is concluded that ganglioside turnover contributes to the maintenance of the intracellular levels of free sphingosine and ceramide.
Modulation of cell growth and differentiation by ceramide
Authors: Bielawska Alicja , Linardic Corinne M. , Hannun Yusuf A.
Published: July 2002

Ceramide has been suggested as an intracellular modulator of cell growth and differentiation [Okazaki, T. el al. (1990) J. Biol. Chem. 265, 15823‐15831]. In this study, parameters that modulate the effects of ceramide on HL‐60 cell growth and differentiation were examined. A short‐chain, cell‐permeable analog of ceramide, C2‐ceramide, induced differentiation of HL‐60 human leukemia cells and inhibited HL‐60 growth in a concentration‐dependent manner. The potency of C2‐ceramide was modulated by the starting cell density such that the concentration of C2‐ceramide producing 50% inhibition of cell growth (IC50%) ranged from 2 μM (for cells suspended at 1 × 105 cells/ml) to 11 μM (for cells at 8 × 105 cells/ml). However, the IC50% showed little variation if the concentration of C2‐ceramide was expressed as fmol of C2‐ceramide per 105 cells. Therefore, the effectiveness of C2‐ceramide appeared to be primarily determined by its cellular rather than molar concentration. Binding of C2‐ceramide to serum proteins resulted in a 10‐fold increase in the IC50%. These results demonstrate that the biologic activity of C2‐ceramide is subject to surface dilution kinetics and is sensitive to the presence of lipid‐binding proteins. In these properties, ceramide behaves as a prototypic lipid second messenger/intracellular mediator.
Biological activity of bovine placental lactogen in 3T3‐F442A preadipocytes is mediated through a somatogenic receptor
Authors: Vashdi Dorit , Elberg Gerard , Sakal Edna , Gertler Arieh
Published: July 2002

Bovine placental lactogen (bPL) exhibited antimitogenic differentiation‐promoting, biological activity in 3T3‐F442A preadipocytes, Competitive binding studies and affinity labelling revealed bPL activity to be mediated through a somatogenic type of receptor that recognizes human growth hormone (hGH) and bovine GH, but not ovine prolactin or human PL. The bioactivity of bPL was sixfold lower than that of hGH despite that bPL is binding to the somatogenic receptors with fivefold higher affinity. This discrepancy may result from the relatively low ability of bPL to induce post‐receptoral effects such as receptor dimerization.
Vitamin E dietary supplementation inhibits transforming growth factor β1 gene expression in the rat liver
Authors: Parola Maurizio , Muraca Roberto , Dianzani Irma , Barrera Giuseppina , Leonarduzzi Gabriella , Bendinelli Paola , Piccoletti Roberta , Poli Giuseppe
Published: July 2002

Overexpression of transforming growth factor β1 (TGFβ1) and increased transcription of pro‐collagen type I, are known to represent major events implicated in the development of liver fibrosis under either experimental or clinical conditions. Here we report that long‐term dietary vitamin E supplementation in animals undergoing an experimental model of liver fibrosis (induced by chronic treatment of rats with carbon tetrachloride) results in a net inhibition of both hepatic TGFβ1 and α2 (I) procollagen mRNA levels. Moreover, of striking interest is the observation that vitamin E supplementation per so down‐modulates basal levels of TGFβ1 mRNA in the liver of untreated animals, suggesting that a dietary regimen rich in vitamin E may potentially interfere with both the initiation and progression of the fibrosclerotic processes.
‘All‐or‐none’ mechanism of the molten globule unfolding
Authors: Uversky V.N. , Semisotnov G.V. , Pain R.H. , Ptitsyn O.B.
Published: July 2002

The Gdm‐HCl‐induced unfolding of bovine carbonic anhydrase B and
S.
aureus β‐lactamase was studied at 4°C by a variety of methods. With the use of FPLC it has been shown that within the transition from the molten globule to the unfolded state the distribution function of molecular dimensions is bimodal. This means that equilibrium intermediates between the molten globule and the unfolded states are absent, i.e. the molten globule unfolding follows the ‘all‐or‐none’ mechanism.
Inactivation of synovial fluid α1‐antitrypsin by exercise of the inflamed rheumatoid joint
Authors: Zhang Zhi , Farrell Adrian J. , Blake David R. , Chidwick Keith , Winyard Paul G.
Published: July 2002

α1‐Antitrypsin (α1AT) is known to be oxidised by reactive oxygen species both in vitro and in vivo, leading to its inactivation. We report here that synovial fluid (SF) α1AT is inactivated during exercise of the knee‐joints of rheumatoid arthritis (RA) patients. Sequential SF sampling from exercised RA patients showed a marked decrease in the mean activity of α1AT after exercise with no change in the molecular forms of α1AT. No such inactivation was found in the control (continuously resting) RA patients. We suggest that oxidation may contribute to α1AT inactivation as a consequence of ‘hypoxic‐reperfusion’ injury after exercise of the inflamed joint.
Evidence for three binding sites for C3 (hemolytically inactive), C3b and C3d on a CR2‐positive Burkitt lymphoma‐derived cell line (Raji)
Authors: Barile Giuseppe , Di Renzo Livia , Lipari Marcella , Frati Luigi , Faggioni Alberto
Published: July 2002

The present investigation shows that C3 (hemolytic inactive) as well as C3b and C3d bind Raji, a CR2‐positive Burkitt lymphoma‐derived cell line. Pretreatment of the cells with OKB‐7 inhibited the binding of C3, whereas pretreatment with HB‐5 inhibited the binding of C3b. Furthermore, the cells coated either with OKB‐7 or HB‐5 bound high amounts of C3d. TPA‐treated cells showed binding for C3b and weak binding for C3 and C3d. Taken together, the data suggest that Raji cells may express three binding sites for C3, C3b and C3d which can be differently modulated by anti‐CR2 MoAbs and TPA.
Cloning, expression and characterization of human kininogen domain 3
Authors: Auerswald Ennes A. , Rössler Dieter , Mentele Rainer , Assfalg-Machleidt Irmgard
Published: July 2002

The internal domain 3 of the heavy chain of human kininogen, a cysteine proteinase inhibitor, was amplified by a polymerase chain reaction from the kininogen cDNA clone phKG36. The DNA fragment was expressed in Escherichia coli using the ompA expression vector pASK.40 and the resulting protein was isolated from periplasm, purified by S‐carboxymethylpapain affinity‐ and ion‐exchange chromatography. The recombinant human kininogen domain 3 is 92% pure, reacts with anti‐kininogen antibodies and is actively inhibitory. The expected amino acid sequence of ANSM‐[G253‐S377] kininogen was confirmed; the inhibitor has a molecular mass of 14,396 Da and an isoelectric point of 6.0 (pH). The determined K
i values of the complexes with papain and cathepsin L are similar to those measured previously with proteolytically liberated kininogen domain 3, and those of single‐domain cystatins, like chicken egg white cystatin. However, recombinant kininogen domain 3 is a weak inhibitor of cathepsin B (K
i = 63 nM) as it has been found for native L‐kininogen (K
i = 340 nM).
Characterization of the hepatic insulin receptor undergoing internalization through clathrin‐coated vesicles and endosomes
Authors: Jose Marta , Biosca Josep A. , Trujillo Ramon , Itarte Emilio
Published: July 2002

Administration of insulin to rats caused a transient increase in the amount of hepatic insulin receptor present in clathrin‐coated vesicles and endosomes. However, the total ‘in vitro’ insulin stimulated tyrosine kinase activity of the receptor present in endosomes did not vary when expressed per mg of protein and decreased when expressed per β‐subunit content. A decrease in the endogenous phospho tyrosine content of the receptor β‐subunit was observed in endosomes in response to insulin. This indicates that a fraction of the internalized receptor is dephosphorylated in endosomes, which renders it unable to become stimulated by insulin ‘in vitro’.
A bacterial enzyme degrading the model lignin compound β‐etherase is a member of the glutathione‐S‐transferase superfamily
Authors: Masai Eiji , Katayama Yoshihiro , Kubota Sachiko , Kawai Shinya , Yamasaki Makari , Morohoshi Noriyuki
Published: July 2002

Cleavage of β‐aryl ether linkages is essential in lignin degradation. We identified another β‐etherase gene (ligF), which contains an open reading frame of 771 bp and lies between genes coding Cα‐dehydrogenase (ligD) and β‐etherase (ligE). The β‐etherase activity of LigF expressed in Escherichia coli was more than 80 times as high as that of LigE. ligF and ligE are homologous to glutathione‐S‐transferase, and upon addition of glutathione a remarkable acceleration of β‐etherase activity was found in E. coli carrying ligF. It is concluded that LigF plays a central role in β‐aryl ether cleavage and that glutathione is the hydrogen donor in this reaction.
Ubiquitous presence of chromogranin A in the inner ear of guinea pig
Authors: Yoo Seung Hyun , Lim David J.
Published: July 2002

Chromogranin A (CGA), which is supposed to be responsible for the calcium storage of secretory vesicles and is also considered to be a marker protein of neurons and endocrine cells, has been found in a variety of organs and tissues. In the present study, soluble proteins from the organ of Corti, saccule, crista, utricle, tectorial membrane, stria vascularis, and the spiral ligament from the inner ear of guinea pig were extracted, and probed with both polyclonal and monoclonal CGA antibodies to determine the presence of CGA. A 75 kDa protein reactve to both antibodies was found in the organ of Corti, saccule, crista, utricle, stria vascularis, and the spiral ligament, suggesting the widespread presence of CGA in the inner ear.
A new UV‐filter compound in human lenses
Authors: Truscott Roger J.W. , Wood Andrew M. , Carver John A. , Sheil Margaret M. , Stutchbury Glen M. , Zhu Jiulin , Kilby Greg W.
Published: July 2002

A new UV‐filter compound, 4‐(2‐amino‐3‐hydroxyphenyl)‐4‐oxobutanoic acid O‐glucoside, has been identified in human lenses. The structure suggests that it is derived biosynthetically from tryptophan. Quantification studies on the new compound show that it is the second‐most abundant UV‐filter compound in the lens with an absorption and fluorescence spectrum similar to that of 3‐hydroxykynurenine glucoside.
Distribution and characterization of immunoreactive rat adrenomedullin in tissue and plasma
Authors: Sakata Junichiro , Shimokubo Toru , Kitamura Kazuo , Nishizono Masatoshi , Iehiki Yoshinari , Kangawa Kenji , Matsuo Hisayuki , Eto Tanenao
Published: July 2002

Adrenomedullin is a new bioactive peptide recently isolated from pheochromocytoma. We report on the rat adrenomedullin distribution and molecular forms in various tissues and plasma. Using a sensitive radioimmunoassay system for rat adrenomedullin, high concentrations of immunoreactive rat adrenomedullin were detected in adrenal gland, lung and cardiac atrium. In lung and atrium, the immunoreactivity concentration in rat was about 6–10 times higher than that in human. The mean plasma concentration of immunoreactive rat adrenomedullin was 3.60 ±0.34 fmol/ml (mean ± S.D.). Analysis in adrenal gland, lung and atrium with reverse‐phase and gel‐filtration high‐performance liquid chromatography showed that most immunoreactive rat adrenomedullin emerged as a single peak at a position exactly identical to that of the authentic rat adrenomedullin peptide, synthesized according to the sequence predicted from the cDNA.
Enhancement of inducible‐type NO synthase gene transcription by protein synthesis inhibitors
Authors: Oguchi Shinobu , Weisz Alessandro , Esumi Hiroyasu
Published: July 2002

Treatment of mouse maerophage‐like RAW 264.7 cells with certain protein synthesis inhibitors is followed by accumulation of the mRNA for the inducible isoiorm of nitric oxide synthase (i‐NOS). The activity of these compounds on the i‐NOS gene in RAW 264.7 cells was analyzed here in detail. Results show that both cycloheximide and anisomycin can efficiently induce i‐NOS mRNA, even when used at concentrations so low () to have only negligible effects on protein synthesis; puromycin, on the other hand, shows only a limited effect on i‐NOS mRNA expression, detectable only when cells are treated with higher concentrations of inhibitor (). In RAW 264.7 cells, low concentrations of cycloheximide trigger an immediate‐early gene response, as indicated by induction of c‐fos and JE mRNAs, and can efficiently activate transcription of transiently transfeeted recombinant reporter genes including either the i‐NOS or the c‐fos gene promoters.
Oxidation of LDL by nitric oxide and its modification by Superoxides in macrophage and cell‐free systems
Authors: Wang Jin-Mei , Chow Song-Nan , Lin Jen-Kun
Published: July 2002

Oxidative modification of low density lipoproteins (LDL) can increase the atherogenicity of LDL. Here we demonstrate the potential role of nitric oxide (NO) in LDL modification. Dose‐dependent alterations of LDL including an increase in electrophoretic mobility, generation of peroxides and degradation of apoprotein B occurred during incubation with NO. Sodium nitroprusside was also shown to increase the electromobility of LDL. Superoxide dismutase (SOD) and catalase did not affect this result. NO treatment also increases the negative charges on other lipoproteins. In addition, we have demonstrated that NO plays a protective role in macrophage or superoxide‐induced LDL modification through its neutralizing action.
Activation of glycogen phosphorylase and glycogenolysis in rat skeletal muscle by AICAR — an activator of AMP‐activated protein kinase
Authors: Young Martin E , Radda George K , Leighton Brendan
Published: July 2002

We determined whether the cell permeable molecule AICAR, whose metabolite activates AMP‐activated protein kinase (AMPK) in cells, affected glycogen metabolism in rat seleus muscle preparations in vitro. The basal and insulin‐stimulated rates of radiochemical lactate formation, net lactate release and glycogen synthesis were determined. AICAR stimulated net lactate release (but not radiochemical lactate formation) only at a basal concentration of insulin. An increased rate of glycogenolysis was the likely cause of increased net lactate release as glycogen phosphorylase activity was significantly increased by AICAR. AICAR‐stimulated net lactate release and phosphorylase activity were potently inhibited by insulin.
Low affinity of Trypanosoma brucei transferrin receptor to apotransferrin at pH 5 explains the fate of the ligand during endocytosis
Authors: Maier Alexander , Steverding Dietmar
Published: July 2002

Uptake of host transferrin (Tf) in Trypanosoma brucei is mediated by a heterodimeric, glycosyl‐phosphatidylinositol‐anchored receptor. After endocytosis, Tf is delivered to lysosomes where it is proteolytically degraded. So far, the sequence of events leading to ligand dissociation and degradation is undefined. We now show by Triton X‐114 phase separation that iron‐free Tf (apo‐Tf) dissociates from the receptor at pH 5.0. The low affinity of apo‐Tf for its receptor at pH 5.0 is confirmed by an apparent dissociation constant of 1.1 μM. The implications of this result on the mechanism of intracellular processing of Tf in trypanosomes are discussed.
Characterization of the chitin biosynthesis process as a compensatory mechanism in the fks1 mutant of Saccharomyces cerevisiae
Authors: Garcı́a-Rodriguez L.J , Trilla J.A , Castro C , Valdivieso M.H , Durán A , Roncero C
Published: July 2002

Deletion of the 1,3‐β‐D‐glucan synthase gene FKS1 in Saccharomyces cerevisiae induces a compensatory mechanism that is reflected in a significant increase in chitin synthase III (CSIII) activity, leading to high rates of chitin synthesis. Deregulation of CSIII activity is mainly due to the intracellular delocalization of Chs3p and Chs4p, the two main components of the CSIII active complex.
Permissive role of protein kinase Cα but not protein kinase Cδ in sphingosine 1‐phosphate‐induced RhoA activation in C2C12 myoblasts
Authors: Meacci Elisabetta , Donati Chiara , Cencetti Francesca , Romiti Elena , Bruni Paola
Published: July 2002

Rho GTPases participate in various important signaling pathways and have been implicated in myogenic differentiation. Here the first evidence is provided that in C2C12 myoblasts sphingosine 1‐phosphate (SPP) rapidly and transiently induced membrane association of RhoA in a pertussis toxin‐insensitive manner. The bioactive lipid preferentially relocalized the GTPase to Golgi‐enriched membrane. Translocation of RhoA was abolished by inhibition or down‐regulation of protein kinase C (PKC). Notably, treatment with Gö6976, an inhibitor of conventional PKCs, which selectively blocked PKCα in these cells, prevented SPP‐induced RhoA translocation. Conversely rottlerin, a selective inhibitor of PKCδ, was without effect, demonstrating that SPP signaling to RhoA involves PKCα but not PKCδ activation. This novel functional relationship between the two proteins may have a role in SPP‐mediated regulation of downstream effectors.
Reversible inactivation of AT2 angiotensin II receptor from cysteine–disulfide bond exchange
Authors: Feng Ying-Hong , Saad Yasser , Karnik Sadashiva S
Published: July 2002

Dithiothreitol (DTT) treatment of angiotensin II (Ang II) type 2 (AT2) receptor potentiates ligand binding, but the underlying mechanism is not known. Two disulfide bonds proposed in the extracellular domain were examined in this report. Based on the analysis of ligand affinity of cysteine (Cys, C) to alanine (Ala, A) substitution mutants, we provide evidence that Cys35–Cys290 and Cys117–Cys195 disulfide bonds are formed in the wild‐type AT2 receptor. Disruption of the highly conserved Cys117–Cys195 disulfide bond linking the second and third extracellular segments leads to inactivation of the receptor. The Cys35–Cys290 bond is highly sensitive to DTT. Its breakage results in an increased binding affinity for both Ang II and the AT2 receptor‐specific antagonist PD123319. Surprisingly, in the single Cys mutants, C35A and C290A, a labile population of receptors is produced which can be re‐folded to high‐affinity state by DTT treatment. These results suggest that the free –SH group of Cys35 or Cys290 competes with the disulfide bond formation between Cys117 and Cys195. This Cys–disulfide bond exchange results in production of the inactive population of the mutant receptors through formation of a non‐native disulfide bond.
Genomic Exploration of the Hemiascomycetous Yeasts: 1. A set of yeast species for molecular evolution studies1
Authors: Souciet Jean-Luc , Aigle Michel , Artiguenave François , Blandin Gaëlle , Bolotin-Fukuhara Monique , Bon Elisabeth , Brottier Philippe , Casaregola Serge , de Montigny Jacky , Dujon Bernard , Durrens Pascal , Gaillardin Claude , Lépingle Andrée , Llorente Bertrand , Malpertuy Alain , Neuvéglise Cécile , Ozier-Kalogéropoulos Odile , Potier Serge , Saurin William , Tekaia Fredj , Toffano-Nioche Claire , Wésolowski-Louvel Micheline , Wincker Patrick , Weissenbach Jean
Published: July 2002

The identification of molecular evolutionary mechanisms in eukaryotes is approached by a comparative genomics study of a homogeneous group of species classified as Hemiascomycetes. This group includes Saccharomyces cerevisiae, the first eukaryotic genome entirely sequenced, back in 1996. A random sequencing analysis has been performed on 13 different species sharing a small genome size and a low frequency of introns. Detailed information is provided in the 20 following papers. Additional tables available on websites describe the ca. 20 000 newly identified genes. This wealth of data, so far unique among eukaryotes, allowed us to examine the conservation of chromosome maps, to identify the ‘yeast‐specific’ genes, and to review the distribution of gene families into functional classes. This project conducted by a network of seven French laboratories has been designated ‘Génolevures’.
A genomic perspective on human proteases
Authors: Southan Christopher
Published: July 2002

Over 400 human proteases documented in secondary databases can already be delineated in genomic sequence. A Genome Ontology annotation of 30 585 sequences in the provisional human proteome set recognises 498 proteases, i.e. 1.6%. Homology searches against finished sequence and comparisons between mouse and zebrafish are likely to increase this total. However, the data already indicate that the mechanistic class, sequence family and domain distribution of the genomic complement of proteases is unlikely to shift significantly from that already observed in the transcript data. Genomically derived novel sequences will require bioinformatic analysis and biochemical verification. The increasing availability of annotated genomic data will enable studies of splice variants, transcriptional control, polymorphisms, pseudogenes, inactive homologues and evolution. Comparative work on complete human protease families should produce a more integrated picture of their biochemistry and physiology. Genomic data will also lead to the identification of new protease involvement in disease processes and their evaluation as drug targets.
Inhibition of carboxypeptidase A by excess zinc: analysis of the structural determinants by X‐ray crystallography
Authors: Gomez-Ortiz Mariola , Gomis-Rüth Francesc X , Huber Robert , Avilés Francesc X
Published: July 2002

Pancreatic metallocarboxypeptidases are inhibited by a millimolar excess of zinc together with other exo‐ and endometalloproteases. We have analyzed the structure of bovine carboxypeptidase A inhibited by an excess of zinc ions using X‐ray crystallography at 1.7 Å overall resolution. Under these conditions, a second zinc is observed to bind to the enzyme active site, establishing a distorted tetrahedrally coordinated complex which involves Glu‐270 (the general base for catalysis), a water molecule, a chloride ion, and a hydroxide ion. This hydroxide ion forms a 114° angular bridge between the inhibitory and the catalytic zinc ions, which are at a distance of 3.3 Å from one another. The inhibitory zinc holds the hydroxide at nearly the same location as a previously observed active site water molecule (W571) and probably perturbs the substrate positioning and stereochemical rearrangements required for substrate cleavage during catalysis.
Tyrosine: an inhibitor of LDL oxidation and endothelial cell cytotoxicity initiated by superoxide/nitric oxide radicals1
Authors: Kapiotis Stylianos , Hermann Marcela , Held Irmtraud , Mühl Adolf , Gmeiner Bernhard
Published: July 2002

Tyrosyl radicals can catalyze LDL oxidation. In addition to their LDL oxidizing ability, superoxide (O2
·−)/nitric oxide (NO·) generate phenoxyl radicals when reacting with tyrosine. Therefore we tested if tyrosine can act as a pro‐oxidant in O2
·−/NO·‐initiated LDL oxidation. When LDL was exposed to O2
·−/NO·, tyrosine exerted a strong inhibitory effect on O2
·−/NO·‐initiated LDL oxidation as measured by TBARS formation and alteration in electrophoretic mobility of LDL. Tyrosine was also able to protect human endothelial cells from the cytotoxic effect of O2
·−/NO·. Because O2
·−/NO· can occur in vivo, the results may indicate that serum‐free tyrosine could act as an efficacious physiological antioxidant in case of O2
·−/NO·‐initiated LDL oxidation and endothelial cell cytotoxicity.
Exchangeability of Qsr1p, a large ribosomal subunit protein required for subunit joining, suggests a novel translational regulatory mechanism
Authors: Dick Frederick A , Eisinger Dominic P , Trumpower Bernard L
Published: July 2002

Qsr1p is a 60S ribosomal subunit protein that is necessary for joining of large and small ribosomal subunits and is also one of the last proteins assembled onto the 60S ribosomal subunit in the cytoplasm. The finding that Qsr1p is identical to L7, a protein previously shown to cycle on and off large ribosomal subunits in the cytoplasm, suggests that the addition of Qsr1p onto the 60S ribosomal subunit could be utilized as a translational regulatory mechanism by limiting the supply of functional 60S subunits.
[1H,13C] NMR determination of the order of lobe loading of human transferrin with iron: comparison with other metal ions
Authors: Sun Hongzhe , Cox Mark C , Li Hongyan , Mason Anne B , Woodworth Robert C , Sadler Peter J
Published: July 2002

Human serum transferrin (hTF) is a single‐chain bilobal glycoprotein (80 kDa) which transports Fe3+ and a variety of other metal ions in blood. Only diferric transferrin, not the apo‐protein, binds strongly to transferrin receptors and is taken up by cells via receptor‐mediated endocytosis. We show here that 2D [1H,13C] NMR studies of recombinant ϵ‐[13C]Met‐hTF allow the order of lobe loading with various metal ions, including Fe3+, to be determined. In particular, the resonance for Met‐464, a residue in the hydrophobic patch of helix 5, is very sensitive to iron binding in the C‐lobe. The selectivity of lobe loading with Fe3+ is compared to loading with Fe2+ (which binds as Fe3+), Al3+, Ga3+ and Bi3+. Similar changes in shifts of the Met residues are observed for these metal ions, suggesting that they induce similar conformational changes in the protein.
Effect of bicarbonate on the S2 multiline EPR signal of the oxygen‐evolving complex in photosystem II membrane fragments
Authors: Hulsebosch R.J , Allakhverdiev S.I , Klimov V.V , Picorel R , Hoff A.J
Published: July 2002

Removal of bicarbonate from spinach photosystem II BBY particles by means of washing in a CO2‐free medium results in the loss of their capability to accumulate the S2 multiline EPR signal upon continuous illumination at 190 K. Addition of 1 mM NaHCO3 before illumination leads to a 50–60% restoration of the multiline signal. Similarly, in BBY particles depleted of Mn by treatment with 1 M Tris‐HCl (pH 8.0) and 0.5 M MgCl2, re‐addition of MnCl2 in the presence of 1 mM NaHCO3 results in a partial restoration (∼30%) of the S2 multiline EPR signal of the Mn cluster, while in the absence of NaHCO3 no restoration is observed. The results provide further evidence that bicarbonate is essential for maintaining the Mn‐containing oxygen‐evolving complex of PS II in a functionally active form.
Amino acid sequence, glycan structure, and proteolytic processing of the lectin of Vatairea macrocarpa seeds
Authors: Calvete Juan J , Santos Cláudia F , Mann Karlheinz , Grangeiro Thalles B , Nimtz Manfred , Urbanke Claus , Sousa-Cavada Benildo
Published: July 2002

VML is a galactose‐binding lectin isolated from Vatairea macrocarpa seeds. By SDS‐polyacrylamide gel electrophoresis, VML is a glycoprotein composed of a major 32–34 kDa double band (alpha‐chain) and minor 22 kDa and 13 kDa bands. N‐terminal sequencing of electroblotted samples showed that the 22 and 13 kDa bands corresponded to C‐(beta) and N‐(gamma) terminal fragments of the alpha‐chain, respectively. The primary structure of VML displays similarity with other leguminous lectins, particularly with Erythrina variegata, Robinia pseudoacacia and Sophora japonica lectins. VML is N‐glycosylated at asparagine residues at positions 111 and 183 with one major glycan structure. Tandem mass spectrometry and methylation analysis indicated the presence of Manα1‐6[(Manα1‐3)(Xylβ1‐2)]Manβ1‐4‐GlcNAcβ1‐4(Fucα1‐3)GlcNAc, a typical plant N‐glycan. Equilibrium sedimentation analysis by analytical centrifugation showed that VML had a mass of 122–130 kDa, which did not change within the pH range 2.5–8.5. These data indicated that VML is a pH‐independent homotetrameric protein and that a small proportion of the alpha‐subunits is cleaved into noncovalently associated N‐ and C‐terminal fragments. Mass spectrometric analysis suggested a mechanism for the proteolytic processing of VML. V. macrocarpa lectin contains a mixture of doubly (28 525 Da) and singly (27 354 Da) glycosylated alpha‐chains. Deglycosylation of Asn‐111 correlates with proteolytic cleavage of the Asn‐114‐Lys‐115 bond yielding glycosylated gamma (residues 1–114, 12 304 Da) and nonglycosylated beta‐ (residues 115–239, 14 957 Da) chains. Some beta‐chain molecules are further deglycosylated and N‐terminally processed yielding products of molecular masses of 13 783 Da and 13 670 Da.
Effect of green and black tea supplementation on lipids, lipid oxidation and fibrinogen in the hamster: mechanisms for the epidemiological benefits of tea drinking
Authors: Vinson Joe A , Dabbagh Yousef A
Published: July 2002

There is considerable epidemiological evidence that tea drinking lowers the risk of heart disease. However, the mechanism by which tea can be protective is unknown. Hamsters were fed a normal or high cholesterol diet for 2 weeks and drank green or black tea ad libitum. The plasma lipid profile was significantly improved by both teas compared to controls. Also in vivo lipid oxidation as measured by plasma lipid peroxides and LDL+VLDL oxidizability were significantly decreased by the teas. In the normal fed tea groups fibrinogen was decreased but not in the high cholesterol groups. Green tea was significantly more effective than the black tea. These results show in the hamster model that black and green tea improve the risk factors for heart disease by both hypolipemic and antioxidant mechanisms and possibly a fibrinolytic effect.
Alzheimer's β‐amyloid vasoactivity: identification of a novel β‐amyloid conformational intermediate
Authors: Crawford Fiona , Soto Claudio , Suo Zhiming , Fang Chunhong , Parker Timothy , Sawar Asad , Frangione Blas , Mullan Mike
Published: July 2002

The β‐amyloid (Aβ) peptide has previously been shown to enhance phenylephrine or endothelin‐1 induced constriction of aortic rings in vitro. The characteristics of Aβ vasoactivity (dose, fragment length, timing) suggest that the mechanism is distinct from Aβ cytotoxicity. To identify which properties of Aβ determine its biological activity on vessels, we investigated a number of Aβ analogues and fragments, individually and in combination, including those that are known to be associated with Alzheimer's disease (Aβ1–42) and hereditary cerebral hemorrhage with amyloidosis – Dutch type (Aβ(22Q)1–40). The vasoactivity appears to be related to the conformation adopted by the peptide in solution. The β‐pleated sheet rich Aβ1–42 and Aβ(22Q)1–40 were each less vasoactive than the mainly random coil wild type Aβ1–40. However, the most vasoactive Aβ peptides were combinations which contain mixtures of random coil and β‐sheet structure. The finding that peptides containing low or high levels of β‐pleated conformation are less vasoactive than those containing intermediate amounts of this structural motif allows us to propose the existence of a transitional form between random coil and β‐pleated that is the vasoactive species of Aβ. This is the first time that Aβ conformational intermediates have been identified and a biological activity associated with them.
Alternative conformations of human replication protein A are detected by crosslinks with primers carrying a photoreactive group at the 3′‐end
Authors: Lavrik O.I. , Kolpashchikov D.M. , Nasheuer H.-P. , Weisshart K. , Favre A.
Published: July 2002

To analyze the influence of single‐stranded template extension of DNA duplex on the conformation of human replication protein A (RPA) bound to DNA we have designed two template‐primer systems differing by the size of the single‐stranded template tail (9 and 19 nucleotides (nt)). Base‐substituted photoreactive dUTP analogs were used as substrates for elongation of radiolabeled template‐primer by DNA polymerase β in the absence or in the presence of RPA. Following UV‐crosslinking it was demonstrated that the pattern of RPA subunit labeling and consequently RPA arrangement near the 3′‐end of the primer is stronlgly dependent upon the length of the template extension.
The role of the sympathetic nervous system in the regulation of leptin synthesis in C57BL/6 mice
Authors: Evans Bronwyn A. , Agar Larissa , Summers Roger J.
Published: July 2002

The objectives of this study were to determine whether leptin synthesis is regulated by the sympathetic nervous system and if so whether β‐adrenergic receptors mediate this effect. We show that sympathetic blockade by reserpine increases leptin mRNA levels in brown but not white adipose tissue, while acute cold‐exposure decreases leptin expression 10‐fold in brown adipose tissue and 2‐fold in white adipose tissue. The cold‐induced reduction in leptin mRNA can be prevented by a combination of propranolol and SR 59230A but not by either antagonist alone, indicating that β3‐adrenergic receptors and classical β1/β2‐adrenergic receptors both mediate responses to sympathetic stimulation. Circulating leptin levels reflect synthesis in white adipose tissue but not in brown adipose tissue.
Revisiting the S2 specificity of papain by structural analogs of Phe
Authors: Lecaille Fabien , Serveau Carole , Gauthier Francis , Lalmanach Gilles
Published: July 2002

Papain characteristically has a strong preference for encoded l‐aromatic amino acids (Phe>Tyr) at P2 position. We re‐examined papain S2 specificity using structural analogs of Phe, in fluorogenic substrates of the series: dansyl‐Xaa‐Arg‐Ala‐Pro‐Trp (Xaa=P2 residue). Kinetic analyses showed that the S2 pocket accommodates a broad spectrum of Phe derivatives. Papain is poorly stereoselective towards Dns‐(d/l)‐Phe‐Arg‐Ala‐Pro‐Trp and binding is not critically affected by replacement of the benzyl ring by the non‐aromatic lateral chain of cyclohexylalanine. The K
m was significantly improved by mono‐ and di‐chlorination of Phe, or by its substitution by an electronegative group‐like NO2, but the specificity constant was unchanged. Shortening or lengthening the side chain by adding or removing a methylene group impairs the P2/S2 interactions significantly, as do constrained structural analogs of Phe. Incorporation of benzyl‐substituted phenylalanyl amino acid could help to design peptide‐derived inhibitors with greater affinity and bioavailability.
Functional analysis of the evolutionarily conserved proline 53 residue in Proteus mirabilis glutathione transferase B1‐1
Authors: Allocati Nerino , Casalone Enrico , Masulli Michele , Ceccarelli Ilaria , Carletti Erminia , Parker Michael W , Di Ilio Carmine
Published: July 2002

The role of the evolutionarily conserved residue Pro‐53 in Proteus mirabilis glutathione transferase B1‐1 has been examined by replacing it with a serine residue using site‐directed mutagenesis. The effect of the replacement on the activity, thermal stability and antibiotic binding capacity of the enzyme was examined. The results presented support the view that Pro‐53 participates in the maintenance of the proper conformation of the enzyme fold rather than playing a direct role in the catalytic reaction. Furthermore, this residue appears to be an important determinant of the antibiotic binding to the enzyme. Experiments with wild type and mutated enzymes provide evidence that glutathione transferases may play an important role in antibiotic resistance exhibited by bacteria.
Human stromelysin gene promoter activity is modulated by transcription factor ZBP‐89
Authors: Ye Shu , Whatling Carl , Watkins Hugh , Henney Adriano
Published: July 2002

Matrix metalloproteinase expression is under strict regulation in physiological conditions. Disruption of the regulatory mechanisms can lead to tissue destruction and is associated with tumour invasion and metastasis. Using the one‐hybrid assay technique with a cis‐element in the promoter region of the stromelysin (matrix metalloproteinase‐3) gene, a cDNA encoding a transcription factor termed ZBP‐89 was obtained. The interaction between ZBP‐89 and the stromelysin promoter element was confirmed by electrophoretic mobility shift assays with a recombinant ZBP‐89. Reporter gene expression under the control of the stromelysin promoter in transiently transfected cells was significantly increased when the cells were co‐transfected with a ZBP‐89 expression construct. These results indicate that ZBP‐89 interacts with the stromelysin promoter and upregulates its activity. As ZBP‐89 expression is known to be increased in gastric carcinoma cells, induction of stromelysin expression may be a significant factor in tumour metastasis.
Molecular cloning and sequencing of two ‘short chain’ and two ‘long chain’ K+ channel‐blocking peptides from the Chinese scorpion Buthus martensii Karsch
Authors: Zhu Shunyi , Li Wenxin , Zeng Xianchun , Jiang Dahe , Mao Xin , Liu Hui
Published: July 2002

Five full‐length cDNAs encoding the precursors of two ‘short chain’ scorpion non‐toxic peptides active on Ca2+‐activated K+ channels (BmP02 and BmP03) and two novel putative long chain K+ channel‐blocking peptides (named BmTXKβ and BmTXKβ2) were first isolated from the venom gland cDNA library of the Chinese scorpion Buthus martensii Karsch (BmK). BmTXKβ2 showed a high similarity with AaTXKβ, while BmTXKβ was completely different in the deduced primary structure from the long chain and short chain scorpion toxins already characterized. Thus, BmTXKβ expands the scorpion long chain K+ channel‐blocking peptide family. Although little sequence similarity exists between the above two short and two long peptides, they are similar at the positions of six cysteines, suggesting that they should all share a similar scaffold composed of an α‐helix and a three‐stranded β‐sheet.
Laparoscopic colposuspension for total vaginal prolapse
Authors: Ostrzenski A.
Published: July 2002

Objective: To present and evaluate a new technique of modified translaparoscopic colpopexy for total vaginal prolapse following hysterectomy as an alternative surgical mode of therapy to abdominal sacral suspension or transvaginal sacrospinous ligament vaginal vault suspension. A modest modification (Group II) of this author's initial surgical technique (Group I) and postoperative observations from 1989 to 1996 are presented. Methods: Twenty‐seven patients with iatrogenic total vaginal prolapse subsequent to hysterectomy enrolled in this clinical study, which was conducted from September 1989 through February 1996. Posteriorly, vaginal apex was suspended to the deep layer of the uterosacral ligaments. The latero‐posteriors vaginal cuff was suspended to the cardinal ligaments. Anteriorly, the vaginal vault was approximated to the pubocervical fascia. These procedures were performed in both groups retroperitoneally. Additionally, and only in Group II, retrpubically, at the mid‐pelvic level, the pubocervical fascia and lateral superior vaginal sulci were suspended to the tendinous arch and to the fascia covering the muscles of the pelvic sidewall. This additional part of the operation constituted a modification of the initial translaparoscopic colpopexy (Group I). Results: In Group I, fifteen patients had good outcome of the operation. However, patient number 16 had a recurrent total vaginal vault prolapse within 6 months following the initial laparoscopic colpopexy. Preceding an initial laparoscopic colpopexy, sacrospinous ligament suspension was performed as one of multiple corrective surgeries. This patient was subjected to the modified laparoscopic colpopexy and has been observed for over three years. The vaginal vault remains well suspended in this particular case as well as in all remaining patients in Group II. There were neither intraoperative, immediate, nor delayed postoperative complications in all 27 patients, in both groups. Conclusions: (1) When Group I results are compared with Group II results, the new, modified translaparoscopic retroperitoneal‐retropubic colpopexy appeared to be a superior technique and very promising, and offers: (a) vaginal cuff suspension by using natural neighbouring genital pelvic structures, (b) reconstruction of pelvic gross/functional anatomy, placement of the vagina adequately in midline position, and alignment of the vagina parallel to the rectum, and re‐constitution of proper relationship between the newly suspended vagina and pelvic viscera. (2) It is a safe operation, simple to learn, and easy to execute; however, retroperitoneal‐retropubic‐mid‐pelvis surgical experience as well as advanced operative laparoscopic skill is needed to meet technical demands of the operation.
Identification of caveolae and caveolin in C6 glioma cells
Authors: Silva W.I. , Maldonado H.M. , Lisanti M.P. , DeVellis J. , Chompré G. , Mayol N. , Ortiz M. , Velázquez G. , Maldonado A. , Montalvo J.
Published: July 2002

Caveolae (CAV) constitute a novel subcellular transport vesicle that has received special attention based on its proven and postulated participation in transcytosis, potocytosis, and in cell signaling events. One of the principal components of CAV are caveolin protein isoforms. Here, we have undertaken the immunochemical identification of CAV and the known caveolin isoforms (1α, 1β, 2 and 3) in cultured rat C6 glioma cells. Immunoblot analysis revealed that particulate fractions from rat C6 glioma cells express caveolin‐1 and caveolin‐2. The relative detergent‐insolubility of these caveolin isoforms was also determined by Western blot analysis. Indirect immunofluorescence analysis with caveolin‐1 and B‐2 antibodies revealed staining patterns typical of CAV's known subcellular distribution and localization. For both caveolin isoforms immunocytochemical staining was characterized by intensely fluorescent puncta throughout the cytoplasm and diffuse micropatches at the level of the plasmalemma. Perinuclear staining was also detected, consistent and suggestive of caveolin's localization in the trans Golgi region. The caveolin‐1 and B‐2 immunoreactivity seen in Western blots and immunocytochemically is related to structurally relevant CAV as supported by the isolation of caveolin‐enriched membrane complexes using two different methods. Light‐density, Triton X‐100‐insoluble caveolin‐1‐ and caveolin‐2‐enriched fractions were obtained after fractionation of rat C6 glioma cells and their separation over 5–40% discontinuous sucrose‐density gradients. Similar fractions were obtained using a detergent‐free, sodium carbonate‐based fractionation method. These results further support the localization of CAV and caveolins in glial cells. In addition, they demonstrate that cultured C6 glioma cells can be useful as a model system to study the role of CAV and caveolins in subcellular transport and signal transduction events in glial cells and the brain.
The effects of psychoactive drugs and neuroleptics on language in normal subjects and schizophrenic patients: a review
Authors: Salomé F , Boyer P , Fayol M
Published: July 2002

The aim of this survey is to present an overview of research into psychopharmacology as regards the effects of different psychoactive drugs and neuroleptics (NL) on language in normal subjects and schizophrenic patients. Eighteen studies that have investigated the effects of different drugs (alcohol, amphetamines, secobarbital, L-dopa, psilocybin, ketamine, fenfluramine) and neuroleptics (conventional and atypical) on language are reviewed. There are no studies concerning the effects of neuroleptics on language in healthy subjects. The results of the effects of other molecules indicate that language production can be increased (alcohol, amphetamine, secobarbital), rendered more complex (d-amphetamine), more focused (L-dopa) or more unfocused (psilocybin) and clearly impaired (ketamine). For schizophrenic patients, most studies show that conventional neuroleptic treatments, at a therapeutic dosage and in acute or chronic mode, reduce language disorders at all levels (clinic, linguistic, psycholinguistic). In conjunction with other molecules, the classical NL, when administered at a moderate dosage and in chronic mode, modify language in schizophrenia, either by improving the verbal flow and reducing pauses and positive thought disorder (NL + amphetamine) or by inducing an impairment in the language measurements (NL + fenfluramine). Clinical, methodological and theoretical considerations of results are debated in the framework of schizophrenic language disorders.