bluuebunny/crossref_metadata_2025 huggingface dataset:
Transfer of ubiquinol from the reaction center to the bc1 complex in Rhodobacter sphaeroides chromatophores under oxidizing conditions
Authors: Drachev L.A. , Mamedov M.D. , Mulkidjanian A.Ya. , Semenov A.Yu. , Shinkarev V.P. , Verkhovsky M.I.
Published: July 2002

The mechanism of interaction between the photosynthetic reaction center (RC) and bc
1 complex has been investigated in chromatophores of Rhodobacter sphaeroides. The kinetics of cytochrome b
h reduction and formation of the transmembrane electric potential were measured at high E
h, a condition where ubiquinol is formed in the RC only on the second light flash. In the presence of antimycin A, the kinetics of cytochrome b
h reduction have been shown to be sensitive neither to the amount of ubiquinol produced nor to the number of active bc
1 complexes. It is concluded that the reaction between the ubiquinol produced on the second flash and the bc
1 complex is monomolecular. To explain the monomolecular pattern of this reaction under oxidizing conditions (the present work) and the previously described bimolecular pattern under reducing conditions [(1983) Biochim. Biophys. Acta 723, 202–218], it is proposed that (i) quinone exchange between the RC and bc
1 complex occurs via a local quinone pool and (ii) the rate of exchange between the quinone pools is very much slower than cytochrome b
h reduction.
The effect of ATP, GTP and cAMP on the cGMP‐dependent conductance of the fragments from frog rod plasma membrane
Authors: Filatov G.N. , Jainazarov A.B. , Kolesnikov S.S. , Lyubarsky A.L. , Fesenko E.E.
Published: July 2002

Using a ‘patch‐clamp’ method in the ‘inside‐out’ configuration, ATP, ADP, AMP‐PCP and AMP‐PNP have been shown to increase the cGMP‐dependent component of the rod plasma membrane conductance 2–4‐fold and GTP, GDP but not GMP or nonhydrolyzable GTP analogs GMP‐PNP and GTP‐γ‐S to abolish the ATP action. The ATP and GTP effects were observed at [EDTA] = 1 mM when magnesium and calcium ions were absent. In about half of the experiments the cGMP‐dependent conductance was shown to be increased by cAMP in the micromolar concentration range by 10–50%, the cAMP action did not depend on the presence of nucleoside triphosphates. In vivo ATP, GTP and cAMP are assumed to modulate the sensitivity of the photoreceptor plasma membrane to cGMP.
GTP‐dependent Ca2+ release from rat liver microsomes Vesicle fusion is not required
Authors: Kleineke Jochen , Schröder Andrea , Söling Hans-Dieter
Published: July 2002

The GTP‐dependent calcium release from rat liver microsomes is known to be promoted in the presence of colloids like polyethyleneglycol (PEG), polyvinylpyrrolidine, or albumin. Dawson et al. [(1987) Biochem. J. 244, 87–92] using the ‘fusogen’ PEG have concluded that both GTP‐induced calcium efflux and the enhancement of InsP3‐promoted calcium release in the presence of GTP could be attributed to a GTP‐dependent vesicle fusion. Here, using the more physiological colloid albumin we report that GTP‐induced calcium release from rat liver microsomes may not be linked to vesicle fusion.
Characterization of vesicles, containing an acylated oligopeptide, released by human colon adenocarcinoma cells
Authors: Masella R. , Cantafora A. , Guidoni L. , Luciani A.M. , Mariutti G. , Rosi A. , Viti V.
Published: July 2002

RNA‐containing vesicles, recovered from the supernatant of high‐density cell samples of human colon carcinoma, produce a high‐resolution 1H NMR spectrum of lipids characterized by isotropic tumbling; these vesicles contain large amounts of triglycerides and cholesterol esters. Both findings have strict analogies to what is displayed by the proteolipid complexes isolated from the sera of tumor‐bearing patients [(1985) Proc. Natl. Acad. Sci. USA 82, 3455–3459; (1986) FEBS Lett. 203, 164–168]. Lipid analysis and enzymatic tests indicate that these vesicles are selected micromaps of plasma membranes, analogous to those that can be recovered from culture media in which tumor cells are grown [(1985) Dev. Biol. 3, 33–57]. Peculiar lipids, an acylated oligopeptide and a modified phospholipid, are also present in the vesicles.
Production of pro‐opiomelanocortin (POMC) by a vaccinia virus transient expression system and in vitro processing of the expressed prohormone by POMC‐converting enzyme
Authors: Andreasson K.I. , Tam W.W.H. , Feurst T.O. , Moss B. , Loh Y.P.
Published: July 2002

Pro‐opiomelanocortin (POMC) was expressed in CV‐1 (green monkey kidney) cells using a vaccinia virus transient expression system [(1986) Proc. Natl. Acad. Sci. USA 83, 8122]. The system involved infection of cells with a recombinant vaccinia virus carrying the T7 RNA polymerase gene and transfection with a plasmid containing the mouse POMC sequence flanked by the T7 RNA polymerase promoter at its 5′‐end and the T7 RNA polymerase terminator at its 3′‐end. Assay of the medium from transfected cells showed that 1–2 μg of immunoreactive ACTH was produced/106 cells. Analysis of the same medium by SDS‐PAGE/Western blots revealed a band of 30–36 kDa, which was immunostained with both ACTH and β‐endorphin antisera. Labeling the transfected cells with [3H]Arg, followed by immunoprecipitation and SDS‐PAGE showed the synthesis of a major peak of POMC, 33 kDa. Purified [3H]POMC expressed by CV‐1 cells was cleaved in vitro by bovine intermediate lobe secretory vesicle pro‐opiomelanocortin‐converting enzyme to ACTH intermediates (19–25 kDa), β‐lipotropin and β‐endorphin. Thus, this work has demonstrated a technique for expressing microgram quantities of prohormones in mammalian cells, suitable for use as substrates for prohormone‐converting enzymes in vitro.
Inhibition of myeloid differentiation by inhibitors of ADP‐ribosylation
Authors: Wu Ming-chi , Zaun M.Rebecca , Wu Fu-Mei
Published: July 2002

Inhibitors of ADP‐ribosylation inhibited the myeloid differentiation of murine myelomonocytic leukemia WEHI‐3BD+ cells induced by granulocyte colony‐stimulating factor. Benzamide, at 2.0 mM, inhibited 50% of the WEHI‐3BD+ cell differentiation but had no significant effect on the proliferation. However, benzylaminododecylguanine hydrochloride and p‐methoxylbenzylaminodecamethylene guanidine sulfate at 2.0 and 2.2 μM, respectively, inhibited 50% of proliferation but had no effect at all on differentiation. The differential effects of inhibitors provide a model to study the role of ADP‐ribosylation in myeloid differentiation.
Evidence for a factor promoting the conversion of VWF from low and intermediate to high molecular mass polymers on the platelet membrane
Authors: Ferri A. , Guerra S. , Gemmati D. , Chiozzi P.
Published: July 2002

On the membrane surface of resting platelets exists a factor capable of promoting the conversion of plasma von Willebrand factor from low and intermediate molecular mass to high molecular mass polymers. The process is accompanied by a parallel increase in the von Willebrand factor activity. This could be a regulatory system for the molecular mass distribution of von Willebrand factor during its lifetime in plasma. Glycoprotein Ib of the platelet membrane appears not to be involved in the process.
Interaction of cytosol fractions containing activated glucocorticoid‐receptor complexes from rat liver and thymus with heterologous nuclei: effects on transcription
Authors: Tzavaras Theodore J. , Tsawdaroglou Nicolaos H. , Sekeris Constantine E.
Published: July 2002

Two rat liver cytosol fractions containing activated glucocorticoid‐receptor complexes are able to stimulate the transcriptional activity of rat liver nuclei; the respective fractions from the cytosol of thymocytes inhibit the capacity of thymus nuclei for RNA synthesis. A similar inhibitory effect on thymus nuclei is exerted by the presence of rat liver cytosol fractions. Spot hybridization using a tyrosine aminotransferase (TAT) probe demonstrates that TAT gene expression is stimulated by the liver cytosol fractions acting on homologous nuclei whereas it is inhibited, in thymus nuclei, by the addition of thymus cytosol fractions. No effect on transcription is observed if the liver or thymus cytosol is heat activated in the presence of the glucocorticoid antagonist, cortexolone. Treatment of liver nuclei, previously subjected to the action of thymus cytosol fractions with the respective liver ones, restores transcriptional activity to control or higher levels. We conclude that rat thymocyte nuclei and cytosol contain transcriptional factors, which in the presence of the glucocorticoid‐receptor complex, irrespective of its source, inhibit gene expression, whereas in the absence of such factors, the glucocorticoid‐receptor complex positively regulates the respective genes.
Histamine and serotonin inhibit induction of ornithine decarboxylase by ornithine in perifused Ehrlich ascites tumour cells
Authors: Matés J.M. , Sánchez-Jiménez F.M. , García-Caballero M. , de Castro I.Núñez
Published: July 2002

Ornithine induced more than 36‐fold the ornithine decarboxylase activity in confined Ehrlich ascites tumour cells after 3.5 h of continuous perifusion with 0.5 mM ornithine; arginine and glutamine also induced the activity 3‐ and 4‐fold, respectively. The addition of cycloheximide or actinomycin D antibiotics to the perifusion medium confirmed that the regulation of the enzyme synthesis takes place at the level of translation. Perifusion in the presence of 0.5. mM ornithine and 55, 25, and 10 μM histamine suppressed the induction by 91, 53, and 35%, respectively. Similar results were obtained in the presence of serotonin. Histidine also showed inhibitory effect but 5 mM histidine was required to produce 21% inhibition; other basic amino acids were ineffective.
Organization of the thyroid hormone receptor in the chromatin of C6 glial cells: Evidence that changes in receptor levels are not associated with changes in receptor distribution
Authors: Ortiz-Caro J. , Yusta B. , Pascual A. , Aranda A.
Published: July 2002

The association of [125I]T3‐receptor complexes with C6 cell chromatin was analysed after a limited digestion with micrococcal nuclease (MN) or DNase I. Both nucleases solubilized up to 60–70% of receptor and 0.4 M KCl extracted 70%, of the non‐digested receptor, thus showing that only a residual fraction of receptor is associated with the nuclear matrix. With DNase I the receptor was released 2–3‐fold faster than the bulk of chromatin, whereas a preferential release of receptor over total chromatin was not observed with MN. The digestion of receptor with DNase I and MN occurred 14‐ and 6‐fold faster, respectively, than the appearance of PCA‐soluble chromatin. Preincubation for 48 h with 4 nM T3 of 2 mM butyrate significantly altered receptor levels but did not change sensitivity to the nucleases. These results suggest that the thyroid hormone receptor is associated with chromatin highly sensitive to nuclease digestion, and that changes in receptor number are not associated with changes in its distribution in chromatin.
Synthesis and properties of tachyplesin I, a lipopolysaccharide‐binding peptide, from Tachypleus tridentatus
Authors: Shieh Tiee-Cherng , Kohzuma Takuji , Park Nam Gyu , Ohno Motonori , Nakamura Takanori , Iwanaga Sadaaki , Yamamoto Tatsuto
Published: July 2002

Tachyplesin I, a cationic heptadecapeptide amide containing two disulfide bonds isolated from hemocytes of Tachypleus tridentatus, which binds to lipopolysaccharide, was synthesized by a solid‐phase procedure. Reduction and oxidation of the deprotected peptide amide yielded an identical peptide to native tachyplesin I in terms of chemical and biological properties. Tachyplesin 1 was found to bind to alkylphosphorylcholine accompanied by perturbation of its tyrosyl and tryptophyl residues but bound only weakly to N‐acetyl‐D‐glucosamine tetramer.
Na+/H+ exchange is not necessary for protein kinase C‐mediated effects in platelets
Authors: Krishnamurthi Sushila , Patel Yatin , Morgan Winston A. , Wheeler-Jones Caroline P.D. , Kakkar Vijay V.
Published: July 2002

The role of Na+/H+ exchange in protein kinase C‐mediated effects in platelets was investigated by studying the effect of removal of extracellular Na+ ([Na+]e) on the different responses induced by phorbol 12‐myristate 13‐acetate (PMA) and 1,2‐ dioctanoylglycerol (diC8). None of the responses studied, namely, protein phosphorylation, translocation of enzyme activity to the membrane fraction, potentiatory and inhibitory effects on platelet activation ([Ca2+]i, arachidonate and granule release) showed an absolute dependence on [Na+]e. With the exception of dense‐granule release, which was clearly potentiated by the removal of [Na+]e and showed a negative correlation with exchanger activity, the other effects of PMA and diC8 were not affected by [Na+]e removal. It is concluded that Na+/H+ exchange is not essential for protein kinase C activation in platelets.
Early steps in the crystallization process of proteins: An approach by fluorescence anisotropy
Authors: Jullien Magali
Published: July 2002

An investigation of the early steps occurring in the crystallization process of a protein has been attempted by means of fluorescence anisotropy using ribonuclease A with ethanol as precipitating agent. It is found that the apparent fluorescence anisotropy is sensitive to protein‐protein interactions and increases linearly with protein concentration. The virial coefficient for rotational diffusion appears to increase sharply beyond 40% ethanol, the concentration which corresponds to crystallization conditions of the protein. The results reported here demonstrate that fluorescence anisotropy can be used to monitor directly the prenucleation phase of the crystallization process.
High incorporation of [3H]inositol into phosphoinositides of human platelets during reversible electropermeabilisation
Authors: Authi Kalwant S. , Hughes Kenneth , Crawford Neville
Published: July 2002

A new method for high incorporation of [3H]inositol into human platelets is described. The method involves incorporation of [3H]inositol during reversible electropermeabilisation by high voltage discharge, followed by resealing the cells during incubation at 37°C. Between 10‐ and 20‐fold increase of isotope uptake is achieved compared to control intact cells. Permeabilised resealed platelets maintain good responses to thrombin and collagen. Analysis of the incorporation of the label amongst the phosphoinositides shows 70% to be in PI, 20% in PIP, and 10% in PIP2. Stimulation with thrombin and analysis of the formation of IP1, IP2 and IP3 shows the labelling to occur in a hormone‐sensitive pool. These studies indicate that reversible electropermeabilisation can be used to achieve good uptake of non‐membrane penetrating substances such as inositol.
Translational in vitro activity of the 3a gene and the coat protein gene derived from brome mosaic virus RNA 3 by site‐specific cleavage with RNase H
Authors: Smirnyagina E.V. , Karpova O.V. , Miroshnichenko N.A. , Rodionova N.P. , Atabekov J.G.
Published: July 2002

Two translationally active fragments were derived from the dicistronic Brome Mosaic Virus (BMV) RNA 3 by site‐specific cleavage with RNase H from E.coli: the 5′‐proximal (L)fragment encoding the 32 kDa protein and the 3′‐proximal (Sh) fragment carrying the coat protein gene. The translational efficiency of the L‐ and Sh‐fragments was compared with those of the native BMV RNA 3 and RNA 4, encoding the 32 kDa and coat proteins, respectively. The Sh‐fragment template activity was similar to that of RNA 4, although it was uncapped and contained 20–22 additional 5′‐terminal nucleotides in comparison with BMV RNA 4.
Circadian rhythm and phytochrome control of LHC‐I gene transcription
Authors: Tavladoraki Paraskevi , Argyroudi-Akoyunoglou Joan
Published: July 2002

A small amount of translatable mRNA for a nuclear‐coded precursor apoprotein of the LHC‐I (light‐harvesting complex of photosystem I) with a molecular mass of 26 kDa is present in etiolated bean leaves. The expression of this protein is phytochrome‐controlled and follows circadian oscillations, for the appearance of which a red‐light pulse is sufficient. The rhythmical oscillations persist for many hours in the dark following the red‐light pulse or in continuous white light. The similarity of this rhythm in the light‐induced accumulation of LHC‐I mRNA with that of LHC‐II mRNA [(1989) Plant Physiol. 90, 665] suggests that the same oscillator may govern the expression of all chloroplast protein genes regulated by light.
Inactivation of human thrombin by water‐soluble carbodiimides
Authors: Borders C.L. , Chan Vivien W.F. , Miner Lydia A. , Weerasuriya Yohan M.
Published: July 2002

Human thrombin is inactivated by 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide (EDC) or 1‐ethyl‐3‐(4‐azonia‐4,4‐dimethylpentyl)carbodiimide (EAC) by a second order process. A plot of the pseudo‐first order rate constant for inactivation by 20 mM EDC at different pH values from 4.2 to 7.7 at 25°C shows that this inactivation is due to the modification of a protonated carboxyl with a pK
a of 5.6. The rate of inactivation by EDC at pH 6.0 is reduced, but not eliminated by saturating levels of the competitive inhibitor dansyl‐L‐arginine N‐(3‐ethyl‐1,5‐pentanediyl)amide, suggesting that the essential carboxyl modified is one other than Asp‐189 in the substrate specificity pocket of thrombin. Complete inactivation by 14C‐EAC correlates with the incorporation of approximately 2.5–3 EACs per thrombin.
Novel 1H‐31P heteronuclear coherence transfer spectroscopy based on spin locking MLEV‐17 and its application to signal assignment for a short DNA fragment
Authors: Hiroaki Hidekazu , Uesugi Seiichi
Published: July 2002

We have applied the MLEV‐17 proton spin locking pulse to 1H‐31P 2D heteronuclear correlation spectroscopy. Using this technique, long‐range correlations between a proton and phosphorus that is up to 5 bonds distant are observed. Thus, the 3′‐, 4′‐ and 5′‐proton resonances can be traced ‘sequentially’ along the chain on the 2D NMR chart. Application of this technique to the complete assignment of the sugar proton and phosphorus resonances for d(ApGpA) is reported. This technique can also be used for convenient assignment of the phosphorus resonances of oligonucleotides as correlations between the 1′‐proton and its 3′‐side phosphorus are observed.
cDNA cloning and nucleotide sequence of lipocortin‐like 33 kDa protein in guinea pig neutrophils
Authors: Sato Eisuke F. , Tanaka Yoshikazu , Utsumi Kozo
Published: July 2002

cDNA clones of guinea pig neutrophil 33 kDa protein, a lipocortin like‐protein, were isolated from two λgt10 libraries and one primer‐extended λgt10 library of guinea pig neutrophils using synthetic oligonucleotide probes or cDNA fragment probe. The cDNA consists of 1389 nucleotides, and contains 1038 nucleotides encoding 346 amino acids of 33 kDa protein and a poly(A) tail. The deduced amino acid sequence shows high homology with those of lipocortin 1 from human U937 cells (89% homology) and rat lung (86%).
Studies of in vivo phosphorylated proteins in BC3H‐1 myocytes suggest that protein kinase C is involved in insulin action
Authors: Vila María del C. , Cooper Denise R. , Davis John S. , Standaert Mary L. , Farese Robert V.
Published: July 2002

Insulin (10 and 100 nM) and phorbol esters increased the phosphorylation of several proteins, including 40, 47 and 80 kDa proteins, which are markers for protein kinase C activation. Insulin effects were evident at 2 min and increased over 20 min. These findings suggest that insulin activates protein kinase C in BC3H‐1 myocytes.
Effect of a mammary‐derived growth inhibitor on the expression of the oncogenes c‐fos, c‐myc and c‐ras
Authors: Lehmann W. , Strauss M. , Kießling U. , Graetz H. , Koberling A. , Langen P.
Published: July 2002

A mammary‐derived growth inhibitor (MDGI) inhibits the resumption of growth of stationary Ehrlich ascites carcinoma (EAC) cells in vitro. The present study shows that the resumption of growth is accompanied by a rapid increase of the steady state mRNA level of the proto‐oncogenes c‐fos, c‐myc and c‐ras, which is reduced by MDGI. EAC cells from the exponential growth phase insensitive to MDGI did not show a reduced RNA expression. The effect of MDGI represents a novel activity at the level of gene expression and suggests a link to exist between growth inhibition and the reduction of c‐fos, c‐myc and c‐ras expression.
Growth stimulation by bombesin does not involve activation of Na+/H+ exchange in chick embryo otic vesicles in vitro
Authors: Gillespie J.I. , Giraldez F. , Greenwell J.R.
Published: July 2002

The effect of the mitogen bombesin on pHi in cells of the chick otic vesicle was studied using dual wavelength microspectrofluorimetric techniques. The results show that the bombesin did not produce an immediate alkalinization in quiescent otic vesicle cells nor a long‐term change in pHi in vesicles reactivated to grow in culture. The recovery of pHi from an acid load, caused by an NH4 pulse, involved both Na+/H+ exchange and Na+:HCO3 influx mechanisms, neither of which was influenced by bombesin. These observations do not fit with a general model whereby pHi is a universal signal for DNA replication and cell proliferation.
Synthesis of human endothelin‐1 precursors in Escherichia coli
Authors: Watanabe Takuya , Itoh Yasuaki , Ogi Kazuhiro , Kimura Chiharu , Suzuki Nobuhiro , Onda Haruo
Published: July 2002

Endothelin, the most potent vasoconstrictor found in nature, is thought to be important in the regulation of blood pressure and/or local blood distribution. Human placenta cDNA fragment encoding preproendothelin‐1 (preproET‐ 1) and its carboxyl terminal mature precursor (C‐matured precursor) was expressed in E. coli. These products were characterized by both enzyme immunoassay and Western blot analysis.
Heterogeneity of ATP‐hydrolyzing sites on reconstituted CF0F1
Authors: Fromme Petra , Gräber Peter
Published: July 2002

The proton translocating ATP‐synthase from chloroplasts, CF0F1, was isolated and reconstituted into asolectin liposomes. [γ‐32P]ATP hydrolysis was measured under uni‐site conditions. When 1 mM unlabeled ATP was added so that all ATP‐binding sites were occupied, [γ‐32P]ATP bound to the first site, was hydrolyzed with a rate of 0.5 ATP/(CF0F1 s). In a second experiment, first cold ATP was hydrolyzed under uni‐site conditions and then 1 mM [γ‐32P]ATP was added. This allows under otherwise identical conditions the measurement of the rate of ATP hydrolysis catalyzed by the second (and possibly third) site. It resulted in a rate of 80 ATP/(CF0F1 s). It is concluded that the catalytic nucleotide binding sites are heterogeneous: there exists one nucleotide binding site which hydrolyzes ATP with a maximal turnover of 0.5/s and another one (or two) which hydrolyze ATP with a turnover of 80/s. The latter one is the catalytic site for maximal turnover.
New reagent for discrimination of single‐ and double‐stranded regions in DNA
Authors: Mazin A.V. , Kuzminov A.V. , Dianov G.L. , Salganik R.I.
Published: July 2002

It was found that DNA alkylation at the N‐7 guanine with the bulky alkylating reagent, N,N,N'‐tri‐(β‐chloroethyl)‐N‐(p‐formylpheny)propylene diamine‐1,3 (TFP) is much diminished when DNA is double‐stranded. We report here an application of this reaction for probing the hairpin structure in the palindrome‐containing single‐stranded (ss) DNA fragment of 377 bases prepared from the EcoRI‐BamHI fragment of plasmid pBR322. 5'‐Labeled ss fragment was modified with TFP and cleaved by piperidine hydrolysis at the alkylated guanine residues according to the Maxam‐Gilbert procedure. Guanines in the hairpin formed by palindrome of 9 bp were protected from TFP action, while dimethyl sulfate modified all guanines.
Investigations on nucleotide binding sites of isolated chloroplast ATPase by modification with 7‐chloro‐4‐nitrobenzofurazan
Authors: Bickel-Sandkötter Susanne , Strümper Petra
Published: July 2002

Addition of NBD‐Cl to isolated and nucleotide‐depleted CF1 at pH 7.5 leads to binding of 2 NBD molecules on tyrosines of CF1, one in one of the 3 α‐subunits and one in one of the 3 β‐subunits. MgADP reduces the amount of bound NBD in β, but not in a. Modification as a function of time is biphasic, showing an initial rapid and a slow kinetic component. MgATP represses the initial rapid phase of binding. Incubation of O‐Tyr‐NBD‐CF1 at pH 9 causes a time‐dependent shift of the NBD‐molecule from tyrosine to lysine. The O‐Tyr‐NBD‐formation is an obligatory intermediate for specific N‐Lys‐NBD‐formation. ADP reduces the formation of the lysine‐bound NBD drastically.
Developmental regulation of D‐β‐hydroxybutyrate dehydrogenase in rat liver and brain
Authors: Zhang Wei-Wei , Churchill Sharon , Churchill Perry
Published: July 2002

Hydroxybutyrate dehydrogenase, D‐β‐; Developmental regulation
Mössbauer study of red blood cells from patients with erythremia
Authors: Oshtrakh M.I. , Semionkin V.A.
Published: July 2002

Mössbauer spectroscopy; Erythremia; Hemoglobin; Fe2+ electronic structure; Ferritin; (Normal adult red blood cell, Erythremic red blood cell)
Extensive precursor‐product relationship between gangliosides formed from exogenous glucosylceramide in rat liver
Authors: Trinchera Marco , Vallenzasca Carlo , Ghidoni Riccardo , Tettamanti Guido
Published: July 2002

Glucosylceramide, radiolabelled on the glucose residue, was administered to rats and the radioactive gangliosides formed at different time points were chemically characterized. They were identified as GM3, GM1, GD1a and GD1b, each one carrying only radioactive glucose. The time course of each individual ganglioside showed that the simpler gangliosides were formed earlier but were consumed earlier than the more complex ones, resulting in radioactivity patterns that were different at each time point. Only 30 h after injection did it resemble that of endogenous rat liver gangliosides. These results indicate that an extensive precursor‐product relationship actually exists in the course of ganglioside biosynthesis.
Urea cycle disorder in C3H‐H‐2° mice with juvenile steatosis of viscera
Authors: Imamura Yasushi , Saheki Takeyori , Arakawa Hiroyuki , Noda Takashi , Koizumi Tsutomu , Nikaido Hiroko , Hayakawa Jun-ichiro
Published: July 2002

We determined the activities of urea cycle enzymes in the liver of C3H‐H‐2o‐jsv mice. The activities of all urea cycle enzymes decreased in the latter period of lactation. The activities of carbamylphosphate synthetase and ornithine transcarbamylase in some affected mice were undetectable. On the other hand, the activities of enzymes other than urea cycle enzymes were normal. We consider that the decrease in the urea cycle enzymes is caused by an abnormality in the mechanism of gene expression.
Amino acid sequence similarity between the terminal protein of hepatitis B virus and predicted hepatitis delta virus gene product
Authors: Khudyakov Yu.E. , Makhov A.M.
Published: July 2002

The comparative analysis of primary and secondary structures, and hydropathy plots of hepatitis B virus (HBV) and hepatitis delta virus (HDV) proteins was carried out. Two short regions belonging to the HBV terminal protein were shown to be homologous to two regions; one encoded by HDV ORF5, and the other encoded by small ORF of the HDV antigenomic RNA strand. We propose a new protein containing both these regions may be synthesized in HDV infected cells. Stiking structural homology between the terminal protein of HBV and this predicted protein called HDAg' of HDV may indicate a possible functional similarity.'We hypothesize the HDAg' may interact with and inhibit the polymerase activity of HBV.
A genetic polymorphism in a functional domain of human pregnancy zone protein: the bait region
Authors: Marynen P. , Devriendt K. , Van den Berghe H. , Cassiman J.J.
Published: July 2002

Genomic clones containing the exons coding for the bait domain of human pregnancy zone protein and α2 macroglobulin were isolated and fragments containing the bait exons were sequenced. It is shown that the bait domains of both α2 macroglobulin and pregnancy zone protein are encoded by two exons, with conserved exon/intron boundaries. A genetic polymorphism showing either a Met or a Val residue as the sixth amino acid of the pregnancy zone protein bait domain was detected with the rare Met allele showing a gene frequency of 0.065.
Molecular cloning of a human thyrotropin receptor cDNA fragment
Authors: Huang Guo Cai , Page Martin J. , Roberts Andrew J. , Malik Afshan N. , Spence Hugh , McGregor Alan M. , Banga J.Paul
Published: July 2002

Autoantibodies to the thyrotropin (TSH) hormone receptor (TSH‐R) are present in the sera of patients with thyroid autoimmune disease which are pathogenetic leading to hyperthyroidism of Graves' disease. Considerable interest has been focused on the cloning of the human TSH‐R, which has until very recently, proven exceedingly difficult due to the very low receptor level expression on thyroid cells. We have used polymerase chain reaction and highly degenerate, inosine containing oligonucleotides derived from sequence alignments of the transmembrane regions 2 and 7 of a number of G‐binding protein receptors including the lutropin/choriogonadotropin (LH/CG) receptors to amplify various cDNAs from human thyroid cDNA. Sequencing analysis of 27 different clones revealed that they fall into eight different groups. The very recent publication of the complete nucleotide sequence of the human TSH‐R revealed that one of the groups (GT1) containing seven clones which had been sequenced belong to the human TSH‐receptor. The sequence of all 7 GT1 clones was identical and in complete concordance with transmembrane regions 2 and 7 of the published TSH‐R sequence. Our results show that by designing oligonucleotides to common transmembrane regions of G‐binding proteins where the primers are biased in their sequence to the LH/CG receptors it is possible to amplify the TSH‐R receptor sequence.
The primary structure of superoxide dismutase purified from anaerobically maintained Bacteroides gingivalis
Authors: Amano Atsuo , Shizukuishi Satoshi , Tsunemitsu Akira , Maekawa Kazuhiko , Tsunasawa Susumu
Published: July 2002

The superoxide dismutase (SOD) of Bacteroides gingivalis can use either iron or manganese as a cofactor in its catalytic activity. In this study, the complete amino acid sequence of this SOD purified from anaerobically maintained B. gingivalis cells was determined. The proteins consisted of 191 amino acid residues and had a molecular mass of 21 500. The sequence of B. gingivalis SOD showed 44–51% homology with those for iron‐specific SODs (Fe‐SODs) and 40–45% homology with manganese‐specific SODs (Mn‐SODs) from several bacteria. However, this sequence homology was considerably less than that seen among the Fe‐SOD (65–74%) or Mn‐SOD family (42–60%). This indicates that B. gingivalis SOD, which accepts either iron or manganese as metal cofactor, is a structural intermediate between the Fe‐SOD and Mn‐SOD families.
Electrostatic repulsion between molecules of like charge can be misinterpreted as binding
Authors: Stemmer Paul , Klee Claude B.
Published: July 2002

Spectroscopic methods have shown that Ca2+ chelators interact with Ca2+‐binding proteins. These spectral alterations have been interpreted as evidence for the binding of chelator by the proteins. We show by direct examination of EDTA interaction with calmodulin and α‐lactalbumin that these proteins repel EDTA rather than bind it. The repulsion is reduced by increased salt concentration but is unaffected by Ca2+ binding to the proteins. The acidic protein, α‐lactalbumin, repells the negatively charged EDTA and inorganic phosphate whereas the basic protein, lysozyme, repells the positively charged spermine. Thus, spectroscopic changes induced by negatively charged Ca2+ chelators on negatively charged Ca2+‐binding proteins are due to electrostatic repulsion, and not to binding. These observations underscore the possible pitfalls of using spectroscopic methods alone to analyze protein‐ligand interactions.
The thermodynamic characteristic of four‐heme cytochrome c in Rhodopseudomonas viridis reaction centers, as derived from a quantitative analysis of the differential absorption spectra in α‐domain
Authors: Shinkarev V.P. , Drachev A.L. , Dracheva S.M.
Published: July 2002

A method of decomposing of the absorption spectrum of four‐heme cytochrome of a Rhodopseudomonas viridis reaction center preparation into spectra of individual components was used to estimate the degree of the reduction of hemes as a function of redox potential in the medium. The method enables an evaluation of the shape of redox‐titration curves of each heme. The redox‐titration curves derived by this approach are approximated well by a Nernst equation with n = 1 and Em
‐values of 360 mV, 312 mV, 20 mV and less than −50 mV. For all of the redox species the values of midpoint potential estimates by the above method are in good agreement with those determined earlier using another procedure [Dracheva et al. (1988) Eur. J. Biochem. 171, 253‐264]. The accuracy of deconvolution of data is within the experimental errors of the redox potential measurement.
Phenethyl alcohol disorders phospholipid acyl chains and promotes translocation of the mitochondrial precursor protein apocytochrome c across a lipid bilayer
Authors: Jordi Wilco , Nibbeling Ria , de Kruijff Ben
Published: July 2002

The interaction of phenethyl alcohol with model membranes and its effect on translocation of the chemically prepared mitochondrial precursor protein apocytochrome c across a lipid bilayer was studied. Phenethyl alcohol efficiently penetrates into monolayers and causes acyl chain disordering judged from deuterium nuclear magnetic resonance measurements with specific acyl chain‐deuterated phospholipids. Translocation of apocytochrome c across a phospoholipid bilayer was stimulated on addition of phenethyl alcohol indicating that the efficiency of translocation of this precursor protein is enhanced due to a disorder of the acyl chain region of the bilayer.
Polyamines improve Ca2+ transport system of the yeast mitochondria
Authors: Votyakova Tatyana V. , Bazhenova Elena N. , Zvjagilskaya Renata A.
Published: July 2002

Spermine at concentrations of 12–100 μM considerably activates the Ca2+ transport system of the Endomyces magnusii yeast mitochondria. As a result, in the presence of spermine the mitochondria are able to decrease extramitochondrial Ca2+ to the physiological level. At Ca2+ concentrations up to 200 μM, spermine enhances the initial rate of Ca2+ uptake (a half‐maximal effect at 12 μM spermine). The Ca2+ concentrations required for half‐maximal Ca2+ uptake rate to be achieved were 160 and 60 μM Ca2+ without and with spermine, respectively. Spermidine is shown to be less effective (a half‐maximal effect at 50–100 μM spermidine). The polyamines do not change the parameters of energy coupling of mitochondria. The data obtained enabled the yeast mitochondria to be considered to take part in regulation of cytoplasmic and matrix Ca2+.
Externally added DNA molecules support initiation of transcription in isolated nuclei from petunia
Authors: Zenvirth Drora , Ginzberg Dalia , Hershkovitz Maty , Loyter Abraham
Published: July 2002

Nuclei isolated from protoplasts transfected with the pUC8CaMVCAT and pDO432 plasmids were able to support, in run off experiments, the synthesis of specific transcripts as was evident from analysis by dot blot hybridization. Also the addition of the above plasmids to nuclei, prepared from non‐transfected protoplasts, supported the synthesis of specific transcripts. Dot blot analysis showed that most of the transcripts obtained were complementary to the relevant gene sequences. α‐Amanitin, at concentrations which are known to block the activity of RNA polymera II, significantly inhibited the synthesis of specific transcripts by the isolated nuclei. The transcription activity was found to be predominantly associated with the nuclear fraction while the transcription products (RNA molecules) appeared in the supernatant obtained following sedimentation of the nuclei.
Interaction of dNTP, pyrophosphate and their analogs with the dNTP‐binding sites of E. coli DNA polymerase I Klenow fragment and human DNA polymerase
Authors: Potapova I.A. , Nevinsky G.A. , Veniaminova A.G. , Khomov V.V. , Lavrik O.I.
Published: July 2002

The 3',5'‐exonuclease center of the Klenow fragment of E. coli DNA polymerase I (FK) was selectively blocked by NaF. The latter was shown to forbid the binding of nucleotides and their analogs to the enzyme exonuclease center. In the presence of poly(dT) · r(pA)10 template · primer complex and NaF, we observed AMP, ADP, ATP, PPi and dATP to be competitive inhibitors of the FK‐catalyzed DNA polymerization. The interactions of the nucleotides with FK and human DNA polymerase α were compared to reveal similarity of binding to the DNA polymerizing centers. Structural components of dNTP and PPi playing key roles in forming complexes with pro‐ and eukaryotic DNA polymerases were identified.
PCR amplification of CGRP II mRNA
Authors: Lasmoles F. , Minvielle S. , Cohen R. , Guliana J.M. , Delehaye M.C. , Segond N. , Calmettes C. , Milhaud G. , Moukhtar M.S.
Published: July 2002

Two genes code for calcitonin gene‐related peptides (CGRPs). One expresses by tissue‐specific alternate splicing calcitonin and CGRP I mRNAs, the other CGRP II mRNA. Calcitonin is the marker of sporadic or hereditary human medullary thyroid carcinoma (MTC). CGRP II expression is not well established in normal or tumoral thyroid. After amplification by polymerase chain reaction, CGRP I and II mRNAs were detected in six cases of MTC associated with other endocrine neoplasia (MEN IIa) and in two cases of isolated MTC. CGRP I was detected in all non‐C cell tumoral thyroids (6 samples), CGRP II was barely detectable in three out of six cases. CGRP II could be a specific tumoral marker of MTC.
X‐Ray analysis of 2′,3′‐lyxoanhydrothymidine, a conformationally restricted inhibitor of retroviral reverse transcriptases
Authors: Gurskaya Galyna V. , Bochkarev Alexey V. , Zdanov Alexander S. , Papchikhin Alexander V. , Purygin Piotr P. , Krayevsky Alexander A.
Published: July 2002

2′,3′‐Lyxoanhydrothymidine (LAT), a conformationally restricted inhibitor of retroviral reverse transcriptases, has been studied by X‐ray analysis. The unit cell contains two crystallographically independent molecules A and B. Their sugar moieties have an identical structure: an 04′‐endo pucker of the furanose cycle and a trans conformation about the exocyclic C4′‐C5′ bond. The conformations of A and B molecules differ with respect to the N‐glycosidic bond: χA(04 ‘Cl’ N1C2) = −121.9° which is typical of a common anti conformation whereas χB (04 ‘Cl’ N1C2) = 121.2° corresponds to a rare high‐syn conformation. All the conformation properties of LAT molecules stem from the presence of an epoxide cycle in their molecules.
Restoration of light induced photosystem II inhibition without de novo protein synthesis
Authors: Hundal Torill , Aro Eva-Mari , Carlberg Inger , Andersson Bertil
Published: July 2002

Illumination of isolated spinach thylakoid membranes under anaerobic conditions gave rise to severe inhibition of photosystem II electron transport but did not result in D1‐protein degradation. When these photoinhibited thylakoids were incubated in total darkness the photosystem II activity could be fully restored in vitro in a process that required 1–2 h for completion.
The environments ofTrp‐248 and Trp‐330 in tryptophan indole‐lyase from Escherichia coli
Authors: Phillips Robert S. , Gollnick Paul
Published: July 2002

The two tryptophan residues, Trp‐248 and Trp‐330, in tryptophan indole‐lyase (tryptophanase) from E. coli have been separately mutated to phenylalanine using site‐directed mutagenesis. Both single tryptophan mutant enzymes have full catalytic activity, but exhibit different fluorescence and near‐UV circular dichroism spectra. These results indicate that Trp‐330 is more deeply buried than is Trp‐248, and is in a more asymmetric environment. Neither residue reacts with N‐bromosuccinimide (NBS), although tryptophan indole‐lyase is inactivated by NBS. These results demonstrate that the tryptophan residues in tryptophan indole‐lyase are not catalytically essential.
The predicted molecular structure suggests that CTF/NF‐I may function as a histone acetylase
Authors: Oikarinen Jouko , Mannermaa Riitta-Maaria
Published: July 2002

The primary structure of nuclear factor‐I (CTF/NF‐I), a eukaryotic regulatory DNA‐binding protein involved in both DNA replication and gene transcription, and the secondary structure predictable from it, are compared here with those of a number of prokaryotic acetylases. Hydropathy and Chou‐Fasman analyses reveal that the polypeptide chain of CTF/NF‐I is likely to fold to higher order structures similar to those of the acetylases, and significant conservation of functionally important regions of the acetylases is observed in CTF/NF‐I. It is therefore suggested that CTF/NF‐I may function as a histone acetylase.
A one‐variable topographical descriptor for the β‐turns of peptides and proteins
Authors: Ball Jonathan B. , Andrews Peter R. , Alewood Paul F. , Hughes Richard A.
Published: July 2002

The β‐turn is a common secondary structure in biologically active peptides and globular proteins, where it is widely thought to serve as a molecular recognition site for many biological processes. Although the primary β‐turn recognition requirements are thought to be straightforward, relating mainly to the relative positions of the peptide sidechains, current classifications of β‐turns are complex and are based solely upon the very variable geometry of the peptide backbone. We demonstrate here that β‐turns can be described in terms of a single dihedral angle, which we have called β, which provides a complete description of the spatial relationship between the entry and exit peptide bonds as well as the relative orientations of the intervening sidechains for any β‐turn. This description should prove particularly useful in the development and application of novel peptide mimetic drugs, compounds for which a classification based on a peptide backbone geometry may be entirely irrelevant.
Transcription and translation of the chloroplast atpB‐gene and assembly of ATP synthase subunit β
Authors: Leu Stefan , Weinberg Dvorah , Michaels Allan
Published: July 2002

In vitro transcription and subsequent translation of the cloned Chlamydomonas chloroplast atpB gene was used to study assembly ofATP synthase. Translation in the presence of thylakoids resulted in association of the β subunit with the membrane. The in vitro synthesized polypeptide bound to the membrane copurified with CF1 on sucrose gradients. This provides more evidence for the self‐assembly of CF1.
Cloning and sequencing of a cDNA encoding human milk β‐casein
Authors: Lönnerdal Bo , Bergström Sven , Andersson Yvonne , Hjalmarsson Karin , Sundqvist Anna Karin , Hemell Olle
Published: July 2002

A cDNA of 1065 bp encoding the human milk β‐casein was cloned and sequenced using a synthetic oligodeoxyribonucleotide probe and a human mammary gland library. The nucleotide (nt) sequence contained an open reading frame sufficient to encode the entire amino‐acid (aa) sequence of a β‐casein precursor protein consisting of 210 aa and a signal peptide of 15 aa. The nt sequence shows 45–62% homology to those of bovine, ovine, rat, and mouse β‐caseins. The highly phosphorylated site, which is responsible for the calcium‐binding capacity of β‐casein, the signal peptide, and a sequence encoding for an inhibitor to the angiotensin‐converting enzyme seem highly conserved among the β‐caseins with known sequences.
TPA induces repression of EGF receptor gene expression
Authors: Gardner David P. , Shimizu Nobuyoshi
Published: July 2002

Epidermal growth factor receptor gene expression in response to the tumor promoting phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) was analyzed in the A549 human lung adenocarcinoma cell line using DNA transfection. After 8 h of treatment a definite repression (45%) of EGF receptor gene transcription could be seen. Repression was maintained for up to 24 h. Little or no induction of transcription could be seen prior to the onset of repression for potential enhancer regions both upstream and downstream of the translation start site. The cis‐element(s) responsible for repression were localized between residues −384 and −151 of the EGF receptor gene promoter region using a deletion plasmid series.
Hepatic zonation of insulin‐stimulated tyrosine phosphorylation
Authors: Bianchi Antonella , Quistorff Bjørn , Witters Lee A.
Published: July 2002

Zonal distribution of insulin stimulation of hepatic protein tyrosine phosphorylation, detected by immunoblotting with an anti‐phosphotyrosine antibody, has been studied in the in situ perfused rat liver by dual‐digitonin‐pulse perfusion. Insulin promotes the rapid and sustained tyrosine phosphorylation of two proteins (pp150 and pp69) that are present only in the perivenous hepatocytes, while three others (pp46, pp48 and pp96) are stimulated identically in the periportal and perivenous cells. The ability of insulin to rapidly activate acetyl‐CoA carboxylase is indistinguishable between the hepatic zones. Hepatic zonation of insulin‐stimulated tyrosine phosphorylation could underly differential hepatic insulin responses and might provide clues to the identification of tyrosine phosphorylated proteins linked to insulin regulation of intracellular events.
Novel features of retroviruses associated with human diseases
Authors: Chandra Prakash , Gerber Thomas , Chandra Angelika
Published: July 2002

The last decade has witnessed a new chapter in the history of retrovirology. As of now, four human retroviruses have been identified and molecularly characterized. They are associated with a wide spectrum of human diseases including cancer, immunodeficiency and neurological disorders. By virtue of their clinical relevance, their novel genes and regulatory mechanisms these viruses have become the focal point of research in retrovirology. The study of these viruses is of fundamental importance in understanding the mechanisms leading to transformation of human cells and distortion of the immunological state.
Interaction of small G proteins with photoexcited rhodopsin
Authors: Wieland Thomas , Ulibarri Isabel , Aktories Klaus , Gierschik Peter , Jakobs Karl H.
Published: July 2002

Bovine rod outer segment (ROS) membranes contain in addition to the heterotrimeric G protein transducin, several small GTP‐binding proteins (23–27 kDa). Furthermore, these membranes contain two substrate proteins (about 22 and 24 kDa) for botulinum C3 ADP‐ribosyltransferase known to ADP‐ribosylate small G proteins in any mammalian cell type studied so far. Most interestingly, [32P]ADP‐ribosylation of ROS membrane small G proteins by C3 is regulated by light and guanine nucleotides in a manner similar to pertussis toxin‐catalyzed [32P]ADP‐ribosylation of the α‐subunit of transducin. These findings suggest that not only the heterotrimeric G protein transducin but also the C3 substrate small G proteins present in ROS membranes interact with photoexcited rhodopsin and thus contribute to its signalling action.
A novel NADPH/NADH‐dependent aldehyde reduction enzyme isolated from the tapeworm Moniezia expansa
Authors: Brophy Peter M. , Crowley Paul , Barrett John
Published: July 2002

An aldehyde reduction enzyme has been purified from the cytosol of the tapeworm, Moniezia expansa, by chromatofocusing and Reactive‐Red chromatography. The enzyme is monomeric (subunit 34 kDa) and can utilise NADH and NADPH as co‐factors. Substrates of the enzyme include alkanals, alka‐2,4‐dienals and alk‐2‐enals, established secondary products of lipid peroxidation. The enzyme reduced methylglyoxal, another possible natural substrate (M. expansa lacks glyoxalase I activity). The parasite enzyme may help form a final line of defence against cytotoxic aldehydes arising from host immune initiated lipid peroxidation.