bluuebunny/crossref_metadata_2025 huggingface dataset:
Crystallization of the cpn6O/cpn10 complex (‘holo‐chaperonin’) from Thermus thermophilus
Authors: Lissin N.M. , Sedelnikova S.E. , Ryazantsev S.N.
Published: July 2002

A stable complex of the chaperonins, cpn60 and cpn10 (Escherichia coli GroEL and GroES homologues), from the extremely thermophilic bacterium Thermus thermophilis has been isolated and crystallized. The crystals have dimensions up to 30 × 200 × 200 μm. Ultra‐thin sections of the crystals estimated by electron microscopy showed a rectangular lattice with unit cell parameters of a=17 nm, b=27 nm, γ=90°.
Receptor mediated mineralocorticoid action in alga cell mutants
Authors: Mirshahi M. , Mirshahi A. , Nato A. , Agarwal M.K.
Published: July 2002

The multiplication of Chlamydomonas cells can be arrested by the spirolactone derivative RU 26752 and this is fully reversible by the natural hormone aldosterone. Continuous growth in the presence of RU 26752 led to the isolation of a population subsequently resistant to the action of mineralocortoid analogues, due possibly to the selection of mutant cells. Immunophotochemical evidence is provided for a 52 kDa protein that possesses functional steroid and DNA binding domains. Alga cells therefore appear to respond to steroid hormones in a manner similar to the mammalian systems, possibly via a receptor that may represent a pygmy ancestor of the latter day steroid receptor superfamily.
Altered response to growth factors in rat epithelial liver cells overexpressing human c‐Fos protein
Authors: Lagarrigue S. , Seillan-Heberden C. , Martel P. , Gaillard-Sanchez I.
Published: July 2002

Human c‐fos cDNA was transfected into normal rat liver epithelial (REL) cells to identify cellular modifications associated with high expression of c‐Fos protein. Responses to EGF and TGFβ were examined in the different cell lines, under anchorage‐dependent and ‐independent conditions. Sensitivity to both factors was modified in transfected cells. While parental cells in monolayer did not respond to EGF, c‐fos containing cells growth was stimulated by this factor. Overexpression of c‐Fos protein led to an enhanced TGFβ‐induced growth inhibition under anchorage dependent conditions, and TGFβ abolished spontaneous growth in soft agar of the cell lines containing c‐fos oncogene. The mechanisms underlying, the increased sensitivity to TGFβ in c‐fos transfected cells are still to be determined.
The homodimeric hemoglobin from Scapharca can be locked into new cooperative structures upon reaction of Cys92, located at the subunit interface, with organomercurials
Authors: Colotti Gianni , Boffi Alberto , Verzili Daniela , Coletta Massimiliano , Chiancone Emilia
Published: July 2002

In the cooperative, homodimeric hemoglobin from Scapharca inaequivalvis, HbI, the subunit interface is formed by the heme‐carrying E and F helices and contains the only cysteine residue of the globin chain (Cys92, F2) in an area which changes from hydrophilic to hydrophobic upon oxygenation. Binding of organomercurials to Hbl is cooperative and entails major quaternary rearrangements. The reaction of CYs92 with p‐chloromercuri‐benzoate (PMB) and p‐nitro‐o‐chloromercuriphenol (PN), a sensitive reporter of the cysteine microenvironment at neutral pH values, has been followed in stopped flow experiments. Kinetic evidence for the cooperativity of mercurial binding has been obtained and the rate of the corresponding conformational transition has been estimated. As expected PN, but not PMB, is able to monitor the oxygen‐linked change of the cysteine microenvironment. The modification of Cys92 with PN has unique functional effects. In PN‐reacted Hbl cooperativity is maintained. albeit to a different extent, depending on the ligation state of the protein during mercaptide formation. It may be envisaged that PN locks the protein into new, cooperative, quaternary structures stabilized by hydrogen bonding interactions between the ionized nitrophenol moiety and the contralateral subunit.
The steady state behaviour of cytochrome c oxidase in proteoliposomes
Authors: Nicholls Peter
Published: July 2002

Electron transfer to oxygen catalysed by cytochrome c oxidase is accompanied by spectral changes at the binuclear a
3CuB centre, both in the soluble enzyme and in membranous systems, indicating spin or ligand state transitions of an iron that remains ferric. The other haem group, cytochrome a, does not change its spectral characteristics significantly during the steady state, but remains partially reduced until anaerobiosis. Cytochrome a
3 is fully oxidized in each of its major steady state forms, and reduced upon anaerobiosis to a single ferrous species. Although cytochrome a is normally the immediate electron donor to the binuclear centre, its redox state does not alter under conditions in which the flux through the enzyme is changing significantly. A second electron transfer pathway to the binuclear centre may therefore exist, possibly one in which direct reduction of the binuclear a
3, CuB centre by CuA occurs. Both cytochrome a and CuA behave as simple electron transfer centres. The energy‐conserving chemistry takes place at the binuclear centre in concert with the four‐electron reduction of molecular oxygen.
Physical map of the extremely thermophilic bacterium Thermus thermophilus HB27 chromosome
Authors: Tabata Kazuyuki , Kosuge Takehide , Nakahara Tadaatsu , Hoshino Takayuki
Published: July 2002

The physical map of the chromosome of Thermus thermophilus HB27 was constructed using three restriction enzymes; EcoRI, SspI and MunI by applying pulsed‐field gel electrophoresis techniques. Although the genome size of 1.82 Mb was almost the same as that (1.74 Mb) reported for T. thermophilus HB8 [Borges, K.M., and P.L. Bergquist. (1993) J. Bacteriol. 175, 103‐110], the MunI cleavage maps were different. A 240 kb plasmid was detected in HB27, and its physical map was also constructed. In addition, several genes were located on the chromosomal physical map.
Different target‐site specificities of the hairpin ribozyme in cis and trans cleavages
Authors: Fujitani Kazuko , Sasaki-Tozawa Noriko , Kikuchi Yo
Published: July 2002

The hairpin ribozyme cleaves a phosphodiester bond at the 5' side of a 5'GUC3' sequence of an RNA with high efficiency. An RNA having a 5'GUA3' sequence instead of the GUC sequence is a poor substrate for this ribozyme. Here, we show that this is indeed so in a trans‐acting ribozyme system, but in a cis‐acting ribozyme system this ribozyme cleaves the 5' side of a GUA sequence as efficiently as the wild‐type cleaves the GUC sequence. One base substitution in the ribozyme also affected the target‐site specificity in the cis‐acting system.
Electrogenic steps during electron transfer via the cytochrome bc1 complex of Rhodobacter sphaeroides chromatophores
Authors: Semenov Alexey Yu.
Published: July 2002

The results of the flash‐induced electrometrical investigation on the functioning of the photosynthetic bacterial cytochrome bc
1 complex are discussed. The data suggest possible arrangement of redox centers in the bc
1 complex and propose that the total electrogenesis within the bc
1 complex includes: (i) electron transfer between the low‐ and the high‐potential cytochrome b hemes, (ii) proton binding by doubly reduced Q2− at ubiquinone‐reducing center ‘C’, and (iii) proton release on oxidation of QH2 at ubiquinol‐oxidizing center ‘Z’.
Acidic pH increases the oxidation of LDL by macrophages
Authors: Morgan Janice , Leake David S.
Published: July 2002

We have investigated the effect of pH on LDL oxidation by macrophages (in the presence of iron ions), using a modification of Hanks' balanced salt solution. Increasing the acidity of the medium greatly increased the oxidation of the LDL by the macrophages as measured by thiobarbituric acid‐reactive substances or increased uptake and degradation by a second set of macrophages. The rate of oxidation of LDL by iron ions alone, measured in terms of conjugated dienes, was also increased greatly even at mildly acidic pH. It is quite possible that atherosclerotic lesions have an acidic extracellular pH, particularly in the vicinity of macrophages, and the observation that LDL oxidation by macrophages is increased at acidic pH may therefore help to explain why atherosclerotic lesions are apparently one of the very few sites in the body where LDL oxidation occurs.
Detection of Duchenne muscular dystrophy gene products in amniotic fluid and chorionic villus sampling cells
Authors: Prigojin Hagit , Brusel Marina , Fuchs Ora , Shomrat Ruth , Legum Cyril , Nudel Uri , Yaffe David
Published: July 2002

We have examined the expression of several Duchenne muscular dystrophy (DMD) gene products in amniotic fluid (AF) and chorionic villus sampling (CVS) cells. Variable amounts ofdystrophin could be detected in most CVS and AF samples by immunoprecipitation followed by Western blot analysis. PCR analysis demonstrated the presence of the muscle type dystrophin mRNA in all AF cell cultures. The brain type dystrophin mRNA was also detected in some of these cultures. These DMD gene transcripts are of fetal origin and are produced by most or all clonable AF cells. The results may facilitate the development of a method for prenatal diagnosis of DMD, based on the expression of the gene in AF and CVS cells.
The unusual rps3‐like orf712 is functionally essential and structurally conserved in Chlamydomonas
Authors: Liu Xiang-Qin , Huang Changzhi , Xu Hui
Published: July 2002

The Chlamydomonas reinhardtii chloroplast orf712 is a previously described open reading frame that lacks a detectable transcript but potentially encodes a polypeptide with sequence similarities to ribosomal protein Rps3 only at its N‐ and C‐tennini. Here we report that orf712 is an essential gene, as demonstrated through gene disruption by particle gun‐mediated chloroplast transformation. We also show that an orf712 is present and structurally conserved in all of the two or three major Chlamydomonas lineages. Our results suggest that orf712 is an unusual rps3 gene that contains a large translated intervening sequence.
Analysis of lactase processing in rabbit
Authors: Rossi Mauro , Maiuri Luigi , Salvati Virginia M. , Russomanno Concetta , Auricchio Salvatore
Published: July 2002

The proteolytic processing of rabbit intestinal lactase‐phlorizin‐hydrolase (LPH) was studied by pulse‐chase and continuous labeling experiments in organ culture from 15‐day‐old rabbits in the presence of glycosylation and processing inhibitors. Monensin and brefeldin A inhibited the two proteolytic cleavages of the precursor indicating that they are post‐Golgi events as previously reported for the unique cleavage of LPH in man [1]. The inhibition was not related to a concomitant alteration glycosylation; in fact, if trimming was blocked by MDNM the abnormal glycosylated precursor was proteolytically processed normally. Finally the use of the anti‐microtubular drug colchicine strongly inhibited both cleavages and caused accumulation of the complex‐glycosylated precursor form in the brush border fraction indicating that proteolytic events depend on intact microtubule (transport).
Percoll purification of chromaffin granules inhibits their ability to take up and maintain calcium
Authors: Jones Peter G. , Meneses Joel , Waisman David M.
Published: July 2002

Secretory granules of the adrenal medulla have recently been shown to be able to sequester and release Ca2+, in addition to their previously established role as carriers of secretory products. In order to study the ability of these or any other secretory granules to participate in intracellular calcium homeostasis, it is imperative that they should be free of other contaminating Ca2+ sequestering organelles, and that the Ca2+ uptake and release mechanisms of those granules should remain intact throughout any chosen purification procedure. We report here that chromaffin granules which were purified by the isopycnic gradient medium Percoll, or even incubated with it, showed an attenuated ability to sequester Ca2+.
The existence of pre‐mature 16S rRNA species in plastid ribosomes
Authors: Vera Antonio , Yokoi Fumiaki , Sugiura Masahiro
Published: July 2002

Plastid 70S ribosomes were prepared from heterotrophic cultured cells of tobacco (Nicotiana tabacum, BY2), and the 5' termini of the 16S rRNA molecules present in the ribosomes were analyzed. RNase protection and primer extension experiments showed that a minor fraction of the 16S rRNA species carries a leader sequence of 30 nucleotides, coinciding with a putative RNase III cleavage site. The results suggest that an RNase Ill‐like activity is present in plastids and that ultimate 5' maturation of 16S rRNA takes place within the ribosome.
Synthesis of 2‐deoxy‐d‐galactose containing gangliosides in vivo
Authors: Hwang Yi-han , Kohla Guido , Geilen Christoph C. , Paul Christine , Kannicht Christoph , Grunow Detlef , Pohlentz Gottfried , Egge Heinz , Nuck Rolf , Reutter Werner
Published: July 2002

Incorporation of 2‐deoxy‐d‐galactose into the oligosaccharide moieties of different gangliosides of rat liver was examined. After intraperitoneal administration of 2‐deoxy‐d‐galactose it was shown by GLC/MS analysis that this hexose analogue is metabolized and incorporated into all the gangliosides investigated, and predominantly into GM3 and GD3. In both of these gangliosides, 25–55% of the galactose residues were substituted by 2‐deoxy‐d‐galactose. The epimer, 2‐deoxy‐o‐glucose, was not detectable.
Cytochrome bo from E. coli does not exhibit the same proton transfer characteristics as the bovine cytochrome c oxidase during oxygen reduction
Authors: Hallén Stefan , Svensson Margareta , Nilsson Thomas
Published: July 2002

The reaction where fully reduced cytochrome bo from E. coli partially reduces dioxygen has been characterized with respect to the kinetics of the associated proton uptake, and with respect to the pH‐ and D2O‐sensitivity of the electron transfer reactions. A monophasic proton uptake with a rate constant of about 8 × 103 s−1 and a stoichiometry of 0.8 H+/bo were recorded, using the indicator dye, Cresol red, at pH 8.2. The electron transfer reactions were independent of pH in the range 6.0–9.5 and were not affected by exchanging H2O toD2O as solvent. Comparison of these results with those obtained in an earlier investigation of the bovine cytochrome c oxidase [(1992) Biochemistry 31, 11853‐11859], indicates differences between the two oxidases with respect to the role of protons in oxygen reduction and/or the mechanism of proton uptake from the medium.
Transfer of foreign DNA into the cells of developing mouse embryos by microprojectile bombardment
Authors: Zelenin Alexander V. , Alimov Andrey A. , Zelenina Inessa A. , Semenova Maria L. , Rodova Marina A. , Chernov Boris K. , Kolesnikov Viktor A.
Published: July 2002

Mouse cells of developing embryos at the 2–4 cell, morula and blastocyst stages, were bombarded by high velocity tungsten microprojectiles. About 70% of developing embryos survived the bombardment. The general embryo structure did not change as a result of the bombardment. Penetration of the tungsten microparticles into the embryo cell nuclei was found at all stages being investigated, and tungsten particle localization on mitotic chromosomes was demonstrated. The total DNA of the mice born from the bombarded embryos was analyzed by dot‐blot hybridization and PCR with post‐hybridization. The most important results were obtained in experiments with blastocysts. In three cases of blastocyst bombardment, the presence of transferred plasmid DNA (pSV3‐neo) was revealed. Transfected cells were shown to be located in the fetal membrane as well as in the embryo. The bombardment of mouse culture cells resulted in their transfection and the production of G418‐resistant clones.
Substrate induced changes of the active site electronic states in reduced cytochrome P450cam and the photolysis product of its CO complex
Authors: Greschner S. , Sharonov Yu.A. , Jung C.
Published: July 2002

MCD spectra of camphor‐free and camphor‐bound reduced cytochrome P450cam have been recorded for the near UV and visible spectral regions at temperatures from 300K down to 2.1K and compared with those of the carbon monoxide photoproducts generated at 4.2K. In the absence of camphor, the reduced P450 is spectroscopically different from the photoproduct. In the presence of camphor, however, the spectra of the reduced P450 and of the photoproduct are almost similar and behave like the photoproduct of the camphor‐free enzyme. This behavior indicates that substrate binding induces a higher active site rigidity. From the significant alteration of the temperature dependence of the MCD intensity for the reduced enzyme induced by camphor binding it is concluded that the near degeneracy of the electronic ground state in the substrate‐free enzyme is removed by substrate binding.
Electrical stimulation partly reverses the muscle insulin resistance caused by tenotomy
Authors: Langfort J. , Czarnowski D. , Budohoski L. , Górski J. , Kaciuba-Uściłko H. , Nazar K.
Published: July 2002

It was shown that 15‐min electrical stimulation of the rat sciatic nerve greatly increases the in vitro measured sensitivity of lactate formation, glucose transport, and glycogen synthesis to insulin, impaired by previous tenotomy. The insulin sensitivity of all these processes was, however, still below that found in the stimulated intact soleus muscle. Extending the stimulation up to 30 min did not cause any further changes in insulin sensitivity either in tenotomized or in intact muscles.
A peptide corresponding to the N‐terminal 13 residues of T4 lysozyme forms an α‐helix
Authors: McLeish Michael J. , Nielsen Katherine J. , Wade John D. , Craik David J.
Published: July 2002

Solid‐phase methods have been used to synthesize LYS(1–13), a peptide corresponding to the first 13 residues of T4 lysozyme. 2D 1H NMR techniques were used to investigate its solution structure in the presence of SDS micelles. The identification of numerous medium‐range NOESY crosspeaks and several slowly exchanging NH protons indicated the presence of an α‐helical structure. This was confirmed by simulated annealing calculations performed using XPLOR.
Detection of minor adducts in cisplatin‐modified DNA by transcription footprinting
Authors: Decoville Martine , Schwartz Annie , Locker Daniel , Leng Marc
Published: July 2002

Two DNA restriction fragments containing either a d(GC)5 or a d(TTGCTTGATTAGTTGTGTT) insert were subjected to reaction with cis‐diamminedichloroplatinum(II) and were then used as templates for RNA synthesis by T7 RNA polymerase. Within the d(GC)5 insert, interstrand cross‐links are preferentially formed. Within the second insert, the reactivity order of the potential binding sites is d(ApG) > d(GpC/ GpC) = d(GpA) > d(GpTpG). In the presence of cyanide ions, the adducts are much less stable at the d(GpA) sites than at the d(GpCpG) sites, in double‐stranded DNA.
Negative growth control by a novel low Mr phosphotyrosine protein phosphatase in normal and transformed cells
Authors: Ruggiero Marco , Pazzagli Claudia , Rigacci Stefania , Magnelli Lucia , Raugei Giovanni , Berti Andrea , Chiarugi Vincenzo P. , Pierce Jacalyn H. , Camici Guido , Ramponi Giampietro
Published: July 2002

Having determined the complete amino acid sequence of a cytosolic phosphatase purified from bovine liver, we studied the role of this enzyme (referred to as ‘PTPase’) in the control of cell proliferation. We used NIH/3T3 fibroblasts, both normal and transformed by the oncogenes v‐erbB, v‐src, and v‐raf: a synthetic gene coding for PTPase was transfected into, and overexpressed in, normal and transformed NIH/3T3 cells with resulting inhibition of cell growth. Inhibition of proliferation correlated with the level of foreign PTPase; growth in soft agar was also inhibited in transformants overexpressing the enzyme. However, PTPase overexpression did not inhibit the rapid turnover of inositol lipids stimulated by platelet‐derived growth factor. We conclude that this novel PTPase is active on cell type‐specific signalling substrates that control normal and transformed fibroblast proliferation.
pp60ν‐src kinase overexpression leads to cellular resistance to the antiproliferative effects of tumor necrosis factor
Authors: Aggarwal Bharat B. , Totpal Klara , Ali-Osman Francis , Budde Raymond J.A. , Pocsik Eva
Published: July 2002

While some tumor cells are sensitive to the antiproliferative effects of tumor necrosis factor (TNF), others are resistant. The molecular basis for cellular resistance to TNF is not completely understood. Previously we have shown that transfection of cells with an oncogene HER2/neu/erb B2, a receptor tyrosine kinase, leads to resistance to the anticellular effects of TNF [(1988) Proc. Natl. Acad. Sci. USA 85, 5102‐5106]. In the present study, we demonstrate that the overexpression of another oncogenic tyrosine kinase, pp60
v‐src
also induces resistance to TNF. In contrast to HER2, however, pp60
v‐src
transfection of cells did not lead to down‐modulation of TNF receptors but rather to decreased intracellular glutathione levels. The pp60
v‐src
‐induced cellular resistance to TNF could be abrogated by interferon‐γ. Thus, these results indicate that the resistance of certain tumors to TNF may also be due in part to the overexpression of pp60
v‐src
oncogene.
Energetic aspects of intramolecular coupling between the nucleotide binding site and the distal switch II region of the yeast RAS2 protein
Authors: De Vendittis Emmanuele , Fasano Ottavio
Published: July 2002

We have studied the interaction of the yeast RAS2 protein with guanine nucleotides using energetic parameters for the dissociation of RAS·nucleotide complexes. The results indicated that a Gly → Ser substitution at position 82 led to an altered interaction with GppNHp and, to a lesser extent, also with GDP. It was also possible to conclude that structural perturbation of Gly82 can stimulate nucleotide release by decreasing the energetic barrier for nucleotide dissociation. This, together with the observation that residues 80 and 81 are involved in the response of RAS to nucleotide exchange factors without affecting GDP binding per se, suggests a potential mechanism for exchange factor‐stimulated GDP release.
Motility and chemotaxis in Bacillus sphaericus dependence upon stage of growth
Authors: Andreev J. , Dibrov P.A. , Braun S.
Published: July 2002

Chemotaxis and motility of Bacillus sphaericus 2362 were monitored as a function of the batch culture age. It was found that both functions changed independently during growth of the culture. Motility was low until the late logarithmic stage ensued, whereafter it increased sharply. The ability of cells to respond to chemo‐effectors peaked at the mid‐logarithmic phase. A major methyl‐accepting chemotaxis protein (P53, M
r = 53 kDa) was identified. The extent of label incorporation in this protein from l‐[methyl‐3H]methionine was maximal in mid‐ and late‐logarithmic phases of the growth. Cells in stationary cultures incorporated very low amounts of the label. At any stage, the labeling was maximal in starved cells; it was almost abolished in cells pre‐incubated with amino acids. Although extents of P53 labeling in mid‐ and late logarithmic cells were similar, late logarithmic cells demonstrated a considerably impaired chemotaxis. Supermotile sporulating cells were practically insensitive to environmental stimuli. The difference in development of sensory and locomotive functions may be interpreted as an adaptive response. A well developed sensory apparatus would allow vegetative cells to adapt efficiently to fluctuating attractant gradients. Insensitive sporulating cells would tend to disperse randomly from the nutrient‐exhausted area. Thus, spore formation would occur in larger volume of the habitat, increasing the chance of microbial population to survive.
An increase of the energy coupling capacity of submitochondrial particles by lanthanides
Authors: Grivennikova V.G. , Gavrikova E.V. , Vinogradov A.D.
Published: July 2002

NADH and succinate oxidase activities of inside‐out submitochondrial particles treated with excess oligomycin are inhibited by lanthanides (La3+ and Dy3+). Both inhibition by oligomycin and oligomycin plus lanthanides are completely relieved by an uncoupler. The respiratory control measured as the stimulation of NADH or succinate oxidation caused by the addition of uncoupler to the oligomycin‐treated particles is thus increased in the presence of lanthanides. The coupling effect of lanthanides is completely prevented and rapidly reversed by excess EDTA. La3+ increases the extent of the aerobic energy‐linked succinate‐supported NAD+ reduction catalyzed by the oligomycin‐treated submitochondrial particles. Lanthanides seem to be useful tools to increase the energy coupling capacity of the submitochondrial particles.
Wortmannin and 1‐butanol block activation of a novel family of protein kinases in neutrophils
Authors: Ding Jiabing , Badwey John A.
Published: July 2002

Neutrophils contain four uncharacterized protein kinases with molecular masses of ca. 69, 63, 49 and 40 kDa that are rapidly activated upon stimulation of these cells with the chemoattractant fMet‐Leu‐Phe [Ding, J. and Badwey, J.A. (1993) J. Biol. Chem. 268, 17326–17333]. We now report that wortmannin and 1‐butanol block activation of all four of these kinases. These reagents are known to inhibit superoxide generation in neutrophils stimulated with this agonist. Wortmannin inhibits phosphatidylinositol 3‐kinase and blocks activation of phospholipase D, whereas 1‐butanol can reduce the generation of phosphatidate in cells by serving as a substrate for phospholipase D. These data suggest that phosphatidylinositol 3‐kinase and phospholipase D may be involved in the activation of several novel protein kinases in neutrophils and that one or more of these kinases is/are involved in superoxide release.
Dependence of thymocyte apoptosis on protein kinase C and phospholipase A2
Authors: Shaposhnikova Vera V. , Dobrovinskaya Oksana R. , Eidus Lazar Kh. , Korystov Yuri N.
Published: July 2002

The effect of inhibitors and activators of protein kinase C and phospholipase A2 on radiation‐induced apoptosis of rat and mouse thymocytes has been studied. It is shown that the apoptosis is prevented by the protein kinase C inhibitor 1‐(5‐isoquinolinylsulfonyl)‐2‐methylpiperasine dihydrochloride and is potentiated by protein kinase C activator phorbol 12‐myristate 13‐acetate, calcium ionophore A23187 and concanavalin A. The protein kinase C activators initiate apoptosis in mouse but not in rat thymocytes. The inhibitor of phospholipase A2 prevents apoptosis induced by all the factors. The results obtained indicate that both protein kinase C and phospholipase A2 are involved in the thymocyte apoptosis.
An increase in the energy coupling capacity of submitochondrial particles in the presence of lanthanides
Authors: Grivennikova V.G. , Gavrikova E.V. , Vinogradov A.D.
Published: July 2002

NADH and succinate oxidase activities of inside‐out submitochondrial particles treated with excess oligomycin are inhibited by lanthanides (La3+ and Dy3+). Inhibition by both oligomycin and oligomycin plus lanthanides is completely relieved by an uncoupler. The respiratory control, measured as the stimulation of NADH or succinate oxidation caused by the addition of uncoupler to the oligomycin‐treated particles, is thus increased in the presence of lanthanides. The coupling effect of lanthanides is completely prevented and rapidly reversed by excess of EDTA. La3+ increases the extent of the aerobic energy‐linked succinate‐supported NAD+ reduction catalyzed by the oligomycin‐treated submitochondrial particles. Lanthanides seem to be a useful tool to increase the energy coupling capacity of the submitochondrial particles.
Chemotaxis, sporulation, and larvicide production in Bacillus sphaericus 2362 the influence of l‐ethionine, and of aminophenylboronic acid
Authors: Andreev J. , Dibrov P.A. , Klein D. , Braun S.
Published: July 2002

4‐Aminophenylboronic acid (APBA), a known inhibitor of sporulation in Bacilli, as well as l‐ethionine, a known inhibitor of chemotaxis in Enterobacteria, inhibited both sporulation and chemotactic behavior but not growth of Bacillus sphaericus. Both compounds also inhibited the methyl group turnover on the methyl‐accepting chemotaxis protein (P53) in this microorganism. Sporulation of B. sphaericus was inhibited only when APBA was added to the growing culture before the late logarithmic stage. It was previously demonstrated that the ability of B. sphaericus to respond to chemoattractants sharply declines at the same age of the culture. Thus, it seems plausible that the action of both inhibitors upon sporulation may be attributed to the inhibition of some regulatory pathway common to chemotaxis and sporulation and involving protein methylation. Possible exchange of the nutrient depletion‐related sensory information between chemotaxis and sporulation systems at the level of methyl group transfer is discussed.
Comparison of the in vitro translated polypeptides from maize shoot, pollen and germinated pollen mRNAs
Authors: Hussey Patrick J. , Wakeley Philip R.
Published: July 2002

We have compared the two‐dimensional gel (2D gel) profiles of in vitro synthesised polypeptides from maize shoot with that of pollen, and the latter with that of germinated pollen. In the former comparison, extensive quantitative and qualitative differences in the polypeptides are apparent. We describe an increase in the abundance of the major cytoskeletal protein, α tubulin, in pollen compared to shoot. We also show that the electrophoretically separable α tubulin polypeptides in ungerminated pollen are not obviously post‐translationally modified. In the comparison between the 2D gel profiles of the in vitro synthesised polypeptides from pollen and germinated pollen a number of notable differences are apparent, the possible origins of which are described.
Expression of a novel human myotonin protein kinase (MtPK) cDNA clone which encodes a protein with a thymopoietin‐like domain in COS cells
Authors: Sasagawa Noboru , Sorimachi Hiroyuki , Maruyama Kei , Arahata Kiichi , Ishiura Shoichi , Suzuki Koichi
Published: July 2002

A full‐length cDNA of human myotonin protein kinase (MtPK) was cloned and expressed in COS‐1 cells. MtPK is recovered from the cytosolic fraction of the COS extract as a 70 kDa protein, which coincides with the size deduced from the predicted amino acid sequence. The sequence has a significant homology to thymopoietin, a peptide hormone of the thymus. Biochemical characteristics of MtPK expressed in COS‐1 cells and its expression in rat tissues are investigated.
Formation of charge separated state P+Q−A and triplet state 3P at low temperature in Rhodobacter sphaeroides (R‐26) reaction centers in which bacteriopheophytin a is replaced by plant pheophytin a
Authors: Shkuropatov A.Ya. , Proskuryakov I.I. , Shkuropatova V.A. , Zvereva M.G. , Shuvalov V.A.
Published: July 2002

Low temperature optical and photochemical properties of Rhodobacter sphaeroides (R‐26) reaction centers, in which bacteriopheophytin a has been replaced by plant pheophytin a, are reported. Modified reaction centers preserve the ability for photoinduced electron transfer from the primary electron donor P to the primary quinone acceptor QA at 80K. The triplet state ESR signal of modified reaction centers with prereduced QA at 10K shows an electron spin polarization pattern and ZFS parameters analogous to those for the triplet state 3P in non‐treated reaction centers. It was found that at low temperature both P+Q−
A and 3P states are formed via a precursor radical pair P+I− in which I is the introduced plant pheophytin molecule. This shows that acceptor systems of bacterial and plant (photosystem II) reaction centers are mutually replacable in structural and functional aspects.
Supercooperativity in platelet aggregation: Substituted pyridyl isoxazoles, a new class of supercooperative platelet aggregation inhibitors
Authors: Vrzheshch Peter V. , Demina Olga V. , Shram Stanislav I. , Varfolomeev Sergey D.
Published: July 2002

The phenomenon of supercooperativity in platelet aggregation is manifested by the occurence of clear‐cut thresholds in dose—response relationships; in such cases the Hill coefficient has unusually high values. Approximation, by the Hill equation, of the relationship of the rate of arachidonate‐induced platelet aggregation to the concentrations of either the inducer or inhibitors such as substituted pyridyl isoxazoles (synthesized by us), indomethacin, and pinane thromboxane A2, demonstrated that the Hill coefficients ranged from 30 to 100. 3‐(3‐Pyridyl)‐5‐phenylisoxazole, which exhibited maximal anti‐aggregatory activity among the synthesized compounds, inhibited neither cyclooxygenase nor thromboxane synthase. The compounds affected the signal transduction pathway at/or posterior to the stage of thromboxane A2 reception.
Neopterin derivatives together with cyclic guanosine monophosphate induce c‐fos gene expression
Authors: Überall Florian , Werner-Felmayer Gabriele , Schubert Christian , Grunicke Hans H. , Wachter Helmut , Fuchs Dietmar
Published: July 2002

We have previously shown that neopterin enhances hydrogen peroxide and chloramine T activity in a luminol‐dependent chemiluminescence assay and strengthens toxicity of these agents against bacteria at slightly alkaline pH (pH 7.5), while 7,8‐dihydroneopterin was shown to be a scavenger independent of the pH value. Besides various oxidants, phenolic antioxidants were shown to specifically induce expression of the c‐fos and c‐jun mRNAs. Using an inducible cfosCAT reporter transactivation system we studied the function of the pteridine derivatives on c‐fos transactivation. For the first time, we demonstrate that neopterin and 7,8‐dihydroneopterin, particularly together with cyclic guanosine monophosphate, induce c‐fos gene expression. In humans, interferon‐γ induces the release of neopterin and 7,8‐dihydroneopterin and also the synthesis of nitric oxide radical which in turn stimulate the formation of cGMP. Thus, in certain situations all three substances, namely neopterin, 7,8‐dihydroneopterin and cGMP, may be present locally and even in the circulation at the same time. Based on our findings this constellation would significantly enhance the risk of c‐fos gene expression and therefore promote tumour growth and development.
The crystal structure of the iron‐free cytochrome c peroxidase and its implication for the enzymatic mechanism
Authors: Su Xiao-Dong , Yonetani Takashi , Skoglund Ulf
Published: July 2002

We report the refined structure of an iron‐free form of cytochrome c peroxidase (CcP) at 2.3 Å resolution. The backbone comparison between native CcP and iron‐free CcP shows that the two structures have the same protein fold within experimental error. The only difference noted is in the heme pocket where the distance between the proximal histidine and the center of the protoporphyrin has increased. The results show that the iron‐free CcP should be a good substitute for native CcP in fluorescence studies and thus also validate previous studies using iron‐free CcPs as efficient fluorescent probes in electron transfer studies.
d‐Val22 containing human big endothelin‐1 analog, [d‐Val22]Big ET‐1[16–38], inhibits the endothelin converting enzyme
Authors: Morita Akihito , Nomizu Motoyoshi , Okitsu Misako , Horie Kenji , Yokogoshi Hidehiko , Roller Peter P.
Published: July 2002

Endothelin converting enzyme (ECE) is essential for generation of the biological effects of endothelin‐1 (ET‐1) from a precursor, big endothelin‐1 (Big ET‐1). We synthesized four analogs of human Big ET‐1[16–38], substituted with single d‐amino acids at P1, P2, P1′and P2′ positions. ECE activity was determined using an ET‐1 specific radioimmunoassay system. None of the d‐amino acid containing Big ET‐1 analogs were apparently cleaved by ECE, however, one of the synthetic peptides, [d‐Val22]Big ET‐1[16–38], strongly inhibited the ECE activity. Furthermore, when this d‐Val22 containing peptide was preadministrated to rat striatum, it was found to inhibit the dopamine release induced by Big ET‐1. This result suggests that the d‐Val22 containing peptide inhibits the ECE activity in vivo. The d‐Val22 containing inhibitor offers hope of developing more potent and highly specific ECE inhibitors of therapeutic significance.
Analysis of the conformation and stability of rat TTF‐1 homeodomain by circular dichroism
Authors: Damante Giuseppe , Tella Gianluca , Leonardi Antonio , Fogolari Federico , Bortolotti Nadia , Di Lauroa Roberto , Formisano Silvestro
Published: July 2002

The conformational stability of TTF‐1HD has been determined by CD‐monitored thermal denaturation and isothermal urea unfolding studies. The Gibbs free energy of stabilization found are 1.44 and 1.26 kcal·mol−1, respectively. TTF‐1HD exhibits a T
m of 42°C and a δC
p of 80 cal·mol−1·K−1 indicating that TTF‐1HD, when free in solution, is a mobile flexible segment folded into loose helices. Such a flexibility would be relevant for the DNA‐binding function of this homeodomain. In fact, a small reduction of the α‐helical content of TTF‐1HD significally modifies its DNA‐binding activity.
N‐terminal domain of dystrophin
Authors: Bonet-Kerrache Armelle , Fabbrizio Eric , Mornet Dominique
Published: July 2002

Contro‐versial experiments have been published on calmodulin binding of dystrophin. In this study, we used recombinant proteins and the techniques of affinity chromatography and ELISA to show that the N‐terminal part of dystrophin binds calmodulin specifically in a calcium‐dependent manner. The calcium‐dependent interaction of calmodulin and dystrophin does not directly regulate binding of actin to dystrophin, but may regulate dystrophin interactions with other associated proteins.
Conformation of the C‐terminus of endothelin‐1 in aqueous solution studied by Monte‐Carlo simulation
Authors: Kuroda Masataka , Yamazaki Kazuto , Taga Tooru
Published: July 2002

The conformation of the C‐terminus of endothelin‐1 in an aqueous solution has been analyzed by a Monte‐Carlo simulation including the hydration energy term. The C‐terminus may adopt multiple or flexible conformations in the solution, but the classification of the conformations shows that a comparably large number of conformers take a similar folding form in which the C‐terminal chain is extended along the α‐helix of the N‐terminal core, and hydrophobic clusters are formed between the side groups of the C‐ and N‐terminus. The aromatic ring of Trp21 is located at a certain distance from the two charged side groups of Asp8 and Glu10, and the functional groups of the key residues in the N‐terminus are uncovered with the C‐terminus tail.
Transforming growth factor β1, insulin and prostaglandin E1 enhance prostaglandin F2α mitogenic action in Swiss 3T3 cells via separate events
Authors: de Alzaga Maria Gomez , Goin Mercedes , Ortiz Marcela , de Asua Luis Jimenez
Published: July 2002

Transforming growth factor β1 (TGFβ1) had no mitogenic effect in Swiss 3T3 cells, but could increase prostaglandin F2α (PGF2α)‐induced DNA synthesis. Insulin, but not prostaglandin E1 (PGE1), further enhanced PGF2α action at low TGFβ1 concentrations. TGFβ1 also acted concertedly with the protein kinase C (PKC) activator 1‐oleoyl‐2‐acetylglycerol to induce mitogenesis. Thus, it appears that TGFβ1 and insulin act via separate signals, while TGFβ1 and PGE1 might share a common pathway not involving TGFβ1‐mediated prostaglandin synthesis. These results suggest that TGFβ1 might elicit various signalling mechanisms to enhance PGF2α‐triggered events.
Nucleotide sequence and analysis of the mouse SPC3 promoter region
Authors: Hanabusa Tadashi , Ohagi Shinya , LaMendola Joseph , Chan Shu Jin , Steiner Donald F.
Published: July 2002

Insulin is converted from the higher molecular weight proprotein, proinsulin by highly specific proteolytic cleavage at two dibasic amino acid sites. SPC3 and SPC2, two recently identified prohormone convertase that are specifically expressed in β cells and other neuroendocrine cells, appear to be responsible for those cleavages. We have sequenced the 5′‐upstream region of the SPC3 gene and examined its promotor/enhancer activity and most of several deletion mutants in several cell lines. This region contains no CAAT box but has several non‐functional TATA‐like sequences and several putative transcriptional regulatory elements, including AP‐1, Spl and cAMP response elements. These features are not unlike those of the human SPC2 upstream region. In βTC3 insulinoma cells, the sequence between theEcoRI (620 bp) and NsiI (702 bp) sites seems to be important for gene expression, while the sequence between the NsiI and DraI (775 bp) sites may contain strong enhancer element(s).
Endurance training alters alanine and glutamine release from muscle during contractions
Authors: Hood David A. , Terjung Ronald L.
Published: July 2002

The release of alanine and glutamine from perfused muscle of trained and control animals was investigated. Release rates did not differ between trained and control muscle at rest. During contractions in trained muscle, alanine release was higher than at rest, while glutamine release was transiently increased. Phenylalanine release did not differ between trained and control muscle, implying that protein degradation was not increased in trained muscle. The muscle cellular adaptations to training include a selective modification of amino acid output, which could potentially influence gluconeogenic flux and alter muscle ammonia levels during contractions.
Both neurotoxin II from venom of Naja naja oxiana and its endogeneous analogue induce apoptosis in tumor cells
Authors: Strizhkov B.N. , Blishchenko E.Yu. , Satpaev D.K. , Karelin A.A.
Published: July 2002

Both neurotoxin II (NT II from venom of Naja naja oxiana) and 20–30 kDa proteins partially purified from pig brain (NTlm) cross‐reacting with antibodies to NT II were cytotoxic for L929 and K562 tumor cells at concentrations of 10−6–10−8 M. Modification of Cys residues significantly reduced cytotoxicity of NT II. Both NT2 and NTlm induced apoptosis in L929 and K562 cells.
Small GTP‐binding proteins of squid photoreceptor
Authors: Petrov Vasily M. , Artamonov Igor D. , Lipkin Valery M.
Published: July 2002

Squid photoreceptor membranes contain several small GTP‐binding proteins (22–27 kDa) in addition to heterotrimeric G‐proteins. In addition, these membranes contain several proteins which can be ADP‐ribosylated by exoenzyme C3 from Clostridium
botulinum (C3). These proteins are similar but not identical to C3 substrates from bovine rod outer segments. It is shown that substrates of C3 in squid photoreceptor interact with rhodopsin in a light‐dependent manner. These data suggest involvement of small GTP‐binding proteins in phototransduction processes in invertebrates.
Expression and characterization of VIP and two VIP mutants in NIH 3T3 cells
Authors: Wulff Birgitte S. , Georg Birgitte , Fahrenkrug Jan
Published: July 2002

Prepro‐vasoactive intestinal peptide (preproVIP) was expressed in NIH 3T3 cells, and the preproVIP‐derived peptides produced by the cells were analyzed by chromatography combined with sequence‐specific radio‐immunoanalysis. In accordance with what has previously been reported on processing in non‐endocrine cell lines, the VIP precursor was processed poorly in these non‐endocrine cells. Mainly an extended form of VIP could be detected in the media from the cells, and no immunoreactivity specific for amidated VIP was found. However, by changing the dibasic cleavage site positioned N‐terminal to the VIP sequence in the precursor into the consensus sequence (Arg,X,Lys/Arg,Arg) for the ubiquitous processing enzyme furin, thought to process, e.g. insulin receptors, factor VII, and by deleting residues 156–170 in the VIP precursor, expression of amidated VIP was obtained in this fibroblast cell line. Peptides from the wild‐type VIP precursor as well as peptides from the mutated VIP precursor were found to be able to stimulate the adenylate cyclase in cells expressing the VIP receptor.
Analysis of an insulin gene transcription control element
Authors: Stein Roland , Henderson Eva , Cordle Susan R.
Published: July 2002

Pancreatic β‐cell‐type‐specific transcription of the insulin gene is controlled by cis‐acting sequence elements lying within its enhancer region. An essential element required for expression is the insulin control element (ICE). The activity of this element is regulated by both positive‐ and negative‐acting transcription factors. In this study, we have identified the nucleotide sequences within the ICE that are required for repression in noninsulin producing cells. Our results indicate that the cis‐acting sequences involved in negative control are distinct from those required in activating expression in β cells.
Studies on the proton‐translocating NADH:ubiquinone oxidoreductases of mitochondria and Escherichia coli using the inhibitor 1,10‐phenanthroline
Authors: Finel Moshe , Majander Anna
Published: July 2002

Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is uncompetitively inhibited by 1,10‐phenanthroline (OP). EPR spectroscopy of submitochondrial particles indicates that OP, similarly to rotenone, inhibits electron transfer between the Fe‐S clusters of complex I and the ubiquinone pool. The proton‐translocating NADH dehydrogenase (NDH1) of E. coli is more sensitive to OP than is NDH1 of Paracoccus. EPR spectroscopy of membranous E. coli NDH1 shows that two slow‐ and one fast‐relaxing Fe‐S clusters become detectable upon reduction by NADH in the presence of OP. However, none of them resembles the mitochondrial cluster 2.
Site‐directed mutagenesis of the P2 residue of human antithrombin
Authors: Sheffield William P. , Blajchman Morris A.
Published: July 2002

Antithrombin (AT) is the principal inhibitor of thrombin in human plasma, and a member of the serine proteinase (serpin) family of proteins. Previously, we have described a point mutation in the human AT gene that converted amino acid 392 from glycine to aspartic acid which was associated with thrombotic disease in a Swedish family [(1992) Blood 79, 1428‐1434]. This observation prompted us to investigate the consequences of other substitutions at this position, termed P2 with respect to the reactive centre. Site‐directed mutagenesis was employed to generate seven mutants (Pro, Met, Gln, Val, Lys, Glu, and Asp), whose properties were compared with wild‐type recombinant AT, following in vitro transcription and cell‐free expression in a rabbit reticulocyte lysate system. With only one exception, the variant forms were less active than the wild‐type in forming complexes with either α‐thrombin, factor Xa, or trypsin. Hydrophobic (Val) or negatively charged (Asp or Glu) substitutions were particularly disruptive, in that these variants exhibited less than 10% wild‐type antithrombin or antitrypsin activity. In contrast, the formation of complexes with the various proteases of the Pro variant was essentially unimpaired. We conclude that the P2 residue of AT plays a role in optimal presentation of the reactive centre to its cognate protease, and propose that the observed requirement of Gly or Pro at this position is suggestive of a bend in the polypeptide backbone that aids in this presentation.
The myeloid‐lineage specific enhancer of the mouse myeloperoxidase gene consists of three cis‐elements defined as in vitro DNase I footprints
Authors: Zhu J.D
Published: July 2002

The myeloid‐lineage specific enhancer at 3.4–3.1 kb upstream of the mouse myeloperoxidase gene [1] has been further characterised. In vitro DNase I footprinting experiments revealed three protected sequences (FT‐I, ‐II and ‐III) in the enhancer, associated with the proteins that are enriched in WEHI 3BD+ cells, at which the MPO gene is highly expressed; but not in two non‐MPO expressing lymphocytic cell lines. Site‐specific mutations at each element severely reduced the level of the reporter gene activity in a non‐additive manner. This is parallel with either abolishment or alteration of the corresponding wild‐type protein‐DNA interaction in vitro. Consideration of the sequence motifs present in the enhancer suggests that the cis‐elements denned as the in vitro DNase I footprints are likely to be novel.