bluuebunny/crossref_metadata_2025 huggingface dataset:
Specific increase of a mitochondrial RNA transcript in chronic ethanol‐fed rats
Authors: Enriquez J.A. , Pérez-Martos A. , Fernández-Silva P. , López-Pérez M.J. , Montoya J.
Published: July 2002

An in vitro transcription system utilizing isolated mitochondria has been used to study the effect of chronic ethanol consumption on liver mitochondrial DNA transcription. The results obtained showed an overall increase of RNA synthesis and a dramatic accumulation of a discrete polyadenylated RNA species. This effect is a consequence of the chronic ethanol consumption since these changes do not occur when isolated control mitochondria are incubated in the presence of ethanol.
Bradykinin degradation pathways in human blood plasma
Authors: Mirgorodskaya O.A. , Shevchenko A.A.
Published: July 2002

The kinetics of proteolysis of bradykinin and its metabolites in human blood plasma was studied, the limiting stages were determined and each stage of the degradation was correlated with the appropriate plasma enzyme. A scheme of the pathways of major and minor degradation processes has been proposed.
Characteristic differences in the mode of quinone reduction and stability between energy‐coupled and ‐uncoupled NADH‐quinone reductases from bacterial respiratory chain
Authors: Unemoto Tsutomu , Miyoshi Tohoru , Hayashi Maki
Published: July 2002

Bacterial respiratory chain has two types of NADH‐quinone reductase (NQR): one is energy‐coupled (type‐1) and the other has no energy‐transducing capacity, that is, energy‐uncoupled (type‐2). Each of the NADH‐reacting flavoprotein subunits of NQR‐1 from Escherichia coli and the marine Vibrio alginolyticus reduced quinone to semiquinone radicals by the one‐electron transfer pathway and was very sensitive to preincubation with NADH. On the other hand, the NQR‐2 from these bacteria reduced quinone to quinol by the two‐electron transfer pathway and was insensitive to preincubation with NADH. Since the NQR‐1 from E. coli functions as a proton pump, whereas that from the marine V. alginolyticus functions as a sodium pump, the formation of semiquinone radicals as an intermediate is likely to be a common mechanism to functioning as either proton or sodium pump.
Differential sensitivity of CG and CCG DNA sequences to ethionine‐induced hypomethylation of the Nicotiana tabacum genome
Authors: Bezdĕk Milan , Koukalová Blaz̆ena , Kuhrová Viera , Vyskot Boris
Published: July 2002

Plant DNA is distinguished from the DNA of all other organisms by its high content of 5‐methylcytosine (5mC). 5mC levels may amount to 30% of total cytosines, distributed between the sequences CG and CXG. The results presented here show that the methylation status of CXG sequences could be influenced by culturing tobacco tissues on subtoxic concentrations of ethionine. The hypomethylating effect of ethionine, evaluated as the capability of MspI or HpaII to cleave the DNA, proved to be rather specific for CCG and differed from that or 5‐azacytidine which did not discriminate between CG and CXG sequences.
The effect of the UL42 protein on the DNA polymerase activity of the catalytic subunit of the DNA polymerase encoded by herpes simplex virus type 1
Authors: Hart Graham J. , Boehme Richard E.
Published: July 2002

The effect that the UL42 protein of herpes simplex virus type 1 has on the DNA polymerase activity of the DNA polymerase catalytic subunit (Pol) of the same virus has been investigated. The observed effects are critically dependent on the salt used and its concentration, such that the UL42 protein may inhibit, have little or no effect on, or activate the Pol activity, depending on the conditions used. The observed effects are due to the values for K mapp for activated DNA and V maxapp for Pol and the Pol‐UL42 protein complex differently varying with salt concentration.
Human immunodeficiency virus type 1 reverse transcriptase Affinity labeling of the primer binding site
Authors: Mitina R.L. , Doronin S.V. , Dobrikov M.I. , Tabatadze D.R. , Levina A.S. , Lavrik O.I.
Published: July 2002

Affinity modification of the primer site of HIV1‐RT was performed with an oligonucleotide derivative containing a photoreactive azido group at the 5′ end of d(pT)10. The affinity of HIV1‐RT for d(pT)10 and for its derivative was first estimated by measuring the Michaelis constants of these two oligonucleotides acting as primers in the retrotranscription of poly(rA). The enzyme was then inactivated under UV‐irradiation at 303–365 nm in the presence of ArN3‐d(U*T9); the dependence of the rate of inactivation on primer concentration was found to be consistent with the K
m value. Last, selectivity of affinity modification was demonstrated through elongation of the covalently bound primer and selective protection of inactivation by d(pT)10 or tRNALy5.
Retinoic acid enhances the secretion of plasminogen from cultured rat microglia
Authors: Nakajima Kazuyuki , Takemoto Nagisa , Kohsaka Shinichi
Published: July 2002

To determine the amount of plasminogen in microglial conditioned medium, a highly sensitive and specific enzyme‐linked immunosorbent assay (ELISA) for rat plasminogen was established. Weak cross‐reactivity with human serum plasminogen was observed, while no reactivity was detected with frog and carp plasminogen. The specificity of the immunosorbent assay was confirmed by Western blotting. The secretion of plasminogen into the microglial culture medium was quantified by using the established ELISA and was found to be increased depending on the culture time and number of microglia. The secretion was increased about 5‐fold by stimulation with retinoic acid, while interleukin‐1, and basic fibroblast growth factor showed no significant effect.
Barley (1→3,1→4)‐β‐glucanase isoenzyme EI gene expression is mediated by auxin and gibberellic acid
Authors: Slakeski Nada , Fincher Geoffrey B.
Published: July 2002

Treatment of young barley leaves with indole acetic acid (IAA) or gibberellic acid (GA3) results in a dramatic increase in levels of (1→3,1→4)‐β‐glucanase isoenzyme El transcripts. In young roots of comparable age, levels of isoenzyme El mRNA are high; IAA inhibits expression while GA3 has no effect on mRNA levels. The addition of both abscisic acid and GA3 to leaves, roots and aleurone layers leads to higher levels of (1→3,1→4)‐β‐glucanase isoenzyme El mRNA than is found with Ga3 alone. Little or no expression of (1→3,1→4)‐β‐glucanase isoenzyme EII is detected in vegetative tissues, but in isolated aleurone layers GA3 enhances levels of isoenzyme EII transcripts, as does IAA. Thus, the two barley (1→3,1→4)‐β‐glucanase genes respond quite differently to phytohormone treatment, depending on the tissue and its stage of development.
A functional tomato ACC synthase expressed in Escherichia coli demonstrates suicidal inactivation by its substrate S‐adenosylmethionine
Authors: Li Ning , Wiesman Zeev , Liu Derong , Mattoo Autar K.
Published: July 2002

1‐Aminocyclopropane‐1‐carboxylate (ACC) synthase is a key enzyme in the biosynthesis of the plant hormone, ethylene. We have isolated, sequenced and expressed a functional tomato (cy Pik‐Red) ACC synthase gene in Escherichia coli. ACC synthase expressed in E. coli was inactivated by incubation with S‐adenosylmethionine (SAM), the half—time of which was concentration dependent. Mixing the tomato fruit protein extract with the cell‐free extract from transformed E. coli did not affect SAM‐dependent inactivation of ACC synthase activity. Thus, single isoforms of the ACC synthase enzyme, which demonstrate the biochemical features expected of the tomato fruit enzyme, can be expressed in E. coli and their structure—function relationships investigated.
Elevated cytosolic concentrations of SecA compensate for a protein translocation defect in Escherichia coli cells with reduced levels of negatively charged phospholipids
Authors: Kusters Ron , Huijbregts Richard , de Kruijff Ben
Published: July 2002

Cellular extracts from cells with reduced synthesis of negatively charged phospholipids were found to support in vitro translocation of the precursor of the outer membrane protein PhoE with increased efficiency. Analysis of these extracts revealed that they contain increased levels of SecA. SecA depletion resulted in a loss of the translocation stimulatory activity, which could be restored by re‐addition of purified SecA. We conclude that elevated cytosolic levels of SecA counteract the reduction of translocation efficiency due to low levels of negatively charged phospholipids in the inner membrane.
Conformation and length of the signal sequence affect processing of secretory protein
Authors: Kohara Atuko , Yamamoto Yoshio , Kikuchi Masakazu
Published: July 2002

Processing of human lysozyme with artificially designed signal sequences was examined in an in vitro translation—translocation system and compared with their secretory capabilities in yeast. It has been shown that the conformation of the C‐terminal region of the signal sequence and the length of the hydrophobic segment are important factors for efficient cleavage of the signal sequence.
Osteoclasts express high levels of p60c‐src, preferentially on ruffled border membranes
Authors: Tanaka Sakae , Takahashi Naoyuki , Udagawa Nobuyuki , Sasaki Takahisa , Fukui Yasuhisa , Kurokawa Takahide , Suda Tatsuo
Published: July 2002

Expression of p60c‐src
, the normal cellular counterpart of the transforming protein of Rous sarcoma virus (RSV), p60c‐src
, was examined in mouse and rat authentic osteoclasts and mouse osteoclast‐like multinucleated cells (MNCs) formed in vitro. In co‐cultures of mouse osteoblastic cells and spleen cells, the expression of p60c‐src
strikingly increased on day 5 in parallel with the appearance of MNCs in the presence of 1α,25‐dihydroxyvitamin D3 (1α,25(OH)2D3). Immunohistochemical examination confirmed the high level expression of p60c‐src
in both mouse authentic osteoclasts and MNCs. Electron microscopic examination revealed that p60c‐src
was primarily localized on ruffled border membranes and vacuoles, but not on the clear zone in rat authentic osteoclasts. These results suggest that p60
c‐src
is important in osteoclastic bone resorption.
The length of the interdomain region of the L7/L12 protein is important for its function
Authors: Bubunenko M.G. , Chuikov S.V. , Gudkov A.T.
Published: July 2002

Several mutated L7/L12 proteins with changed interdomain regions were obtained. The results showed that the flexible region comprising the 39–52 amino acid residues is functionally important. Its length, but not its amino acid composition, is crucial for the function.
ATP‐activated Ca2+‐permeable channels in rat peritoneal macrophages
Authors: Naumov Alexander P. , Kuryshev Yuri A. , Kaznacheyeva Elena V. , Mozhayeva Galina N.
Published: July 2002

The patch‐clamp technique was used to study mechanisms of ATP‐induced Ca2+ influx in rat peritoneal macrophages. The experiments on whole‐cell and patch membranes have shown that extracellular ATP activates channels permeable to di‐ and monovalent inorganic cations. Ratios of unitary channel conductances in 105 mM Ca2+, Sr2+, Mn2+, Ba2+ and normal sodium solutions were 1.0, 0.95, 0.75, 0.55 and 0.85, respectively. The channels could open in the presence of non‐hydrolyzable GTP analogues in artificial intracellular solution. The data are consistent with the hypothesis that a GTP‐binding protein is involved in receptor‐to‐channel coupling.
Identification of two genes of Rhodobacter capsulatus coding for proteins homologous to the ND1 and 23 kDa subunits of the mitochondrial Complex I
Authors: Dupuis Alain
Published: July 2002

A region of the genome of Rhodobacter capsulatus has been sequenced and shown to encode proteins homologous to the NDI subunit and the 23 kDa subunit of the mitochondrial NADH:CoQ oxidoreductase (Complex I). The association of these two open reading frames in the R. capsulatus genome parallels the organisation of the chloroplast genome. It suggests that genes encoding subunits of the NADH:CoQ oxidoreductase must be clustered in the genome or R. capsulatus.
Single cell observation of ligand‐induced desensitization of B‐cell membrane immunoglobulin‐mediated calcium signals
Authors: Yamada Hiroshi , Mizuguchi Junichiro , Noji Masahide , Nakanishi Mamoru
Published: July 2002

Using a digital imaging fluorescence microscope we have observed the membrane immunoglobulin (mIg)‐induced desensitization of calcium signals in individual BAL17 B lymphoma cells which express two kinds of antigen receptors, mIgM and mIgD. The mIgD‐mediated desensitization was partly abrogated by pretreating the cells with phorbol 12‐myristate 13‐acetate (PMA) for 24 h, however, the mIgM‐mediated one was not affected by the pretreatment. This supports the idea that at least two mechanisms are operative for mIg‐induced desensitization in B cells.
Tyrosine phosphorylation is an early and requisite signal induced by interferon‐γ in HL‐60 cells
Authors: Offermanns Stefan , Schultz Günter
Published: July 2002

Interferon‐γ (IFNγ) is a potent immunomodulatory cytokine. However, the early mechanisms which mediate the pleiotropic effects of IFNγ on different cells are as yet poorly understood. Therefore, we tested the role of tyrosine phosphorylation in signalling induced by IFNγ. IFNγ was found to induce rapid tyrosine phosphorylation of several proteins in HL‐60 cells. This effect was detectable by 2 min, reached a maximum by about 4–16 min and thereafter declined. Tyrosine phosphorylation was dependent on receptor occupation and was maximally stimulated by 10 ng/ml IFNγ. Treatment of HL‐60 cells with the tyrosine kinase inhibitors, genistein and herbimycin A, inhibited both IFNγ‐stimulated tyrosine phosphorylation and IFNγ‐induced Fc receptor expression. Thus, increased tyrosine phosphorylation appears to be an obligatory early and proximal signal mediating at least some of the later cellular responses induced by IFNγ in HL‐60 cells.
Cloning and sequencing of a rat type II activin receptor
Authors: Shinozaki Hiromitsu , Ito Ikuro , Hasegawa Yoshihisa , Nakamura Kazuto , Igarashi Shigeo , Nakamura Manabu , Miyamoto Kaoru , Eto Yuzuru , Ibuki Yoshito , Minegishi Takashi
Published: July 2002

A full‐length cDNA for a rat type II activin receptor was cloned by hybridization from a rat ovary cDNA library. The deduced amino acid sequence (513 residues) containing a single membrane‐spanning domain and an intracellular kinase domain with predicted serine/threonine specificity. The amino acid sequence is 99.8% and 99.4% identical in the coding region with the previously cloned mouse and human type II activin receptor, and only 66.7% identical in the coding region with the previously cloned rat type IIB activin receptor. We examined the effect of PMSG‐hCG on the mRNA level of type II activin receptor in immature rat ovaries. Northern blot analysis of ovarian RNA revealed two mRNAs (3.0 kb and 6.0 kb).
Bovine α2‐antiplasmin N‐Terminal and reactive site sequence
Authors: Christensen Søren , Sottrup-Jensen Lars
Published: July 2002

Bovine α2‐antiplasmin (α2AP) has been purified and partially characterized. The amino acid composition is very similar to that of human α2AP, and the N‐terminal (23 residues determined) and reactive site loop sequences (42 residues determined) are highly homologous to those of the human protein. Compared with human α2AP, bovine α2AP has an 18‐residue N‐terminal extension, homologous with part of the pre‐sequence of human α2AP. A re‐investigation of the N‐terminal sequence of freshly prepared human α2AP reveals a new form extended by 12 residues.
Evidence for binding of extrachromosomal DNA sequences to nuclear matrix proteins in multidrug‐resistant KB‐V1 cells
Authors: Thiebaut Franz , Hanauske Axel-R. , Von Hoff Daniel D.
Published: July 2002

Multidrug‐resistant KB‐V1 cells carry amplified mdrl gene sequences located in an extrachromosomal compartment (on episomes). Since episomes do not contain centromeric or telomeric sequences it is unclear whether they are able to bind to nuclear matrix proteins that may regulate episomal gene expression. Using high salt treatments followed by in situ hybridization and dot blot analyses we found evidence for direct binding of episomal DNA to nuclear matrix proteins. This binding could only be reversed after incubation with trypsin or proteinase K as determined by contour‐clamped homogeneous electric field (CHEF) electrophoresis. Our findings are consistent with the concept that circular extrachromosomal DNA may not only reintegrate into nuclear DNA but may also be subject to functional control by regulatory proteins within the nuclear matrix.
Ca2+ binding of latent transforming growth factor‐β1 binding protein
Authors: Colosetti Pascal , Hellman Ulf , Heldin Carl-Henrik , Miyazono Kohei
Published: July 2002

Latent transforming growth factor‐β1 binding protein (LTBP) is a constituent of the latent high molecular weight complex of TGF‐β1 in human platelets. In the present communication, we show that LTBP binds Ca2+ in its free form as well as in the latent TGF‐β1 complex. The binding of Ca2+ induces a structural change which protects the molecule against proteolysis and changes its elution position when analyzed by anion exchange chromatography. The in vitro activation of TGF‐β1 is not influenced by the presence of Ca2+. The possible significance of Ca2+ binding of LTBP is discussed.
Trans‐activation of the murine dystrophin gene in human‐mouse hybrid myotubes
Authors: Noursadeghi M. , Walsh F.S. , Heiman-Patterson T. , Dickson G.
Published: July 2002

Myotube cultures of the myogenic cell line, C2, produce significantly lower levels of dystrophin than primary mouse cultures. We demonstrate that expression of the C2 dystrophin gene increases 10‐fold in hybrid myotubes formed by fusion of C2 and dystrophin‐deficient human myoblasts from a Duchenne muscular dystrophy patient. These results indicate that C2 cells are deficient in endogenous gene regulatory factors which enhance dystrophin expression, and that the C2 cell line may therefore be used to identify putative trans‐acting factors involved in the regulation of dystrophin gene expression.
Effect of ouabain, digoxin and digitoxigenin on potassium uptake and histamine release from rat peritoneal mast cells
Authors: Knudsen T. , Ferjan I. , Johansen T.
Published: July 2002

Rat peritoneal mast cells were used to study the effects of digitalis glycosides on potassium uptake and histamine release induced by compound 48/80, substance P and egg‐albumin (immunological release). In the absence of calcium all glycosides inhibited potassium uptake. Ouabain and digoxin enhanced the histamine release while digitoxigenin either had no effect or was slightly inhibitory. In the presence of calcium, the glycosides only affected potassium uptake and histamine release slightly. In the presence of lithium or lanthanum the enhancement of the histamine release was counteracted. Hydrophilic digitalis glycosides seem to enhance histamine release secondary to an increase in intracellular sodium. Lipophilic glycosides have no effect on the release.
On the role of intracellular concentration of Ca2+ and H+ in thymocyte death after irradiation
Authors: Shaposhnikova Vera V. , Dobrovinskaya Oksana R. , Gukovskaya Anna S. , Trepakova Elena S. , Korystov Yuri N.
Published: July 2002

The role of intracellular Ca2+ and H+ concentrations in radiation‐induced interphase death of rat thymocytes has been studied. In response to concanavalin A treatment in the Ca2+‐containing medium, or to the CaCl2 treatment in the Ca2+‐free medium, the [Ca2+]i rise in irradiated cells was as in the non‐treated cells. No changes in the level of [Ca2+]i and pHi were found within l h after irradiation of thymocytes with a dose of 6 Gy. 15 μM 5‐(N‐ethyl‐N‐isopropyl)‐amiloride. an inhibitor of Na+/H+ exchange, did not affect the DNA fragmentation. The fragmentation was prevented by 2–4 μM (1 ‐[bis(4‐chlorophenyl)methyl]‐3‐[2‐(2,4‐dichlorophenyl)]‐2‐[(2,4‐dichlorophenyl)‐methoxy]‐ethyl)‐1 ‐H‐imidazoliumchloride, an inhibitor of calmodulin. The above data indicate that triggering of interphase death in irradiated thymocytes is not mediated by changes in either [Ca2+]i or pHi. Such changes seem to be involved in intermediate steps of the interphase death process.
The inhibition of human duodenal adenylate cyclase activity by Ca2+ and the effects of EGTA
Authors: Smith Julie A. , Griffin Martin , Mireylees Stewart E. , Long Richard G.
Published: July 2002

This study demonstrates that the inhibition of adenylate cyclase activity by Ca2+ is enhanced in the presence of increasing [EGTA] (0, 0.3, 1, 2.5 mM) by 2 orders of magnitude. It has been established that this effect is not because of poor Ca2+ buffering by low [EGTA] or high Ca2+ binding by the membrane preparation. It is present irrespective of stimulus. We suggest the enhanced sensitivity of adenylate cyclase to Ca2+ induced by EGTA is caused by the Ca‐EGTA complex being a more inhibitory species than Ca2+. Thus consideration of the effects of the Ca‐EGTA complex should be made when interpreting the results from experiments involving Ca2+ and EGTA.
Alternative splicing of a previously unidentified CFTR exon introduces an in‐frame stop codon 5' of the R region
Authors: Melo Carlos A. , Serra Cristina , Stoyanova Violeta , Aguzzoli Cristina , Faraguna Dino , Tamanini Anna , Berton Giorgio , Cabrini Giulio , Baralle Francisco E.
Published: July 2002

The cystic fibrosis transmembrane conductance regulator (CFTR) has been extensively characterized as the carrier of the basic defect in cystic fibrosis. CFTR is part of a growing family of proteins encoded by a single gene, the variant isoforms of which are generated by alternative splicing or RNA editing. We have analyzed the CFTR mRNA in the region of exons 10–11 in T84 cells and detected an alternatively spliced exon (10b) accounting for about 5% of the CFTR mRNA. The exon lOb found in both the human and mice genomes, introduces an inframe stop codon. The resulting mRNA is translated into a truncated CFTR protein, identified in T84 cells by immunoprecipitation with the CFTR‐specific monoclonal antibody MATG 1061. The insertion of a differentially spliced exon carrying an inframe stop codon is a novel cellular mechanism for the production of a protein sharing common sequences with another, but having different properties and functions.
Kinetics of glucose oxidase catalyzed electron transfer mediated by sulfur and selenium compounds
Authors: Kulys J. , Buch-Rasmussen T. , Bechgaard K. , Marcinkeviciene J. , Christensen J.B. , Hansen H.E.
Published: July 2002

Unusually high electron transfer rates in Aspergillus niger glucose oxidase catalyzed oxidation of glucose using 5,6:11,12‐Bis(dithio)tetracene (TTT), 1,2‐dimethyltetraselenafulvalene (DMTSF) and tetrathiafulvalene (TTF) were observed. At pH 7.0 oxidation rate constants (TN/K
m) in the range from 1.0 · 107 to 8.7 · 107 M · s−1 were deduced from experimental data. One of the investigated mediators, DMTSF, has been used for electrocatalytical glucose oxidation on graphite at a potential of 0.3 V vs. a standard calomel electrode (SCE). The prepared bioelectrodes have a sensitivity of 1.3 μA/(cm2 · mM), a pH optimum at 6.5‐7.0, and a linear range which covers the relevant range for monitoring physiological levels of glucose. The bioelectrodes are stable for more than one month.
5‐Methylcytosine in genes with methylation‐dependent regulation
Authors: Volpe Pietro , Iacovacci Paolo , Butler Richard H. , Eremenko Tamilla
Published: July 2002

An asymmetric distribution of deoxy‐5‐methylcytidylic acid‐inhibiting restriction sites (dcm‐sites) takes place in ten human genes regulated by 5‐methylcytosine. These genes are dcm‐site enriched upstream and dcm‐site poor downstream. Along them, there is a scattering of hypermethylatable introns and hypomethylatable exons with a common code: the 5mCpG dinucleotides characterize promoters; Gp5mCs characterize introns; Tp5mCs and Cp5mCs are in small concentrations in exons. Housekeeping genes contain more dcm‐sites when compared with tissue‐specinc genes. This depends on the higher number of dcm‐sites in their promoters and introns. In exons, the relatively lower number of dcm‐sites is almost the same in both housekeeping and tissue‐specific genes. Going from 5' to 3', the average frequency of occurrence of these sites per nucleotide units decreases in introns and increases in exons. This difference is highly discriminated for tissue‐specinc and less discriminated for housekeeping genes.
Constitutive over‐production of DNA‐damage recognition proteins and acquired UV resistance in prolonged culture of F9 teratocarcinoma stem cells
Authors: Chao Chuck C.-K.
Published: July 2002

An ultraviolet (UV)‐resistant F9 variant cell line, termed F9Vc, was established from a prolonged culture of murine F9 embryonic stem cells. A 6‐fold UV resistance was detected in F9V2 cells compared to the F9 parental cells, as determined by ID50 (36 J/m2
vs. 6 J/m2
), the UV dose causing 50% growth inhibition. Using a DNA mobility‐shift assay, a nuclear protein (termed UVDRP) that preferentially binds to UV‐damaged DNA was detected in F9 and F9Vc cell extracts. The UVDRP in F9Vc cells was present at a 7‐fold higher concentration than that of F9 cells. Interestingly, the F9 UVDRP was transiently induced following cellular differentiation by retinoic acid (RA)/cAMP, with optimum induction (15‐fold) at 6 days. Although constitutively over‐produced, UVDRP also remained inducible in F9Vc cells in response to RA/cAMP. Indirect DNA repair measurement by host cell reactivation of UV‐damaged plasmid DNA demonstrated that F9Vc cells exhibited a slight increase or a similarity in repair ability compared to the F9 cells. Parallel experiments using the repair‐defective xeroderma pigmentosum (XP) group A fibroblasts and the normal VA 13 fibroblasts also indicated that over‐production of UVDRP binding activity was associated with enhanced DNA repair and UV resistance. The findings indicate that prolonged culture of F9 cells can establish a condition sufficient to cause constitutive over‐production of UVDRP binding activity and UV resistance. The results also suggest that the RA/cAMP‐inducible UVDRP in F9 stem cells may be important for the sensitivity or resistance of the cells to UV damage.
The hinge region of chicken annexin I contains no site for tyrosine phosphorylation
Authors: Sidis Yisrael , Horseman Nelson D.
Published: July 2002

Annexin I (AnxI) is a calcium‐dependent membrane binding protein which has been implicated in various physiological activities. The region of the chicken anxI cDNA encoding the first 130 amino terminal residues was cloned by reverse transcription PCR in order to determine the relationship of its variable ammo‐terminal regulatory region with other known annexins. This nucleotide sequence shows 86% identity with pigeon Anxl isoforms, and 57% with its human homolog. The protein encoded by the chicken anxI cDNA lacks the canonical epidermal growth factor receptor/kinase phosphorylation site, which is present in Anxl of other species. In contrast, the putative protein kinase C phosphorylation site of the amino‐tenninus is present in the chicken AnxI. Whereas the pigeon genome contains two anxI genes, genomic Southern analysis shows that in the chicken Anxl is encoded by only a single gene. These data suggest that AnxI has undergone significant sequence variation in the avians, and clarifies the relationships of the avian anxI genes with their ancestral homologs.
Purification and reconstitution of the δ opioid receptor
Authors: Gomathi K.G. , Sharma Shall K.
Published: July 2002

The δ opioid receptor has been purified, in an active form, by succinylmorphine affinity chromatography. The receptor was purified partially from bovine frontal cortex and to apparent homogeneity from neuroblastoma × glioma hybrid NG108‐15 cells as observed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis followed by silver staining. Antiserum to the purified bovine receptor inhibited ligand binding to membranes and immunoprecipitated a 58 kDa protein from NG108‐15 cells. Reconstitution of the receptor with lipids enhanced binding by 9‐fold. The 58 kDa band protein after electroelution and reconstitution with lipids also showed specific binding, indicating that the receptor could be renatured even after SDS‐PAGE in an appropriate lipid environment.
The pH‐dependent anion‐conducting channel of the mitochondrial inner membrane is potently inhibited by zinc ions
Authors: Selwyn Michael J. , Ng Lay Tin , Choo Hui Lim
Published: July 2002

Zinc is a potent reversible inhibitor of the pH‐dependent anion‐conducting channel in the mitochondrial inner membrane, 50% inhibition was produced by 1.5 μM added Zn2+ at which point free Zn2+ was ⩽ 10−8 M. Inhibition by Zn2+ is rapid but can be prevented or rapidly reversed by excess EDTA. Concentrations of Zn2+ higher than 4 μM caused reversal of inhibition to a variable extent depending on the anion. Under these conditions Zn2+ did not inhibit ribose entry, the phosphate transporter, or the pH‐insensitive component of the NO3
− uniport.
Effect of the general anesthetic halothane on the activity of the transverse tubule Ca2+‐activated K+ channel
Authors: Beeler Troy , Gable Kenneth
Published: July 2002

The effect of the general anesthetic halothane on the activity of the rat skeletal muscle Ca2+‐activated K+ channel in planar lipid bilayers was investigated. Halothane concentrations in the clinical range (1.0–0.2 mM) alter the regulation of the channel by both Ca2+ and membrane potential. At Ca2+ concentrations between 10 and 250 μM and membrane potentials between 0 and −30 mV, halothane significantly decreases the open state probability without changing the channel conductance. The results demonstrate that halothane can act directly on the Ca2+‐activated K+ channel or its lipid environment to alter the channel gating kinetics.
The dephosphorylation characteristics of the receptors for epidermal growth factor and platelet‐derived growth factor in Swiss 3T3 cell membranes suggest differential regulation of receptor signalling by endogenous protein‐tyrosine phosphatases
Authors: Böhmer Frank-D. , Böhmer Sylvia-Annette , Heldin Carl-Henrik
Published: July 2002

Comparison of the phosphotyrosine‐specific dephosphorylation of the autophosphorylated receptors for epidermal growth factor (EGF) and platelet‐derived growth factor (PDGF) in Swiss 3T3 cell membranes by the endogenous phosphatases revealed striking differences. EGF receptor dephosphorylation was clearly faster than PDGF receptor dephosphorylation and strongly inhibited by Triton X‐100 and octylglucoside, whereas PDGF receptor dephosphorylation was to a lesser extent detergent‐susceptible. PDGF receptor dephosphorylation was effectively inhibited by phenylarsineoxide, protamine and poly‐lysine and partially by N‐ethylmaleinimide, whereas EGF receptor dephosphorylation was not affected by these agents. We suggest that these differences in dephosphorylation of EGF and PDGF receptors are due to their differential interaction with membrane‐associated protein‐tyrosine phosphatases and important for differential regulation of receptor signalling.
Negative correlation with liver cell division of a 38 kilodalton protein whose phosphorylation is enhanced by ras and G‐proteins
Authors: Hegde Ashok N. , Swamy Ch.V.B. , Murali Krishna Bh. , Das M.R.
Published: July 2002

We showed earlier that the phosphorylation of a 38 kDa protein (p38) from rat liver plasma membrane is stimulated by ras or endogenous G‐proteins. We have now estimated the level of expression of p38 in liver tissues from embryos at different stages of development, regenerating liver and also in tumor cell lines of hepatic origin. Our results indicate that the expression of p38 is negatively correlated with cell division. It is suggested that the phosphorylation of p38, an event which is regulated by ras proteins and G‐proteins, could be involved in signal transduction processes associated with the inhibitory regulation of cell division.
Neurogranin, a B‐50/GAP‐43‐immunoreactive C‐kinase substrate (BICKS), is ADP‐ribosylated
Authors: Coggins Philip J. , McLean Kim , Zwiers Henk
Published: July 2002

Neurogranin is a neurone‐specific, B‐50‐immunoreactive C‐kinase substrate that has limited homology to, but considerable biochemical similarity to B‐50/GAP43. The most significant differences between these two proteins are their cellular localisation and molecular mass (Neurogranin, 7.5 kDA cytosolic; and B‐50, 25 kDa membranal). An understanding of the similarities and differences between Neurogranin and B‐50 may facilitate the elucidation of their hitherto elusive functions in the nervous system. The results of the present study demonstrate that, in common with B‐50, Neurogranin is a substrate for ADP‐ribosyltransferases. This finding is discussed with regard to the concept of molecular flexibility of B‐50‐like proteins as the basis of their putatively diverse roles in the nervous system.
cDNA cloning of rat proteasome subunit RC7‐I, a homologue of yeast PRE1 essential for chymotrypsin‐like activity
Authors: Nishimura Chihiro , Tamura Tomohiro , Tokunaga Fuminori , Tanaka Keiji , Ichihara Akira
Published: July 2002

The nucleotide sequence of a cDNA that encodes a new subunit, named RC7‐I, of the 20 S proteasome of rat hepatoma cells has been determined. The polypeptide predicted from the open reading frame consists of 201 amino acid residues with a calculated molecular weight of 22,912 and isoelectric point of 7.25. Approximately 80% of the partial amino acid sequences of several fragments of RC7‐I, determined by protein chemical analyses, were found to be in excellent accordance with those deduced from the cDNA sequence. Computer analysis showed that RC7‐I belongs to the β‐type subgroup of proteasomes with similarity to the β‐subunit of the archaebacterial proteasome, differing clearly from α‐type subunits of the proteasome gene family. The overall structure of RC7‐I was found to be homologous to that of yeast PRE1, which is necessary for chymotryptic activity.
Residues 1 to 80 of the N‐terminal domain of the β subunit confer neuronal bungarotoxin sensitivity and agonist selectivity on neuronal nicotinic receptors
Authors: Wheeler Susan V. , Chad John E. , Foreman Richard
Published: July 2002

Standard two electrode voltage clamp techniques were used to investigate the response of neuronal nicotinic acetylcholine receptors, expressed in Xenopus oocytes, to various agonists and neuronal bungarotoxin (NBT). The β subunit is an important determinant of the receptor's pharmacological profile. Co‐expression of α4 and β2 subunits produced a receptor that was relatively insensitive to cytisine and nicotine and inhibited by NBT, whilst the α4β4 combination produced a receptor that was highly sensitive to cytisine and nicotine but resistant to toxin. The first 80 amino acids of the N‐terminal domain of the β subunit are implicated in these characteristics, since the combination of α4 with a hybrid β subunit comprising amino acids 1 → 80 of β2 and 81 → 416 of β4 became relatively insensitive to nicotine and cytisine and resistant to inhibition by neuronal bungarotoxin.
Solvent oxygen is not incorporated into N10‐formyltetrahydrofolate in the reaction catalyzed by N10‐formyltetrahydrofolate synthetase
Authors: Song Sophie , Jahansouz Hossain , Himes Richard H.
Published: July 2002

The mechanism of the reaction catalyzed by N
10‐formyltetrahydrofolate synthetase involves the formation of formyl phosphate as an intermediate which then formylates tetrahydrofolate at the N‐10 position. Previous studies demonstrated that the non‐enzymic formylation of tetrahydrofolate by formyl phosphate occurs exclusively at the more nucleophilic 5‐nitrogen in the reduced pyrazine ring. The experiments described in this report were designed to determine whether N
5‐formyltetrahydrofolate might be the first product to be formed on the enzyme, followed by formyl transfer to the 10‐nitrogen via the cyclic intermediate n
5,10‐methenyltetrahydrofolate. If this were the case, oxygen from solvent H2O would be incorporated into the formyl group of the N
10‐derivative. By conducting the reaction in a 1:1 mixture of [16O]H2O and [18O]H2O and using 13C NMR spectroscopy we show that no 18O is incorporated into the product and conclude that the reaction proceeds via a direct formylation of the N‐10 position by formyl phosphate.
Analysis of poly(A)+RNA patterns in human tissues
Authors: Chenchik Alexander A. , Diachenko Ludmila B. , Beabealashvilli Robert Sh.
Published: July 2002

A novel method for analysing and comparing the relative amounts of the most abundant (higher than 0.1% abundance) individual mRNAs present in different poly(A)+RNA preparations has been developed. This method is based on the synthesis of short (10–30 nucleotide) cDNA termination products, by reverse transcription of poly(A)+RNA primed with a 5'‐labeled oligonucleotide. A set of 30 different oligonucleotides are used as primers in separate reactions, their length and sequences having been chosen to provide more than a 90% probability of initiating synthesis from any individual RNA present in the poly(A) +RNA. Each primer produces about 10–60 bands per track, following polyacrylamide gel electrophoresis under denaturing conditions. Data presented reveals poly(A)+RNA pattern differences for a number of human tissues and identifies changes in RNA patterns between normal tissues and neoplastic human tumors (myoma of the uterus) from several individuals.
Human α2‐macroglobulin structure
Authors: Gettins Peter G.W. , Crews Brenda C.
Published: July 2002

To localize the pair of thiol‐ester‐derived cysteine‐949 residues within a half‐molecule of α2M and estimate the internal diameter of this α2‐macroglobulin trap, we have measured the separation between these cysteines. We unexpectedly found that the two cysteines of intermediate form α2M had different reactivity, which permitted selective modification of one cysteine with dansyl, and the other with fluorescein fluorophores. From fluorescence energy transfer measurements, we calculated a separation of 41 ± 10 Å between these fluorophores. This indicates that the Cys‐949 residues are probably located at the perimeter of the trap with an internal diameter at least as large as this separation.
High‐affinity binding of a protein‐lipopolysaccharide phytotoxin from Verticillium dahliae to cotton membranes
Authors: Meyer Riaan , Dubery Ian A.
Published: July 2002

The presence of a specific binding site for a protein‐lipopolysaccharide (PLP) phytotoxin isolated from culture filtrates of Verticillium dahliae has been demonstrated in plasma membranes from cotton seedlings. The 125I‐labelled PLP was used as a ligand in binding assays with plasma membrane enriched fractions. Root tissue exhibited the highest amount of binding activity compared to hypocotyl and cotyledon tissue. Binding of the [125I]PLP was saturable, reversible, and with an affinity (K
d = 1.42 × 10−8 M) comparable with the concentration required for biological activity. A single class of binding site was found, and the maximal number of binding sites were estimated as 5.4 × 10−15
protein.
1H assignment and secondary structure determination of human melanoma growth stimulating activity (MGSA) by NMR spectroscopy
Authors: Fairbrother Wayne J. , Reilly Dorothea , Colby Timothy , Horuk Richard
Published: July 2002

The solution structure of melanoma growth stimulating activity (MGSA) has been investigated using proton NMR spectroscopy. Sequential resonance assignments have been carried out, and elements of secondary structure have been identified on the basis of NOE, coupling constant, chemical shift, and amide proton exchange data. Long‐range NOEs have established that MGSA is a dimer in solution. The secondary structure and dimer interface of MGSA appear to be similar to those found previously for the homologous chemokine interleukin‐8 [Clore et al. (1990) Biochemistry 29, 1689‐1696]. The MGSA monomer contains a three stranded anti‐parallel β‐sheet arranged in a ‘Greek‐key’ conformation, and a C‐terininal α‐helix (residues 58 69).
Efficient processing and export of human growth hormone by heat labile enterotoxin chain B signal sequence
Authors: Ghorpade Anuja , Garg Lalit C.
Published: July 2002

The heat‐labile enterotoxin chain B (LTB) signal sequence was used for the processing and export of human growth hormone (hGH). The protein was completely processed and exported across the cell membrane to accumulate in the periplasmic space in Escherichia coli. The human growth hormone cDNA was cloned as a PCR amplified fragment under the control of tac promoter and translationally fused to the LTB signal sequence. The rate of processing of hGH under the control of the LTB signal sequence was equal to or more than the rate of induction of expression, indicating efficient processing. The receptor binding activity of the processed periplasmic protein was established in a radio receptor assay.
Site‐directed mutagenesis of the tick‐borne encephalitis virus NS3 gene reveals the putative serine protease domain of the NS3 protein
Authors: Pugachev Konstantin V. , Nomokonova Natalia Yu. , Dobrikova Elena Yu. , Wolf Yuri I.
Published: July 2002

Several mutations were introduced into the putative serine protease domain of the tick‐borne encephalitis virus NS3 protein and into a possible internal cleavage site within the protein. The influence of these mutations on proteolytic activity of NS3 protein and NS3' protein formation was tested in vitro. It was found that NS3' formation was not dependent on the activity of the NS3 N‐terminal serine protease. Mutations affecting the Ser‐138 residue of the NS3 protein prohibited cleavage between NS2B and NS3 proteins when the NS2B‐NS3 part of the viral genome was expressed in vitro, suggesting the key role of Ser‐138 in viral serine protease functioning.
Characterization and purification of a hydrogenase from the eukaryotic green alga Scenedesmus obliquus
Authors: Schnackenberg J. , Schulz R. , Senger H.
Published: July 2002

Several catalytic properties of the hydrogenase from Scenedesmus obliquus have been examined to optimize the purification conditions. The Km
‐value for H2‐evolution in the presence of the most effective electron mediator methylviologen is 0.66 mM. The pH‐optimum is 6.3, the temperature‐optimum is 50°C and the energy of activation is 38.4 ± 2 kJ·mol−1. The soluble hydrogenase from the green alga, Scenedesmus obliquus, was purified 1290‐fold to homogeneity. The enzyme consists of two subunits with molecular masses of 55 kDa and 36 kDa. The molecular weight of the native enzyme, determined by gel filtration, is 150 ± 5 kDa.
Kinetics of cellular permeability of phenoxazine and its dependence on P‐glycoprotein expression
Authors: Wadkins Randy M. , Houghton Peter J.
Published: July 2002

We present here the initial characterization of the mechanism of reversal of cellular resistance to Vinca alkaloids by phenoxazine (PZ). Changes in fluorescence upon cellular accumulation of PZ allowed measurement of the membrane transport kinetics in a sensitive KB‐3‐1 cell line and two multi‐drug resistant (MDR) counterparts. The accumulation of PZ is characterized by two uptake routes, with pseudo‐first order rate constants of 0.3 s−1 and 0.07 s−1, while efflux of PZ from cells revealed rate constants of 0.2 s−1. PZ rapidly reaches steady‐state concentrations within cells, which may make it more clinically useful than modulators that accumulate more slowly (e.g. verapamil).
Presence of the brown fat‐specific mitochondrial uncoupling protein and iodothyronine 5'‐deiodinase activity in subcutaneous adipose tissue of neonatal lambs
Authors: Trayhurn Paul , Thomas Moira E.A. , Duncan Jacqueline S. , Nicol Fergus , Arthur John R.
Published: July 2002

Subcutaneous adipose tissue of neonatal lambs has been examined for the presence of markers diagnostic of thermogenic brown fat. Uncoupling protein, uncoupling protein mRNA, and iodothyronine 5'‐deiodinase activity were each detected in subcutaneous adipose tissue, as well as in the major internal fat depot (perirenal), of newborn lambs. These brown fat markers were not present, however, in adipose tissue of adult sheep. It is concluded that subcutaneous fat in newborn lambs is functionally ‘brown’, and similar to the internal fat; subcutaneous and internal adipose tissues follow a similar developmental path ‐ from ‘brown’ to ‘white’.
Procathepsin L‐specific antibodies that recognize procathepsin L but not cathepsin L
Authors: Ishidoh Kazumi , Takeda-Ezaki Mitsue , Kominami Eiki
Published: July 2002

Procathepsin L was purified to apparent homogeneity from the culture medium of v‐Ha‐ras transformed NIH3T3 (Ras‐NIH) cells in three steps; anion‐exchange chromatography, gel nitration, and re‐gel filtration. SDS‐PAGE analyses revealed that the purified samples contained only the precursor form, procathepsin L, but not the mature enzyme, cathepsin L. Antibodies against purified procathepsin L were raised. These recognized both rat cathepsin L and the purified procathepsin L. To isolate procathepsin L‐specific antibodies that did not recognize cathepsin L, sequential affinity chromatography procedures were carried out. Immunoblot analyses showed that the procathepsin L‐specific antibodies recognized only procathepsin L, but not cathepsin L.
The role of protein tyrosine phosphatases in density‐dependent growth control of normal rat kidney cells
Authors: Rijksen Gert , Völler Maureen C.W. , van Zoelen Everardus J.J.
Published: July 2002

In normal rat kidney cells protein tyrosine phosphatases (PTPases) play a role in attaining density‐dependent growth arrest after stimulation with mitogens. The PTPase inhibitor sodium orthovanadate prevents density‐dependent growth inhibition of EGF‐treated cells and mimicks in that respect the action of TGFβ and retinoic acid. However, enhanced PTPase activity is not obligatory for maintaining cells in a density‐arrested state. In contrast to TGFβ and retinoic acid, vanadate is unable to restimulate density‐inhibited cells, indicating that different mechanisms are operating. Yet, vanadate is strongly potentiating the effect of low concentrations of TGFβ but not of retinoic acid, implicating that tyrosine phosphorylation is linked to TGFβ action and that PTPase may represent a negative control element in the TGFβ signaling pathway.
Inhibition of protein phosphatases activates glucose‐6‐phosphatase in isolated rat hepatocytes
Authors: Claeyssens S. , Chedeville A. , Lavoinne A.
Published: July 2002

Incubation of hepatocytes in the presence of microcystin‐LR, okadaic acid, calyculin A (inhibitors of protein phosphatases PP1 and PP2A) or microcystin‐RR (a specific inhibitor of PP2A) activated glucose‐6‐phosphatase both in the supernatant and in intact or disrupted microsomes. Puromycin, an inhibitor of protein synthesis, totally suppressed this activating effect, suggesting the involvement of protein phosphatases in the regulation of glucose‐6‐phosphatase synthesis.
The DNA‐binding properties of an artificial 42‐residue polypeptide derived from a natural repressor
Authors: Hehlgans Thomas , Stolz Monica , Klauser Stephan , Cui Taian , Salgam Prathima , Verca Stefano Brenz , Widmann Margit , Leiser Andreas , Städler Kurt , Gutte Bernd
Published: July 2002

Bacteriophage 434 repressor recognizes the operator sequences ACAAG and ACAAT. As the same or similar sequences occur in the enhancer region of HIV‐1, 434 repressor was a potential HIV enhancer‐binding protein. We found that the interaction of the DNA‐binding domain of 434 repressor with a 57‐bp HIV enhancer DNA was very weak whereas a 42‐residue construct, comprising the recognition helix and four copies of a positively charged segment of the repressor, bound strongly. The results of footprint and cell‐free in vitro transcription studies showed that the 42‐residue peptide bound preferably to the enhancer region of HIV‐1 and acted as an artificial repressor. Replacement of an essential glutamine of the recognition helix by glutamic acid resulted in a partial shift of the sequence specificity of the 42‐residue peptide.