bluuebunny/crossref_metadata_2025_split huggingface dataset:
Induction of translational thermotolerance in liver of thermally stressed rats
Authors: DE MAIO Antonio , BECK Stephen C. , BUCHMAN Timothy G. 
Published: March 2005

Heat‐shock gene expression in cultures of single cell types has been well characterized but little is known about the heat‐shock response of intact organs in vivo. In this study, the kinetics of hepatic heat‐shock gene expression and the induction of thermotolerance were characterized in rats. Animals were subjected to a defined, reversible stress by increasing the core body temperature to 41°C or 42°C for 30 min. New synthesis of the inducible form of the heat shock‐70 family of proteins (hsp‐72) peaked simultaneously with the maximal level of hsp‐72 transcripts at both temperatures. These data are consistent with previous observations in cultures of hepatoblastoma cells after thermal stress [De Maio, A., Beck, S. C. & Buchman, T. G. (1993) Circ. Shock 40, 177–186].The incorporation of radioactive amino acids into polypeptides by the liver was blocked during the first hour of recovery after heat shock at 42°C. This inhibition of protein synthesis by thermal stress could be prevented by prestressing rats at 42°C for 30 min and allowing the rats to recover for 24 h at normal body temperature (37°C). This phenomenon, previously defined as ‘translational thermotolerance’, correlates with the hepatic content of hsp‐72; maximal protection occurs 24 h after a 42°C thermal stress when hsp‐72 (protein) is also maximum and decreases with the clearance of hsp‐72 from the liver. These data suggest that the presence of hsp‐72 within the liver may modulate the organ response to subsequent stresses and may be important to organ and animal survival after repeated insults.
Interferon‐γ up‐regulates a unique set of proteins in human keratinocytes
Authors: HONORÉ Bent , LEFFERS Henrik , MADSEN Peder , CELIS Julio E. 
Published: March 2005

Treatment of proliferating and quiescent primary human keratinocytes with interferon‐γ (IFN‐γ) (100 U/ml, 23.5 h) followed by two‐dimensional gel analysis revealed three proteins, IGUP I‐3421 (Mr= 48 200, pI= 6.06); IGUP I‐3524 (Mr= 56 900, pI= 5.92), a protein homologous to peptidechain‐release factor and tryptophanyl‐tRNA syntetase; and IGUP I‐5111 (Mr= 30400, pI= 5.76) recorded in the keratinocyte protein database (Celis et al., 1991, 1992) that are highly and specifically up‐regulated by IFN‐γ among several agents tested including 14 other cytokines, second messengers [dibutyryl cAMP (Bt2cAMP), dibutyryl cGMP (Bt2cGMP)] and compounds known to affect keratinocytes [4β‐phorbol 12‐myristate 13‐acetate (PMA), retinoic acid, Ca2+, dexamethasone, lipopolysaccharides, foetal calf serum]. Protein IGUP I‐5111 was selected for further studies as its level is affected by simian‐virus‐40 transformation and because peptide sequences were available in the microsequence database. The cDNA was cloned from a fibroblast cDNA library using degenerate oligodeoxyribonucleotides and expressed in AMA cells using the vaccinia virus expression system. Database searches indicated that the predicted protein, which migrated with the AMA variant of keratinocyte protein IEF SSP 5111, is novel although it exhibits weak similarity to cytoskeletal proteins. IGUP I‐5111 contains the RGD sequence found in many extracellular glycoprotein ligands of the integrin receptor family and it is found at least partially in the culture supernatant.Considering the presence of IFN‐γ in psoriatic plaques as well as its putative involvement in the pathophysiology of the disease it was of interest to determine whether the set of proteins was up‐regulated in these cells. Two‐dimensional gel analysis of the protein phenotype of non‐cultured, unfractionated psoriatic keratinocytes failed to reveal up‐regulation of any of the three IFN‐γ‐induced proteins suggesting that the effect of IFN‐γin vivo may be modulated by the activity of other cytokine(s) or growth factor(s). Psoriatic keratinocytes were equally sensitive to IFN‐γ as their normal counterparts.
Photoaffinity labeling of the human red‐blood‐cell urea‐transporter polypeptide components
Authors: NEAU Philippe , DEGEILH Françoise , LAMOTTE Hervé , ROUSSEAU Bernard , RIPOCHE Pierre 
Published: March 2005

The tritiated urea analogue 1‐(3‐azido‐4‐chlorophenyl)‐3methyl‐2‐thiourea ([3H]MeACPTU) was used as a probe to photolabel the human red‐blood‐cell membrane facilitated urea transporter. On irradiation, [3H]MeACPTU incorporated irreversibly into white ghost membranes. SDS/gel electrophoresis of membranes revealed radioactive incorporation in five major bands of 200, 110, 60, 40 and 14 kDa. The labeling of the 40‐kDa and 60‐kDa bands was partly prevented by the presence of a high concentration of other urea analogues such as thiourea and 1‐(3,4‐dichlorophenyl) 2‐thiourea (DCPTU). The photolabeling pattern obtained with white ghosts of the Kidd blood‐group type Jk(a−,b−) showed no labeling of the 40‐kDa polypeptide. Protecting experiments carried out with anti‐Jka, anti‐Jkb and anti‐Jk3 sera prevented radioactive incorporation in the 60‐kDa band and in the 110‐kDa band. Urea permeability of pink ghosts of blood type Jk(a+,b+) measured in the presence of Jk3 antibodies was 19% lower than the control values. However, urea permeability of frog urinary bladder epithelial cells was not affected by the presence of Jk‐reactive antibodies.These results support the hypothesis that the Kidd antigen and the facilitated urea transporter are the same protein. Our estimation of the number of copies in each cell is close to that of the previously published value of 14000.
Synergistic binding of hydrophobic probes and zinc ligands to thermolysin
Authors: PFUETZNER Richard A. , CHAN William W.‐C. 
Published: March 2005

The strong synergism previously observed in the binding of inhibitors to two Zn‐proteases, has also been found for thermolysin. As in earlier cases, the effects are produced by a small Zn‐ligand (e.g. a hydroxamate) in the presence of another compound which contains the key structural features of specific substrates (a specificity probe). For thermolysin, the most effective specificity probes are hydrophobic derivatives of amines and amino acids (e.g. carbobenzyloxy‐L‐alaninol). Even the simple combination of benzyl alcohol and formohydroxamate displays considerable synergism. The above effects are temperature dependent and correlate well with a thermally induced conformational isomerization reported recently for this enzyme.Our results seem to be related to previous observations of substrate synergism in the reverse reaction and to superactivation by chemical modification of this enzyme. All these effects are consistent with a change in the environment of the catalytically important zinc atom upon binding of the hydrophobic side chain of the substrate. With the inclusion of thermolysin, binding synergism is now known to occur in an endopeptidase as well as in exopeptidases of diverse specificity. The general occurrence of this phenomenon in zinc proteases and its possible significance are discussed in an accompanying study.
Cloning and characterization of the poly(hydroxyalkanoic acid)‐depolymerase gene locus, phaZ1, of Pseudomonas lemoignei and its gene product
Authors: JENDROSSEK Dieter , MÜLLER Beate , SCHLEGEL Hans Günter 
Published: March 2005

Four different DNA fragments each coding for poly(hydroxyalkanoic acid) depolymerase (phaZ1–phaZ4) were isolated in pUC plasmids from a genomic library of Pseudomonas lemoignei in Escherichia coli. All recombinant strains secreted a highly active poly(3‐hydroxybutyric acid) depolymerase and produced large translucent halos on an opaque medium containing poly(3‐hydroxybutyric acid) granules. One DNA region (phaZ1) was present in seven independently isolated clones. Three other cloned DNA fragments were different from phaZ1 and from each other (phaZ2–phaZ4). In phaZ1, an open‐reading frame of 1245 bp was identified from the nucleotide sequence of a 5435‐bp MboI fragment (57 mol G+C/100 mol) of this region and encoded a novel poly(hydroxyalkanoic acid) depolymerase of P. lemoignei, poly(3‐hydroxybutyric acid) depolymerase C. A leader‐sequence peptidase‐cleavage site was predicted from the deduced amino acid sequence between Ala37 and Leu38. The calculated relative molecular masses of the precursor and the putative mature protein were 43468 and 39581, respectively. The polypeptide contains a lipase consensus sequence (Gly‐Xaa‐Ser‐Xaa‐Gly) and an unusually high proportion of threonine residues (22 of 36 amino acids) near the C‐terminus. The N‐terminus of the deduced amino acid sequence of PhaZ1 differed from that of the purified poly(3‐hydroxybutyric acid) depolymerases A, B and the poly(3‐hydroxyvaleric acid) depolymerase of P. lemoignei.The phaZ1 gene product, poly(3‐hydroxybutyric acid) depolymerase C, was partially purified from recombinant E. coli (pUC91 ::phaZ1). The purified protein was specific for poly(hydroxyalkanoic acid) consisting of monomers of four or five carbon atoms and for p‐nithrophenylbutyrate as substrates. The polymer‐hydrolyzing activity, but not the p‐nitrophenylate esterase activity, was inhibited by complex media such as Luria‐Bertani medium and by soluble E. coli proteins. The enzyme protein did not cross‐react with antibodies raised against purified poly(3‐hydroxyvaleric acid) depolymerase of P. lemoignei.
Evidence that the reaction of the UDP‐N‐acetylglucosamine 1‐carboxyvinyltransferase proceeds through the O‐phosphothioketal of pyruvic acid bound to Cys115 of the enzyme
Authors: WANKE Christoph , AMRHEIN Nikolaus 
Published: March 2005

The enzyme UDP–N‐acetylglucosamine 1‐carboxyvinyltransferase (enolpyruvyltransferase, EC 2.5.1.7) catalyses the transfer of the intact 1‐carboxyvinyl moiety of phosphoenolpyruvate to the 3′‐hydroxyl group of the glucosamine moiety of UDP–(2′)‐N‐acetylglucosamine with the concomitant release of inorganic phosphate, the first committed step in the biosynthesis of the bacterial cell wall peptidoglycan. Overexpression of the enzyme from Enterobacter cloacae in Escherichia coli allowed the isolation of large amounts of purified enzyme (approx. 900 mg/20 g fresh mass bacteria). By incubating the enzyme with 14C‐labelled phosphoenolpyruvate, 32P‐labelled orthophosphate and unlabelled UDP–(2′)‐N‐acetyl‐(3′)‐1‐carboxyvinylglucosamine, we were able to isolate and characterise a reaction intermediate, covalently bound to the protein. It contains stoichiometric quantities of the C3 moiety (0.98 mol/mol) as well as of the phosphate moiety (0.95 mol/mol) of phosphoenolpyruvate relative to protein. The rapid turnover of this protein‐bound intermediate in the presence of UDP–(2′)‐N‐acetylglucosamine towards the product UDP–(2′)‐N‐acetyl‐(3′)‐1‐carboxyvinylglucosamine suggests that the intermediate is kinetically competent. We also present evidence that the intermediate is bound as the O‐phosphothioketal of pyruvic acid to Cys115 of the enzyme. This is the same Cys residue to which phosphomycin, an irreversible inhibitor of the UDP–GlcNAc carboxyvinyltransferase, binds covalently. Exchange of Cys115 for a Ser residue resulted in an inactive enzyme, demonstrating the essential role of Cys115 for the reaction.The only other enzyme known to catalyse the transfer of the intact 1‐carboxyvinyl moiety of phosphoenolpyruvate to a substrate is the 3‐phosphoshikimate 1‐carboxyvinyltransferase (EC 2.5.1.19), the sixth enzyme of the shikimate pathway. The reaction of this synthase is known to proceed through a single, tightly but not covalently bound, tetrahedral intermediate. Even though the two enzymes share similarities in their primary amino acid sequences, their reaction mechanisms appear to be substatially different.
Flavin dynamics in oxidized Clostridium beijerinckii flavodoxin as assessed by time‐resolved polarized fluorescence
Authors: LEENDERS Rik , VAN HOEK Arie , VAN IERSEL Martijn , VEEGER Cees , VISSER Antonie J. W. G. 
Published: March 2005

The time‐resolved fluorescence characteristics of flavin in oxidized flavodoxin isolated from the anaerobic bacterium Clostridium beijerinckii have been examined. The fluorescence intensity decays were analyzed using the maximum‐entropy method. It is demonstrated that there exist large differences in fluorescence behaviour between free and protein‐bound FMN. Three fluorescence lifetime components are found in oxidized flavodoxin, two of which are not present in the fluorescence‐intensity decay of free FMN. The main component is distributed at 30 ps, with relative contribution of 90%. Another minor component (4% contribution) is distributed at 0.5 ns. The third component is distributed at 4.8 ns (6%), coinciding with the main distribution present in the fluoresence decay of free FMN. The results allowed us to determine the dissociation constant, Kd= 2.61×10−10 M (at 20°C).Conllisional fluorescence‐quenching experiments revealed that the flavin moiety responsible for the longest fluorescence lifetime is, at least partially, exposed to the solvent. The shortest lifetime is not affected significantly, indicating that it possibly originates from an active‐site conformation in which the flavin is more or less buried in the protein and not accessible to iodide.The fluorescence anisotropy behaviour of free and protein‐bound FMN was examined and analyzed with the maximum‐entropy method. It was found that an excess of apoflavodoxin is required to detect differences between free and protein‐bound FMN. In free FMN one single distribution of rotational correlation times is detected, whereas in flavodoxin the anisotropy decay is composed of more than one distribution.Associative analysis of fluorescence anisotropy decays shows that part of the 4.8 ns fluorescence lifetime present in the flavodoxin fluorescence decay, is coupled to a rotational correlation time similar to that of free FMN in solution, while another part of this lifetime is coupled to a longer correlation time of about 1 ns. This finding is in accordance with earlier studies [Barman, B. G. & Tollin, G. (1972) Biochemistry 11, 4746–4754] in which it was proposed that the first binding step of the flavin to the protein involves the phosphate group rather than another part of the FMN.The two shortest fluorescence lifetimes, which do not carry information on the long‐term rotational behaviour of the protein, seem nonetheless to be associated with a longer rotational correlation time which is comparable to overall protein tumbling. These lifetime components probably originate from a complex in which the flavin‐ring system is more or less immobilized within the protein matrix.
Characterization of prostaglandin‐F 2α‐binding sites on rat hepatocyte plasma membranes
Authors: NEUSCHÄFER‐RUBE Frank , PÜSCHEL Gerhard P. , JUNGERMANN Kurt 
Published: March 2005

Prostaglandin (PG) F 2α has previously been shown to increase glucose output from perfused livers and isolated hepatocytes, where it stimulated glycogen phosphorylase via an inositol‐trisphosphate‐dependent signal pathway. In this study, PGF 2α binding sites on hepatocyte plasma membranes, that might represent the putative receptor, were characterized.Binding studies could not be performed with intact hepatocytes, because PGF 2α accumulated within the cells even at 47deg;C. The intracellular accumulation was an order of magnitude higher than binding to plasma membranes.Purified hepatocyte plasma membranes had a high‐affinity/low‐capacity and a low‐affinity/high‐capacity binding site for PGF 2α. The respective binding constants for the high‐affinity site were Kd= 3 nM and Bmax= 6 fmol/mg membrane protein, and for the low‐affinity site Kd= 426 nM and Bmax= 245 fmol/mg membrane protein.Specific PGF2α binding to the low‐affinity site, but not to the high‐affinity site, could be enhanced most potently by GTP[γS] followed by GDP[βS] and GTP, but not by ATP[γS] or GMP.PGF2α competed most potently with [3H]PGF 2α for specific binding to hepatocyte plasma membrances, followed by PGD2 and PGE2.Since the low‐affinity PGF2α‐binding site had a Kd in the concentration range in which PG had previously been shown to be half‐maximally active, and since this binding site showed a sensitivity to GTP, it is concluded that it might represent the receptor involved in the PGF2α signal chain in hepatocytes. A biological function of the high‐affinity site is currently not known.
Specificity of T‐cell protein tyrosine phosphatase toward phosphorylated synthetic peptides
Authors: RUZZENE Maria , DONELLA‐DEANA Arianna , MARIN Oriano , PERICH John W. , RUZZA Paolo , BORIN Gianfranco , CALDERAN Andrea , PINNA Lorenzo A. 
Published: March 2005

The local specificity determinants for a T‐cell protein tyrosine phosphatase (TC‐PTP) have been inspected with the aid of a series of synthetic peptides, either enzymically or chemically phosphorylated, derived from the phosphoacceptor sites of phosphotyrosyl proteins. The truncated form of T‐cell PTP, deprived of its C‐terminal down‐regulatory domain, readily dephosphorylates submicromolar concentrations of eptapeptides to eicosapeptides, reproducing the C‐terminal down‐regulatory site of pp60c‐src (Tyr527), the phosphorylated loop IV of calmodulin and the main autophosphorylation site of two protein tyrosine kinases of the src family (Tyr416 of pp60c‐src and Tyr412 of p51fgr). However, phosphopeptides of similar size, derived from phosphoacceptor tyrosyl sites of the abl and epidermal‐growth‐factor receptor protein tyrosine kinases, the phosphorylated loop III of calmodulin, and phosphoangiotensin II undergo either very slow or undetectable dephosphorylation, even if tested up to 1 μM concentration.The replacement of either Ser‐P or O‐methylated phosphotyrosine for phosphotyrosine within suitable peptide substrates gives rise to totally inert derivatives. Moreover, amino acid substitutions around phosphotyrosine in the peptides src‐(412–418), src‐(414–418) and abl‐(390–397) deeply influence the dephosphorylation efficiency. From these data and from a comparative analysis of efficient versus poor phosphopeptide substrates, it can be concluded that acidic residues located on the N‐terminal side of phosphotyrosine, with special reference to position –3, play a crucial role in substrate recognition, while basic residues in the same positions act as negative determinants. In any event, the presence of at least two aminoacyl residues upstream of phosphotyrosine represents a necessary, albeit not sufficient, condition for detectable dephosphorylation to occur.By replacing the truncated form of TC‐PTP with the full length TC‐PTP, the dephosphorylation efficiencies of all peptides tested are dramatically impaired. Such an effect is invariably accounted for by a substantial increase in Km values, accompanied by a more or less pronounced decrease in Vmax values. These data support the concept that the C‐terminal regulatory domain of TC‐PTP exerts its function primarily by altering the affinity of the enzyme toward its phosphotyrosyl targets.
A New Structural Transition of Serum Albumin Dependent on the State of Cys34
Authors: Christodoulou John , Sadler Peter J. , Tucker Alan 
Published: March 2005

Reactions of fatty‐acid‐free bovine serum albumin and recombinant human albumin with a range of antiarthritic gold(I) complexes [auranofin, deacetylated auranofin, triethylphosphinegold(I) chloride] and related thiols (thioglucose, tetraacetylthioglucose, glutathione, dithiothreitol) have been investigated using 1H‐NMR spectroscopy.
In reactions of albumin with auranofin, tetraacetylthioglucose and dithiothreitol, release of cystine was detected, whereas for deacetylated auranofin, thioglucose and glutathione, mixed disulphides with cysteine were produced. It has been previously proposed that Cys34 of human and bovine serum albumins is partly blocked by disulphide formation with cysteine and glutathione. The above reactions lead to deblocking by thiol–disulphide interchange reactions. No release of glutathione from albumin was detected.
Changes in the His Hɛ1 regions of the 1H‐NMR spectra show that albumin exists in two structural forms dependent on whether the side‐chain of Cys34 is a free thiolate, or blocked by gold(I)triethylphosphine, by disulphide formation with cysteine or by another form of oxidation. We propose that Cys34 is either in a buried or in an exposed environment; the possible molecular basis of the structural change is discussed.
The relationship between reactions at Cys34, cysteine release, and the observed structural transition are discussed in terms of chrysotherapy, albumin metabolism and the use of gold(I) as a heavy atom derivative in X‐ray crystallographic studies of albumins.
cDNA sequence and structure of a gene encoding trout testis high‐mobility‐group‐1 protein
Authors: Štros Michal , Nishikawa Sandra , Dixon Gordon H. 
Published: March 2005

Perchloric acid extraction of trout testis nuclei revealed the presence of two large high‐mobility‐group (HMG) proteins, HMG‐T1 and HMG‐T2. The sequence of a complete cDNA (1407 bp) for trout testis HMG‐1 protein (referred as to HMG‐T1) has been determined. The deduced HMG‐T1 protein contains 203 amino acids with more than 86% similarity to mammalian HMG‐1 proteins. A single‐sized mRNA for HMG‐T1 has been detected by Northern‐blot analysis consistent with the size derived from the HMG‐T1 cDNA. Amplification of human and trout genomic DNAs by polymerase chain reaction using primers specific for trout and human HMG‐1 cDNAs revealed that unlike the human genome, which contains predominantly intronless HMG‐1 sequences, intronless HMG‐T1 sequences were not found in the fish genome. Southern‐blot analysis suggested that the trout testis HMG‐1 gene is encoded by at least two sequences with high similarity.A gene encoding HMG‐T1 protein has been isolated from a trout testis genomic library and by PCR of trout genomic DNA (3879 bp); The trout testis HMG‐1 gene is organized into five exons (four exons corresponding to the protein‐coding region) and its exon/intron boundaries are identical to those of the human HMG‐2 gene [Shirakawa, H. & Yoshida, M. (1992). J. Biol. Chem. 267, 6641–6645] suggesting the evolution of HMG‐1 and HMG‐2 genes from a common ancestor.
Tyr394 and Tyr505 are Autophosphorylated in Recombinant Lck Protein‐tyrosine Kinase Expressed in Escherichia coli
Authors: Jullien Pascale , Bougeret Cécile , Camoin Luc , Bodeus Monique , Durand Hervé , Disanto James P. , Fischer Siegmund , Benarous Richard 
Published: March 2005

The activity of the Src family protein‐tyrosine kinase P56lck is regulated by phosphorylation and dephosphorylation of two critical tyrosine residues Tyr394 and Tyr505. Tyr394 is autophosphorylated after p56lck activation, whereas phosphorylation of Tyr505 is believed to be due to p50csk which negatively modulates P56lck activity.To determine whether Tyr505 could be autophosphorylated, we used the prokaryotic glutathione S‐transferase expression system to express wild‐type Lck, the mutants [Y394F]Lck and [Y505F]Lck, a kinase‐deficient P56lck with a mutation of the ATP‐binding site [K273E]Lck and a double mutant [Y394F, Y505F]Lck. We studied the kinase activities and the patterns of autophosphorylation for tyrosine residues in these mutants and wild‐type Lck both in vivo and in vitro.Wild‐type Lck, [Y505F]Lck and [Y394F]Lck were phosphorylated on tyrosine. Both the kinase‐deficient mutant[K273E]Lck and the double mutant [Y394F, Y505F]Lck did not react with monoclonal anti‐phosphotyrosine antibody [anti‐Y(P) mAb], thus providing evidence that (a) the bacterial strains used lacked intrinsic protein‐tyrosine kinase activities, and therefore tyrosine phosphorylations of wild‐type Lck, [Y505F]Lck and [Y394F]Lck are due to autophosphorylation occurring in vivo in bacteria, and (b) that p56lck can only be autophosphorylated on two tyrosine residues, namely Tyr394 and Tyr505. Phosphopeptide mapping analysis confirmed that P56lck can undergo autophosphorylation on these two tyrosine residues. We propose that autophosphorylation at Tyr505 of P56lck may represent an accessory mechanism for the down‐regulation of the tyrosine kinase activity of P56lck.