bluuebunny/crossref_metadata_2025_split huggingface dataset:
Photoinactivation studies on adenosine deaminase
Authors: Lupidi G. , Colao C. , Marmocchi F. , Cristalli G. 
Published: July 2007

AbstractReinvestigation of direct modification of histidine residues in calf intestine adenosin deaminase resulted in loss of the activity towards substrates as adenosine, 2′‐deoxyadenosine and 6‐hydroxyl‐aminopurin riboside. Photo‐oxidation of the enzyme in the presence of methylene blue led to a complete loss of enzymatic activity and the presence of inhibitors such as purin riboside, EHNA, coformycin, showed protection against methylene blue oxidation. Kinetic analysis of the inactivation by diethylpyrocarbonate, indicated that enzyme inactivation results from the modification of at most one essential histidine residue. Photoinactivation of adenosine deaminase from Aspergillus Orizae reduces the activity of the enzyme but the accessibility of histidine residues in the active site seems to be lower as compared to that shown from mammalian adenosine deaminase. The results obtained are in agreement with the important role played by the “bridge” between an enzyme histidine residue and the 5′‐OH of the ribose mojety of substrate in the transition state stabilization in the two enzymatic forms observed. Upon ultraviolet irradiation of calf intestine adenosine deaminase in the presence of 2,2,2‐thrichloroethanol, the trasformation of fluorescent tryptophan residues occurs with the reduced enzymatic catalytical activity and confirms the likely location of tryptophans near the binding site of the enzyme.
Variation of phosphoribosylpyrophosphate synthetase activity in human cells
Authors: Okada Naomi Nagatake , Ishizuka Toshiharu , Isogai Eriko , Suzuki Nobuo , Tatibana Masamiti 
Published: July 2007

AbstractThe behavior of phosphoribosylpyrophosphate (PRPP) synthetase during the cell cycle was studied using RSa cells derived from human fetal fibroblasts. The cells were synchronized by the double thymidine block method. PRPP synthetase activity in cell extracts increased 2‐fold in the S phase. Metabolic flux through phosphoribosylpyrophosphate into nucleotides, which represents the in situ activity of PRPP synthetase, increased 3‐fold in the S phase. The mRNA levels of PRPP synthetase subunit I and II genes (PRPS1 and PRPS2 respectively) increased 3‐fold and the increase preceded elevation of enzyme activity in the S phase. Thus variation in PRPP synthetase activity during the cell cycle is ascribed mainly to varying amounts of the enzyme. Degradation of PRPS1 and PRPS2 mRNA was significantly suppressed in the early S phase and was presumably related to periodic accumulation of the two mRNAs. In earlier work, it was found that the expression of PRPP synthetase isoform genes PRPS1 and PRPS2 is regulated in a tissue specific manner, thereby suggesting a functional difference between them. We have now obtained evidence that there is no apparent difference between the expression of PRPS1 and PRPS2 genes in the cell cycle.
Amino acid residue 250 has a functional role in the assembly of rabbit liver microsomal cytochrome P450 2B4
Authors: Schulze Johannes , Lehnerer Michael , Lewis David F. V. , Hlavica Peter 
Published: July 2007

AbstractCytochrome P450 2B4 lacking amino acids 2‐27, CYP2B4 (Δ2‐27), was mutated at position 250 and expressed in E.coli fused toglutathione S‐transferase. Expression of the E250S variant (holo‐plus apoenzyme) proceeded to an extent comparable with that of CYP2B4 (Δ2‐27), while the protein level of the E250P mutant averaged 42% that of the control pigment. Comparison of these data with the corresponding reduced CO difference spectra of the various CYP2B4 (Δ2‐27) forms revealed that, in the control and E250S preparations, about 90% and 44%, respectively, of the total amount of hemoprotein present existed in the form of holoenzyme, whereas the E250P derivative failed to produce a reduced carbonyl complex. Thus, replacement of the negatively charged E250 with an uncharged, polar serine residue substantially hampered assembly of CYP2B4 (Δ2‐27); introduction of an α‐helix‐disrupting proline completely blocked the formation of holoenzyme. These phenomena suggested that the negative charge of E250, residing in the putative G helix, underwent pairing with some positively charged group, possibly H285 located in the I helix. Deletion of the negative charge obviously perturbed the active‐site geometry such as to affect both the incorporation and/or retention of the heme ligand and the spectral binding of substrates such as hexobarbital.
Effect of Vitamin E on antioxidant enzymes and nitric oxide in ischemia‐reperfused kidney injury
Authors: Uysal Fevziye , Girgin Ferhan K. , Tüzün Sevgi , Aldemir Sevil , Sözmen Eser Yildirim 
Published: July 2007

AbstractThe effects of Vitamin E administration on antioxidant enzyme activities and nitrite‐nitrate levels of the reperfused rat kidney tissues were investigated by performing a 60 min ischemia followed by 24 and 72 hours of reperfusion. Vitamin E administration or the placebo (SF) was applied as 100 mg/kg BW. As expected, catalase (CAT) (p<0.05) and superoxide dismutase (SOD) (p<0.05) activities of ischemia/reperfused (I/R) kidney tissue were lower and malondialdehyde (MDA) levels were higher than control kidneys in both SF and vitamin E treated groups following 24 h reperfusion. During reperfusion of long term (72 h), vitamin E triggered a decrease in the MDA levels in the ischemic tissue, while it did not provoke a significant effect on SOD and catalase activities. Total nitrite levels of ischemic tissues in both of the groups were higher than matched control kidneys and this elevation was more clear in the vitamin E treated group. Our results showed that vitamin E has a protective effect on I/R injury, by a direct chain breaking effect on lipid peroxidation (LPO) and hence preventing the nitric oxide (NO) reservoir of ischemic tissue. Alfa‐tocopherol may be a promising agent for the prevention of tissue injury caused by free oxygen radicals.
Soluble guanylate cyclase in Saccharomyces cerevisiae
Authors: Kuo W. N. , Kanadia R. N. , Mcnabb M. 
Published: July 2007

AbstractAfter removing nonspecific immunoreactivities from crude extract by immunoaffinity chromatography, an immunoreactive‐band at 40kDa of soluble guanylate cyclase (SGC) from Saccharomyces cerevisiae was detected by Western blot using rabbit anti‐β1 subunit of SGC. Cyclic GMP level and SGC activity was measured by ELISA. Immunoprecipitated yeast SGC was activated by sodium nitroprusside, whereas inhibited by 1H‐(1,2,4) oxadiazolo(4,3‐A)quinoxalin‐1‐one. Increased cyclic GMP level was also noted when intact yeast cells were incubated with s‐nitrosoglutathione, a NO donor. The result implies that NO can be utilized intracellularly and extracellularly. Moreover, the presence of SGC suggests the significance of NO/cyclic GMP signaling in unicellular eukaryotes.
Luminol luminescence induced by oxidants in antioxidant‐deficient yeasts Saccharomyces cerevisiae
Authors: Jakubowski Witold , Bartosz Grzegorz , Ertel Danuta , Bilinski Tomasz , Kędziora Józef 
Published: July 2007

AbstractLuminal chemiluminescence induced in the presence of yeast cells and yeast cell homogenates was significantly induced by exogenous oxidants (hydrogen peroxide and menadione), tert‐Butyl hydroperoxide did not stimulate chemiluminescence by itself but augmented menadione‐induced chemiluminescence. Comparison of yeast strains deficient in catalase, superoxide dismutase or glutathione showed that only glutathione‐deficient strains showed elevated chemiluminescence in this system. These results support the idea that more reactive species than hydrogen peroxide and superoxide are critical in the induction of luminal chemiluminescence.
Hydroxyl group of insulin A19Tyr is essential for receptor binding: Studies on (A19Phe) insulin
Authors: Du Xin , Tang Jian‐Guo 
Published: July 2007

AbstractOne mutant proinsulin gene was constructed through PCR with the code of A19Tyr changed into that of Phe. The mutant proinsulin, (A19Phe)‐lysproinsulin (F19KPI) was expressed in E. coli and purified. After trypsin and carboxypeptidase B cleavage and ResourceTM Q separation, (A19Phe)‐human insulin (F19HI) was obtained. With native insulin as standard, activity assay and structural analysis were carried out. It was found that the conformation of F19HI is very similar to that of native insulin according to the data from native PAGE, reverse‐phase FPLC and circular dichroism spectrum, which are also in agreement with the result of radioimmune assay. F19HI retains almost full immune activity, but displays only 13.3% of receptor binding activity. These results suggest that the hydroxyl group of insulin A19Tyr is essential for receptor binding.
Purification of recombinant human pepsinogens and their application as immunoassay standards
Authors: Aoki Takashi , Tomaki Emiko , Satoh Miyoko , Takashiro Miho , Onagi Hitoshi , Itoh Masao , Teramoto Tetsuya , Morikawa Junji , Watabe Hiroyuki 
Published: July 2007

AbstractHuman pepsinogen (PG) A and C were cloned in Escherichia coli, but the levels of expression were low and unstable. When these were fused to maltose‐binding protein (MBP), the fusion proteins (MBP‐PGA and MBP‐PGC) were expressed as the major products. Although these fused products were almost totally recovered from the insoluble fraction, the renaturation and purification procedures were easy and simple. MBP‐PGA and the PGA segment obtained by factor Xa digestion (designated as r‐PGA) possessed proteolytic activities equivalent to native PGA purified from gastric tissue (t‐PGA). For PGCs (MBP‐PGC, r‐PGC and t‐PGC) also, the specific activities were almost the same. However, the activities of PGCs were about 3‐ to 4‐hold higher than those of PGAs. In PGA and PGC immunoassay systems, r‐PGs (r‐PGA and r‐PGC) and the EIA kit standard PGs (gastric mucosal PGs) exhibited a good correlation. From these results, r‐PGs would seem to be applicable as assay standards without compromising the sensitivity of the immunoassay systems.
The effects of N‐thiophosphoryl amino acids on the activity of green crab (Scylla Serrata) alkaline phosphatase
Authors: Chen Qing‐Xi , Zhu Chun‐Ming , Zhou Hai‐Meng , Lu Hai‐Yan , Lin Hai‐Ning 
Published: July 2007

AbstractGreen crab (Scylla Serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme which catalyzed the nonspecific hydrolysis of phosphate monoesters. In the present paper, the effects of several N‐thiophosphoryl amino acids on the activity of green crab alkaline phosphatase have been studied. The results show that these derivatives of amino acids can lead to reversible inactivation. The equilibrium constants for inhibitors binding with the enzyme and/or the enzyme‐substrate complexes have been determined. The obtained results show that both N‐thiophosphoryl‐Cys and N‐thiophosphoryl‐Glu were non‐competitive inhibitors, while other five N‐thiophosphoryl amino acids were un‐competitive inhibitors. For the un‐competitive inhibitors, the inhibition strength follows the order N‐thiophosphoryl‐Ile>‐Val>‐Lys>‐Ala>‐Tyr. Compared with respective free amino acids, it can be seen that N‐thiophosphorylation of the amino acids increased their inhibition strength except the N‐thiophosphoryl‐Cys.
Effect of prooxidants on mitochondrail permeability transition and cell death in Ehrlich ascites tumour cells
Authors: Teplova Vera V. , Kudrjavtsev Andrew A. , Odinokova Irina V. , Evtodienko Yuri V. , Saris Nils‐Erik L. 
Published: July 2007

AbstractCa2+ retention in mitochondria, opening of the Cysclosporin A‐ sensitive permeability transition pore and cell death were studied in Ehrlich ascites tumour cells in the presence of different prooxidants. Low concentrations (1‐20 μM) of the prooxidants (menadione, cumenehydroperoxide, t‐butylhydroperoxide) induced pore‐opening in permeabilized cells at threshold Ca2+ load. Incubation of cells with low concentrations of prooxidants was able to induce cell cycle disturbance and cell death. Under the prooxidant effect, mitochondrial membrane potential drop and Ca2+ retention decrease in mitochondria were found to precede death of Ehrlich ascites tumour cells.
Glycated protein‐iron chelate increases lipid peroxide level in cultured aortic endothelial and smooth muscle cells
Authors: Nishigaki Ikuo , Yagil Kunio , Tanimoto Akihide , Sasaguri Yasuyuki 
Published: July 2007

AbstractFormation of an iron chelate of glycated protein was demonstrated by the appearance of an absorption peak at approximately 270 nm after mixing glycated bovine serum albumin with FeCl3. This peak disappeared and a new peak appeared at approximately 420 nm to form an isosbestic point at approximately 340 nm by the addition of deferoxamine mesylate, an ironchelating agent, to the mixture, thus confirming the formation of the iron chelate of the glycated protein in the mixture. The lipid peroxide level was increased markedly in endothelial cells and slightly in smooth muscle cells from bovine aorta incubated in the medium containing glycated fetal bovine serum‐iron chelate. Morphological observation by phase‐contrast microscopy and scanning electron microscopy revealed that the glycated fetal bovine serumiron chelate caused intense damage to the endothelial cells. These results indicate that glycated protein‐iron chelate provokes lipid peroxidation, which explains at least in part the mechanism of atherogenesis found in diabetic patients.
Association of pp60cc‐src with αIIbβ3 in resting platelets
Authors: Kralisz Urszula , Cierniewski Czeslaw S. 
Published: July 2007

AbstractTo detect whether 125I‐αIIbβ3 is associated with tyrosine kinases in platelets, antibodies specific to pp60c‐src, pp54/58lyn, and pp62Fyn were used to precipitate their homologous antigens. In contrast to Lyn and Fyn kinases, pp60c‐src appears to be complexed with αIIbβ3. Both proteins, pp60c‐src and αIIbβ3, coprecipitated when antibodies to pp60c‐src were used in the immunoprecipitation experiments. This conclusion was further supported by immunoprecipitation of αIIbβ3 from Triton X‐100 extracts of nonlabelled platelets with P2 antibodies. There was no pp60c‐src detectable in immunoprecipitates obtained with antibodies specific to α2β1 or GPIb. Since PGE1 was used to prevent platelet activation in buffers throughout all procedures and there was no phosphorylation of pp72syk we assume that the platelets were in the resting state. Therefore, we conclude that αIIbβ3 and pp60c‐src can form a complex in resting platelets suggesting that pp60c‐src is directly involved in initiating the outside‐in signaling cascades in blood platelets.
A proposed estimate of the tumor aggressiveness of human breast cancer using radiorespirometry
Authors: Mayaudon Jacques , Yan Shou‐He , Wallon Joëlle 
Published: July 2007

AbstractThe relationships between carcinomatous aggressiveness and the glucolytic metabolism, namely the rate of 14CO2 production from [U‐14C] glucose, are obtained from human breast tissues using radiorespirometry. The values are estimated as the initial velocity (V) expressed in η14CO2×min‐1×g‐1 of fresh tissues by [U‐14C] glucose metabolism. The aggressiveness of the breast carcinomatous is diagnosed by the SBR grade system. As two control normal tissues, (V) are 0.86 to 0.90 from non‐cancer patients. In carcinomatous tissues (Vc), there is an increase from 1.53 to 3.14, but in the corresponding surrounding non‐cancer tissues (Vn) these show a decrease from 2.20 to 0.22 for SBR I, SNR II to SBR III. The ratio between (Vc) and (Vn) are found, according to carcinomatous aggressiveness, as 1.45 to 1.54, 1.69, 2.35 to 2.86 and 4.82 to 10.38 respectively for SBR I, lobular carcinoma, SBR II and SBR III; while the ratio is 1.04 for the normal tissue which come from non‐cancer patients. The above results suggest the possibility of assessing the carcinomatous aggressiveness by radiorespirometry before a histopathological diagnosis, even in a lower aggressiveness as in SBR I cases which are difficult to diagnose and manage.
Constitutive nitric oxide synthase in Saccharomyces cerevisiae
Authors: Kanadia R. N. , Kuo W. N. , Mcnabb M. , Botchway A. 
Published: July 2007

AbstractAfter removing nonspecific immunoreactivities from crude extract by immunoaffinity chromatography, an immunoreactive‐band at 60kDa of constitutive nitric oxide synthase (cNOS) from Saccharomyces cerevisiae was detected by Western blot using mouse monoclonal anti‐neuronal NOS (cNOS). The activity of yeast cNOS, which was prepared by either histone‐agarose chromatography or anti‐neuronal NOS immunoprecipitation, was monitored by the formation of citrulline. Yeast cNOS was activated in the presence of calmodulin and arginine, whereas it was inhibited by L‐NAME, a mammalian NOS inhibitor. Moreover, actinomycin‐D decreased the extracellular and the intracellular levels of nitrate and nitrite which had been converted from NO. The results suggest that cNOS occurs in unicellular eukaryotes and the enzyme activity can be regulated.
Glutathione metabolism and glutathione S‐conjugate export ATPase (MRP1/GS‐X pump) activity in cancer
Authors: de Bittencourt Paulo Homem , Curi Rui , Williams John F. 
Published: July 2007

AbstractMg2+‐dependent vanadate‐sensitive glutathione S‐conjugate ATPase (GS‐X pump) activity is a common feature of some ATP‐binding cassette (ABC) transporters, such as the multidrug resistance‐associated protein (MRP1) gene product, that exports biologically active electrophiles after their conjugation with intracellular glutathione (GSH) from normal and cancer cells. Antitumor electrophiles (e.g. naturally occurring cyclopentenone prostaglandins and anticancer chemicals) can be intracellularly conjugated with GSH via a glutathione S‐transferase catalyzed reaction and be eliminated through GS‐X pumps thus threatening cancer chemotherapeutics. Since different sensitivities to antitumor electrophiles are shown by different cell types, the ability of several human cancer cell lines to produce and export S‐(2,4‐dinitrophenyl)‐glutathione (DNP‐SG) conjugate through the GS‐X pump, using whole cells and inside‐out membrane vesicle preparations, is investigated. Different cancer cell lines exhibited characteristically different GS‐X pump activity. In particular, HEp‐2 larynx carcinoma cells possess an elevated DNP‐SG export rate through the GS‐X pump compared with HeLa, K562, U937 or HL‐60 cells, which exhibit the lowest activity. The differences in DNP‐SG export rates are not due to decreased glutathione S‐transferase activity or impaired de novo synthesis of GSH. The findings suggest that the GS‐X pump may be involved in the modulation of the biological activity of both naturally occurring electrophiles and anticancer drugs. The differential expression of GS‐X pumps may lead to an improved understanding of multidrug resistance and may be exploited in the development of new therapeutic strategies for the treatment of cancer patients.
Various forms of rabbit plasma α‐1‐antiproteinase
Authors: Saito Akio , Sinohara Hyogo 
Published: July 2007

AbstractAmino acid sequencing of the ficin‐derived C‐terminal fragments of α‐1‐antiproteinase (also called α‐1‐antitrypsin or α‐1‐proteinase inhibitor) prepared from rabbit plasma revealed the presence of the E isoform, which had been confirmed in the cDNA library in addition to the F and S‐1 forms. The S‐2 form was identified in inflamed rabbit plasma. The ficin digest of human plasma α‐1‐antitrypsin resulted in a major fragment of the M type. Multiple forms of α‐1‐antiproteinase in the rabbit plasma implicate the unknown functions other than the inhibition of neutrophil elastase.
Peroxide production and apoptosis in cultured cells carrying mtDNA mutation causing encephalomyopathy
Authors: Zhang Jin , Yoneda Makoto , Naruse Keiji , Borgeld Harm‐Jan W. , Gong Jian‐Sheng , Obata Shu‐ichi , Tanaka Masashi , Yagi Kunio 
Published: July 2007

AbstractWhen cybrids with a point mutation, which locates in the tRNALeu(UUR) gene of mtDNA and causes a mitochondrial encephalomyopathy (MELAS syndrome), were exposed to a high concentration of oxygen (95%), the peroxide production markedly increased by 6 h of oxygen exposure, whereas the peroxide production was similar among the cybrids under a normal concentration of oxygen. The peroxide production by oxygen exposure was enhanced particularly in cybrids with high proportions of the mutant mtDNA and low respiratory capacities. The appearance of apoptotic cells by oxygen exposure was high in cybrids with the impaired respiratory function due to the mutation. An antioxidant NAC successfully suppressed both the peroxide production and apoptosis. These results imply that the peroxide production plays an important role in inducing apoptosis in cells carrying the mtDNA mutation causing encephalomyopathy.
Purification and characterization of a soluble form of lysosome‐associated membrane glycoprotein‐2 (lamp‐2) from rat liver lysosomal contents
Authors: Akasaki Kenji , Tsuji Hiroshi 
Published: July 2007

AbstractLysosomal membrane of rat liver contains a highly glycosylated protein referred to as lamp‐2. Lamp‐2 occurs to a significant extent in a soluble fraction of rat liver lysosomes. The soluble form of lamp‐2 (SF‐lamp‐2) was purified to electrophoretic homogeneity. An apparent molecular weight (Mr) of SF‐lamp‐2 on sodium dodecy sulfate‐polyacrylamide gel electrophoresis was determined to be 91,000 which is 5,000 less than that of the membranous form of lamp‐2 (MF‐lamp‐2). SF‐ and MF‐lamp‐2 were very similar to each other in terms of sialic acid content, NH2‐terminal amino acid sequence and isoelectric point. Gel filtration data indicated that native SF‐lamp‐2 has an Mr=360,000. Taken together, SF‐lamp‐2 forms a tetrameric structure consisiting of a homogenous polypeptide lacking a membrane‐spanning domain and a cytoplasmic tail near the COOH‐terminus.
Angiotensin‐converting enzyme inhibitor increases insulin‐induced pp185 phosphorylation in liver and muscle of obese rats
Authors: Carvalho Daniela S. , Villaça Veridiana , Brenelli Sigisfredo L. , Carvalho Carla R. O. , Saad Mario J. A. 
Published: July 2007

AbstractThe clinical use of angiotensin‐converting enzyme (ACE) inhibitors has been associated with increased insulin sensitivity. However, the molecular mechanism is unknown. The authors examined the early steps in insulin action, i.e., the phosphorylation status of the insulin receptor and of the pp185 in liver and muscle of obese rats treated acutely with captopril, using immunoblotting with antiphosphotyrosine antibodies. Following treatment with captopril there was an improvement in insulin‐induced insulin receptor and pp185 phosphorylation in the liver and muscle of obese rats. This finding contribute to an explanation of the mechanism by which ACE inhibitors appear to improve insulin sensitivity.
Isolation and characterization of the mouse ribosomal protein S7 gene
Authors: Annilo Tarmo , Jelina Jelena , Pata Illar , Metspalu Andres 
Published: July 2007

AbstractWe have isolated and characterized the single intron‐containing gene encoding the mouse ribosomal protein S7. The mouse 129SV S7 gene has seven exons and six introns spanning about 5000 nucleotides. The exon‐intron structure of the gene is similar to other vertebrate homologues. Southern blot analysis showed that in addition to the isolated single‐copy intron‐containing gene, there are 10‐12 members in the mouse S7 gene family, which are all most probably processed pseudogenes. The promoter region of rpS7 contains several evolutionarily conserved putative regulatory elements. The main transcription start site was mapped to a T residue within a polypyrimidine tract, 79 nucleotides upstream from AUG codon.