bluuebunny/crossref_metadata_2025 huggingface dataset:
Correlation between human vascular smooth muscle cell proliferation and protein kinase Cα‐expression: Effect of d‐α‐tocopherol
Authors: Marilley Dominique , Mosieniak Grazina , Boscoboinik Daniel , Azzi Angelo
Published: July 2007

Population doublings of four different human aortic vascular smooth muscle cell strains correlate with the amount of protein kinase Cα present in these cells. d‐α‐Tocopherol inhibits, at different extents, protein kinase C activity in all cells studied. Furthermore, the extent of inhibition positively correlates with the amount of protein kinase Cα expression and not with that of the other isoforms. It is suggested that, in human aortic smooth muscle cells, protein kinase Cα modulates cell proliferation and serves as a target for d‐α‐tocopherol inhibition.
Investigation of the effect of anti‐neoplastic drugs, cyclophosphamide, cisplatin and methotrexate on the turnover kinetics of human erythrocyte acetylcholinesterase
Authors: Kamal Mohammad A. , Nasim Faizul H. , Al‐Jafari Abdulaziz A.
Published: July 2007

The present work addresses the effects of three antineoplastic drugs [cyclophosphamide (CP), cisplatin (CDDP) and methotrexate (MTX) which are in current use for the treatment of various tumors] on turnover kinetics of human erythrocyte membrane‐bound acetylcholinesterase (AcChoEase, EC 3.1.1.7). It was found that CP and MTX significantly decreased kcat and ksp, whereas CDDP decreased only the kcat value, (ksp was non‐significantly affected). In light of these findings, the CP, CDDP and MTX needs particular attention in tumor therapy and their effects on turnover number must be considered at the time of administering these drugs to cancer patients.
Spontaneously hypertensive rats: Eicosa‐8,11,14‐trienoic acid metabolism and arachidonic acid biosynthesis
Authors: de Catalfo Graciela E. Hurtado , Dumm Irma N.T. de Gómez
Published: July 2007

Delta 9, Δ 6 and Δ5 desaturation activity and the thioesterification of eicosa‐8,11,14‐trienoic acid (DGLA) were measured in spontaneously hypertensive rats (SHR) compared to normotensive controls (WKY). SHR exhibited lower levels in the long‐chain fatty acyl‐CoA (LCFA) synthetase and in Δ6 and Δ5 desaturase activities in liver. The enzymatic activity changes were reflected on the fatty acid composition of liver microsomes. In testis, the thioesterification of DGLA and its conversion to arachidonic acid (AA), at the Δ5 desaturation step were also depressed in SHR. We conclude that, in SHR, the alteration in polyunsaturated fatty acid (PUFA) metabolism may influence the synthesis of AA‐derived eicosanoids involved in the control of blood pressure regulation.
The effect of n‐3 pufa rich diet upon macrophage and lymphocyte metabolism and function
Authors: Rosa L.F.B.P. Costa , Safi D. A. , Guimarães A. R. P.
Published: July 2007

A large body of evidence from experimental studies has documented that n‐3 fatty acids can modify a variety of cell functions and disease states. As lymphocytes and macrophages are important cells for the development of the inflammatory and non‐inflammatory immune response and are known to utilize high rates of glucose and glutamine, we have evaluated the effect of n‐3 PUFA rich diet on the metabolisation of glucose and glutamine these cells, as well as the effect of one such diet upon the proliferative response of lymphocytes and the phagocytic capacity and hydrogen peroxide production by macrophages. The diet provoked an increase in the flux of glucose through the Krebs cycle in macrophages as well as a reduction in G6PDh and glutaminase activity in these cells. Lymphocytes from n‐3 PUFA rich diet‐fed rats showed a reduction in glucose and glutamine decarboxylation. Taken together the data show that, at least in part, the functional changes observed in macrophages and lymphocytes from n‐3 PUFA‐rich diet fed rats are related to the effect of this diet upon glucose and glutamine metabolism, leading to immunosupression
Short‐term effects of dietary fats on the lipid composition and desaturase activities of rat liver microsomes
Authors: Girón M. D. , Lara A. , Suárez M. D.
Published: July 2007

Diets supplemented with 10% coconut, olive or sunflower oil were given to rats at weaning. After two, four and six days, the lipid composition and desaturase activities of liver microsomes were measured. The percentage of oleic acid and δ9‐desaturase activity were increased in animals fed an olive oil diet while animals given sunflower oil showed the highest content of linoleic and polyunsaturated n‐6 fatty acids. On day 6, olive oil‐fed rats had the highest levels of 22:6 n‐3 in the phosphatidylethanolamine fraction. Saturated fatty acids were similar among dietary groups, despite the marked differences in the saturated fatty acid content of the three oils. Polyunsaturated n‐6 and n‐3 fatty acids decreased during the six days of feeding with coconut oil. Our results show that liver microsome membranes respond to different dietary fatty acids sources by changes in enzyme activities and relative content of some fatty acids after a period of dietary manipulation as brief as 6 days.
The primary structure of rat ribosomal protein L6
Authors: Chan Yuen‐Ling , Wool Ira G.
Published: July 2007

The amino acid sequence of the rat 60S ribosomal subunit protein L6 was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed by determination of amino acid sequences in the protein. Ribosomal protein L6 has 296 amino acids (the NH2‐terminal methionine is removed after translation of mRNA); the molecular weight is 33,417. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 7 to 10 copies of the L6 gene. The mRNA for the protein is about 1,100 nucleotides in length. Rat L6 is related to ribosomal proteins from other eukaryotes and to histone H1.
The interactions of alpha 2HS glycoprotein with metalloproteinases
Authors: Ochieng Josiah , Green Brenda
Published: July 2007

The objective of the present analyses was to determine whether matrix metalloproteinases interact with alpha 2HS glycoprotein, the human homologue of bovine fetuin. Alpha 2HS glycoprotein was incubated with metalloproteinases at 1:1 and 1:10 molar ratios. The serum glycoprotein was completely degraded by gelatinase‐A and matrilysin at the higher enzyme concentration after an overnight incubation at 37°C. The data show that gelatinase‐A, matrilysin and gelatinase‐B do interact with alpha 2HS glycoprotein and that gelatinase‐B associates most tightly with the serum glycoprotein.
Modification of human neuron‐specific enolase for application to radioimmunoassay
Authors: Aoki Takashi , Kaneta Mitsuhiro , Onagi Hitoshi , Morikawa Junji , Tsubota Nobuyuki , Watabe Hiroyuki
Published: July 2007

A recombinant human neuron‐specific enolase (R‐NSE), isolated from Escherichia coli, could not be used in an RIA system because of instability upon labeling. To apply R‐NSE to RIA and to simplify the purification procedure, the N‐ and C‐terminals of R‐NSE were modified by tyrosine‐ and histidine‐tagging, respectively. SY‐NSE, containing one additional tyrosine residue, was obtained from both soluble and insoluble fractions. More derivatives tagged by two or four tyrosine residues were expressed, but only in the insoluble fraction. SY‐NSE and SY‐NSE.H6 (containing six histidine residues at C‐terminal of SY‐NSE) purified from the soluble fraction were applicable to the RIA system, indicating that the addition of a tyrosine residue at the terminal is effective if the antigen is unstable during labeling.
Design of an artificial hemoprotein based on human serum albumin
Authors: Shumyantseva Victoria V. , Bulko Tatyana V. , Zimin Alexander G. , Uvarov Valentine Yu. , Archakov Alexander I.
Published: July 2007

For modelling cytochrome P450‐catalyzed reactions, an artificial hemoprotein was designed. Upon complex formation of human serum albumin with iron protoporphyrine IX, there occurred the incorporation of heme into the protein and formation of a specific complex with the albumin to heme molar ratio 2:1. The apparent dissociation constant Kd of the complex, as determined by optical absorption spectroscopic technique, was 1.9±0.4 M‐6. Based on spectral studies and molecular modelling of the complex spatial structure, it was assumed that His 31 or His 90 may be the most probable 5th ligand for the heme iron. The artificial hemoprotein was able to catalyze (in the presence of riboflavin as electron carrier) the NADH‐dependent aniline hydroxylation and dimethylaniline and amidopyrine N‐demethylation. The electron transfer pathway from NADH to substrate was demonstrated. Flavin appears to serve as an input center (mediator) for the rapid transfer of electrons from NADH to heme, where substrate is oxidized. The same reactions were accomplished using riboflavin photoreduction in the hemoalbumin ‐ riboflavin system with the unfocussed laser emission at λ=457.9 nm. As electron donor, metallic zinc was used. The artificial hemoprotein obtained was also able to catalyze H2O2‐dependent oxidase reactions.
Purification and characterization of chromogranin‐A from the adrenal gland of human and bovine
Authors: Kumar Ujendra
Published: July 2007

In the present study, we report a simple, highly reproducible procedure for the purification of chromogranin A. A three step column chromatography of supernatant from human and bovine provided a major single band at Mr 67 and two minor bands at low molecular mass. Identity of the major and minor bands of CgA was confirmed by western blotting using LK2H10, a monoclonal antibody against chromogranin A. Recognition of smaller fragments in the final preparation in both species by antibody to chromogranin A may suggest that these are the proteolytic breakdown product of the main protein. Bovine adrenal gland was found to be many fold richer in chromogranin than human adrenal gland. The simplification of the purification procedure for CgA may now help in elucidating further physiological function of this protein.
Activation of the intracellular Ca2 ‐dependent serine protease ISP1 of Bacillus megaterium by purification of by high Ca2 concentrations
Authors: Váchová Libuše
Published: July 2007

The total proteolytic activity of ISP1 determined after its partial purification by size exclusion HPLC increased 3.0, 7.3 and 27.3 times in sporulating, growing and netropsin‐treated cells, respectively, as compared with the corresponding original activity in crude cytoplasmic preparations at the same CaCl2 concentration of 3 mM. A similar rise in proteolytic activity occurred on increasing the Ca2+ concentration in the crude cytoplasm from 3 to 30 mM. This activation in the cytoplasm of netropsin‐treated and sporulating cells was not reversed by subsequent lowering of CaCl2 concentration back to 3 mM by dialysis. Moreover, a similar activation appeared even after the same dialysis of cytoplasm that had not been exposed to 30 mM CaCl2. The activation was probably due to the processing of ISP1, as established by SDS‐PAGE and immunoblotting, but an involvement of additional regulation factor(s), e.g. an inhibitor or other molecules, is possible.
Redox regulation of signal transduction in smooth muscle cells: Protein kinase C inhibition by thiol agents
Authors: Taher Mohiuddin M. , Abd‐Elfattah Anwar S. A.
Published: July 2007

Thiol depleting agents phenylarsine oxide and N‐ethylmaleimide significantly inhibited the phorbol ester induced protein kinase C activation in vascular smooth muscle cells. Phenylarsine oxide is a good protein tyrosine phosphatase inhibitor. Sodium orthovanadate, also a protein tyrosine phosphatase inhibitor, neither activated nor inhibited protein kinase C in vascular smooth muscle cells. Phenylarsine oxide, N‐ethylmaleimide, orthovanadate, hydrogen peroxide and pervanadate [sodium orthovanadate + hydrogen peroxide] all significantly induced mitogen activated protein kinase in vascular smooth muscle cells. Phorbol ester and platelet derived growth factor induced mitogen activated protein kinase was inhibited by phenylarsine oxide pretreatment to vascular smooth muscle cells. However, hydrogen peroxide/pervanadate induced mitogen activated protein kinase was not prevented by phenylarsine oxide. These results suggest that oxidation of the cellular thiols inhibits the protein kinase C and activates mitogen activated protein kinase in vascular smooth muscle cells. In addition, peroxides induced, the activation of mitogen activated protein kinase in vascular smooth muscle cells which is independent of protein kinase C.
Susceptibility of growth factors to degradation by Helicobacter pylori protease: Effect of ebrotidine and sucralfate
Authors: Slomiany B. L. , Piotrowski J. , Slomiany A.
Published: July 2007

In this study, we investigated the susceptibility of the growth factors (EGF, bFGF, TGFß and PDGF) to degradation by the protease elaborated by H. pylori and assessed the effect of antiulcer agents, ebrotidine and sucralfate, on this enzymatic action of the bacterium. The colonies of H. pylori were washed with saline, filtered to retain the bacteria, and the filtrate used as an enzyme source. The assays revealed that while EGF and ßFGF showed only marginal (5‐7%) susceptibility to H. pylori protease, a 61.7% degradation occurred with PDGF and 62.3% with TGFß. Introduction of ebrotidine or sucralfate to the assay system led to the reduction in the rate of growth factors degradation, which at 50µg/ml ebrotidine reached the value of 85.1% for PDGF and 88.6% for TGFß, while with sucralfate the optimal inhibition of PDGF (79.6%) and TGFß (82.7%) degradation occurred at 200µg/ml. The results demonstrate that PDGF and TGFß are susceptible to H. pylori degradation and that antiulcer agents, ebrotidine and sucralfate are capable of counteracting this effect.
Interaction of polyadenylic acid (5′) with histone H1 or protamine
Authors: Kuo Wu‐Nan , Chambers Maxine C. , Jn‐Baptiste Junior B.
Published: July 2007

The interaction between polyadenylic acid (5′) (poly[A]) and histone (or protamine) was analyzed by electrophoretic retardation of poly[A]‐histone (or protamine) complex in agarose gel. The potency of interaction was protamine > histone H1, arginine‐rich histone > other histones. The catalytic subunit of cyclic AMP‐dependent protein kinase effectively decreased the electrophoretic retardation of poly[A]‐histone H1. The interaction between poly[A] and histone H1 was also detected by the drastically enhanced absorbance around 340 nm. The findings may implicate a regulatory role of histone H1 on mRNAs through its binding on poly[A] tails.
Alterations in circulating fatty acids and the compartmentation of selected metabolites in women with breast cancer
Authors: Quevedo‐Coli S. , Crespi C. , Benito E. , Palou A. , Roca P.
Published: July 2007

The presence of the tumor in women with breast cancer provokes a profile of biochemical change characterized by hypoglycemia, hypemremia and high levels of free fatty acids and ketone bodies in plasma. The total circulating levels of amino acids and lactate are slightly higher in patients with breast cancer. Moreover, alterations in the circulating levels of free and total fatty acids are associated with enhanced levels of total free fatty acids and significantly lower levels of esterified arachidonic acid. This profile may indicate a state of moderate catabolic activation in breast cancer patients and may also be associated with a slight mobilization of proteins and fatty acids by some of the peripheral tissues in order to cover the needs of the host and the tumor. However, the alteration in the distribution of different fatty acids (saturated, mono‐unsaturated and poly‐unsaturated) and the different behaviour of the free and esterified fractions may be the result of a greater release of only specific fatty acids by tumor or other host tissues, rather than a higher release of the whole spectrum of free fatty acids. Thus, it is proposed that some of the alterations may be directly related to localized tumor activity.
Spectroscopic characterization of the uptake of essential and xenobiotic metal cations in cells of the soil bacterium Azospirillum brasilense
Authors: Kamnev A. A. , Renou‐Gonnord M. ‐F. , Antonyuk L. P. , Colina M. , Chernyshev A. V. , Frolov I. , Ignatov V. V.
Published: July 2007

The results of flame (FAAS) or graphite furnace atomic absorption spectrometric (GFAAS) analyses are presented and discussed on the accumulation of essential metals (Mg, Ca, Mn and Fe contained in the cultivation medium) and traces of each one of the conventionally xenobiotic elements from the group V, Co, Ni, Cu, Zn or Pb, added to the medium in concentrations (0.2 mM) which do not essentially suppress growth of the bacterial culture, in cells of the plant root‐associated nitrogen‐fixing bacterium Azospirillum brasilense. Along with the essential cations assimilated by the bacterium, Zn and Cu were found to effectively accumulate in the biomass from the environment. The uptake of Co and Ni was significantly less pronounced, whereas Pb and V appeared to be present in cells in much lower concentrations than in the cultivation medium evidently showing no tendency to be assimilated by azospirilla. The effect of the above xenobiotics on the uptake level of the four essential elements provided evidence that they may compete for the formation of biologically active complexes with substances of both intracellular and extracellular localization. The analytical data obtained are compared with Fourier transform infrared (FTIR) spectra of intact vacuum‐dried bacterial cells grown in a standard medium and under the conditions of an increased metal uptake.
Isolation and characterization of an enzyme from sheep seminal vesicles that catalyzes the glutathione‐dependent prostaglandin H2 to prostaglandin F2α
Authors: Burgess John R. , Reddy C. Channa
Published: July 2007

An enzyme which catalyzes the direct 2‐electron reduction of prostaglandin H2 to prostaglandin F2α has been purified from the microsomes of sheep seminal vesicles. This enzyme, called prostaglandin endoperoxide reductase, was found to be a monomer of 16,500 molecular mass. The activity of the enzyme was dependent on reduced glutathione, enhanced by heat‐treatment, and inhibited by sulfhydryl reagents. The enzyme is not a glutathione S‐transferase nor does it utilize prostaglandin D2 as a substrate, and thus is distinct from previously characterized prostaglandin F2α, biosynthetic enzymes. The protein also catalyzes the reduction of cumene hydroperoxide, but not hydrogen peroxide. Thus, this microsomal prostaglandin endoperoxide reductase may play an important role in the synthesis of prostaglandin F2α, in some tissues.
Salt‐induced folding of alkaline denatured creatine kinase under high pH conditions
Authors: Yang Hai‐Peng , Zhong Hai‐Ning , Li Sen , Zhou Hai‐Meng
Published: July 2007

The conformational changes of creatine kinase during alkaline unfolding and salt‐induced folding at high pH have been followed by fluorescence emission and circular dichroism spectra. The results obtained show that at low ionic strength, with increasing pH value, creatine kinase denatured gradually to reach the ultimate unfolded conformation in the vicinity of pH 12.7. With the increase of pH from 9.0 to 12.7, the fluorescence emission maximum red shifted from 337 to 355 nm, indicating complete exposure of the buried tryptophan residues to the solvent. The far‐UV CD spectra show that even at pH 12.7, the apparently fully denatured enzyme retains a great part of ordered secondary structure. At pH 12.7 by adding the salt, the relatively unfolded state of denatured enzyme changes into a compact conformational state by hydrophobic collapsing. Folded state induced by salt bound ANS strongly, indicating the existence of increased hydrophobic surface. The above results suggest that the salt‐induced folded state at high pH may be the folded intermediate which exists in the general protein folding, and the larger residual ordered secondary structure might become folded being point on the salt‐induced folding.
Aloesin up‐regulates cyclin E/CDK2 kinase activity via inducing the protein levels of cyclin E, CDK2, and CDC25A in SK‐HEP‐1 cells
Authors: Lee Kwang Youl , Park Jeong Hill , Chung Myung Hee , Park Young In , Kim Kyu Won , Lee Young Joo , Lee Seung Ki
Published: July 2007

In the present study, we show that aloesin, which is a low molecular weight ingredients present in Aloe vera, stimulates the proliferation of cultured human hepatoma SK‐HEP‐1 cells. The incorporation of [3H] thymidine into DNA in the cell cultures was significantly increased at a dose of 10 μM aloesin. The aloesin‐induced DNA synthesis appears to require newly synthesized proteins because cycloheximide treatment blocked the DNA synthesis evoked by this compound. We then examined whether this compound increases the intracellular levels of cell cycle regulators by immunoblotting. The data showed that aloesin increased the levels of cyclin E, CDK2, and CDC25A in SK‐HEP‐1 cells. In addition, immuno‐complex kinase assays showed that aloesin up‐regulated the enzyme activity of cyclin E/CDK2 kinase in a dose‐dependent manner. Collectively, these results suggest that aloesin stimulates the proliferation of SK‐HEP‐1 cells by inducing the intracellular levels of cyclin E/CDK2 kinase complex and CDC25A, which, together, result in the up‐regulation of cyclin E‐dependent kinase activity.
The antioxidant properties of thioctic acid: Characterization by cyclic voltammetry
Authors: Chevion S. , Hofmann M. , Ziegler R. , Chevion M. , Nawroth P. P.
Published: July 2007

Thioctic acid (TA) level and its antioxidant capacity were monitored by cyclic voltammetry (CV), in solution and in human plasma. A linear correlation between the anodic wave current Ia at 900 mV and [TA] was obtained. This indicates that TA, commonly found in human plasma, is an antioxidant component characterized by a potential E1/2=900 mV and constitute, at least in part, the second anodic wave of the CV tracing. When plasma from diabetic patients was analyzed by CV, those patients under treatment with TA showed higher Ia than others not taking the drug. When the patients under treatment with TA were divided into groups according to the severity of their nephropathy, or according to the severity of their total complication count, Ia levels representing TA were significantly higher in those patients with more severe complications.
Ca2 /calmodulin‐binding proteins in yeast. Catabolite repression and induction by carbon sources
Authors: Santos Carlos F. Tourinho dos , Larson Roy E. , Panek Anita D. , Paschoalin Vânia M. F.
Published: July 2007

Soluble calmodulin‐binding proteins from Saccharomyces carlsbergensis were analyzed in cells grown on glucose, maltose and galactose as carbon source. A large number of polypeptide chains showed affinity for calmodulin by affinity chromatography and overlay techniques. Amongst these, polypeptides of 115, 67 and 45 kDa were only detected during the second exponential phase of growth on glucose or non‐fermentative carbon sources, suggesting that they might be subjected to catabolite repression. Polypeptides of 195 and 22 kDa were only observed in cells grown on maltose, whereas 88 kDa polypeptide was only observed in galactose‐grown cells. Among the calmodulin ‐binding polypeptides, eight were phosphorylated in a Ca2+ /calmodulin ‐dependent manner (220, 200, 175, 100, 62, 55, 31 and 16 kDa). Ca2+/calmodulin dependent [γ‐32P] incorporation was dramatically decreased in yeast cells submitted to a heat treatment.
Mössbauer spectroscopic study of the interaction of indole‐3‐acetic acid with iron(III) in aqueous solution
Authors: Kamnev Alexander A. , Kuzmann Ernö
Published: July 2007

The data of Mössbauer spectroscopic measurements in rapidly frozen 57FeIII nitrate solutions containing indole‐3‐acetic acid (IAA) are presented. The results obtained provide direct evidence that iron(III) is gradually reduced by IAA with the formation of soluble iron(II) complex. It has been found that further drying of the solution in air results in complete re‐oxidation of iron(II) with the formation of iron(III) complex. The structure of the complexes obtained and their possible role in solubilization and transformation of iron species in soil in the presence of IAA secreted by soil microorganisms into the environment are also discussed.
Aspects of glutamine metabolism in human tumour cells
Authors: Colquhoun Alison , Newsholme Eric A.
Published: July 2007

The importance of the non‐essential amino acid, glutamine, to the proliferation of human tumour cells was investigated. All of the cells studied had a high activity of phosphate‐dependent glutaminase and were found to utilise glutamine from the culture medium during long term culture. The rate of cell proliferation, determined by [6‐3H]‐thymidine incorporation, was dependent on glutamine concentration with the exception of Hs578T and MOLT 4 cells. The glutamine concentration giving half maximal growth (ED50 ranged from 0.02‐0.24mM. The glutamine analogue, acivicin [ L.(αS,5S)‐α‐amino‐3‐chloro‐4,5‐dihydro‐5‐isoxazoleacetic acid], markedly inhibited cell proliferation in the absence of glutamine. However, in the presence of glutamine acivicin only caused inhibition of proliferation in certain cell lines. Replacement of glutamine in the culture medium by glutamate resulted in an increase in the rate of cell proliferation when compared with rates in the absence of glutamine with no glutamate supplement. In addition, cells grown in the presence of the glutamine synthetase inhibitor, methionine sulphoximine, showed a marked decrease in proliferation. These data suggested the presence of glutamine synthetase in human tumour cells, which was confirmed by radiochemical assay of maximal glutamine synthetase activity. The breast tumour cells Hs578T, with high glutamine synthetase activity, were able to proliferate at rates similar to that in the presence of glutamine, when glutamine‐deficient medium was supplemented with purine nucleosides. However, the breast tumour cells MCF7, with low glutamine synthetase activity, were unable to proliferate at comparable rates in the presence of either purine or pyrimidine nucleoside supplements.
Increased longevity of kinetin‐fed Zaprionus fruitflies is accompanied by their reduced fecundity and enhanced catalase activity
Authors: Sharma Suraj P. , Kaur Jaspal , Rattan Suresh I. S.
Published: July 2007

Kinetin, a cytokinin plant growth hormone, retards senescence in plants, delays aging in human cells in culture, slows down development of insects and prolongs their lifespan. We have now observed that the increased longevity of Kn‐fed Zaprionus fruitflies was accompanied by an increase in the specific activity of catalase during developmental stages and in adult insects. In addition, the egg laying capacity of Kn‐fed fruitflies was reduced drastically as compared with those kept on a normal diet. These results support the view that improved maintenance of the soma and prolongation of its life is achieved at the cost of decreased reproductive activity.
Thromboxane A2 mediates cisplatin‐induced apoptosis of renal tubule cells
Authors: Jariyawat Surawat , Takeda Michio , Kobayashi Mami , Endou Hitoshi
Published: July 2007

The purpose of this study was to elucidate the role of thromboxane A2 (TXA2) in cisplatin‐induced apoptosis of mouse renal cells derived from the terminal proximal tubule (S3). RT‐PCR analysis revealed that S3 cells express TXA2 receptor mRNA. A TXA2 receptor antagonistKW‐3635, dose‐dependently inhibited cisplatin‐induced apoptosis of S3 cells. Treatment of S3 cells with a TXA2 agonist, STA2, induced a significant decrease in their viability and resulted in the characteristic ladder pattern resulting from intranucleosomal DNA cleavage. Treatment with KW‐3635 resulted in attenuation of the increase in c‐fos mRNA expression level in cisplatin‐treated S3 cells. In conclusion, TXA2 appears to mediate cisplatin‐induced apoptosis of S3 cells by inducing an increase in the level of c‐fos mRNA expression.
Purification, characterization, and expression of rat epididymal β‐D‐galactosidase
Authors: Hall Joseph C. , Jacobetz Dianne R. , LaMarche Mary D. , Kochins John G. , Tubbs Christopher E.
Published: July 2007

The purpose of the present study is to purify, kinetically characterize and measure the amount of soluble acid β‐D‐galactosidase (EC 3.2.1.23) in different anatomical regions (caput, corpus, and cauda) of the adult rat epididymis. Based upon SDS‐PAGE analysis, the subunit molecular mass of the caput and cauda enzyme is ∼85,000 daltons while the corpus enzyme is ∼50,000 daltons. The apparent Km and Vnmax values are 67, 24, and 59 μM and 5.0, 1.88, and 6.3 μM/min./‐mg protein for the enzyme purified from the caput, corpus, and cauda regions of the epididymis, respectively. However, no regional differences in the amount of soluble enzyme protein are observed. These data demonstrates regional differences in the activity of epididymal acid β‐D‐galactosidase and suggest that the observed regional differences in enzyme activity may be due to posttranslational modifications.
Unfolding and inactivation of penaeus penicillatus acid phosphatase during denaturation by guanidine hydrochloride
Authors: Chen Su‐Li , Chen Qing‐Xi , Yang Pei‐Zheng , Qiu Wen‐Jie , Wang Liao‐Teng , Zhou Hai‐Meng
Published: July 2007

The conformational changes of penaeus penicillatus acid phosphatase during denaturation in guanidine hydrochloride solutions were studied by following changes in the intrinsic fluorescence, ultraviolet difference absorption, and circular dichroism spectra. Inactivation of the enzyme in guanidine hydrochloride solutions were compared with unfolding of the enzyme molecule. The results show that the extent of unfolding in guanidine solutions measured by several different methods closely coincide with each other and that slightly lower concentrations of guanidine are required to bring about inactivation than are required to produce significant conformational changes of the enzyme molecule. At the same concentrations, the inactivation rate constants are markedly faster than the rate constants for unfolding of the enzyme. The above results suggest that the active sites of this enzyme display more conformational flexibility than the enzyme malecule as a whole.
Protoporphyrin IX potentiates horseradish peroxidase‐catalyzed oxidation of NADH:Involvement of enzyme‐porphyrin interaction
Authors: Sil Susmita , Chakraborti Abhay S.
Published: July 2007

Protoporphyrin IX potentiates horseradish peroxidase‐catalyzed hydrogen peroxide‐mediated NADH oxidation, but the porphyrin cannot change the enzyme‐catalyzed o‐dianisidine oxidation. Spectrofluorimetric studies reveal that an interaction occurs between horseradish peroxidase and protoporphyrin IX. The interaction is predominantly hydrophobic and entropy‐driven endothermic process. This interaction may influence the potentiation effect of the protoporphyrin IX on horseradish peroxidase‐catalyzed NADH oxidation because the latter has a positive correlation with the extent of binding of the protein with the porphyrin.
NMR characterization of physicochemical properties of rat D‐dopachrome tautomerase
Authors: Yoshida Hitoshi , Nishihira Jun , Suzuki Masaki , Hikichi Kunio
Published: July 2007

D‐dopachrome tautomerase (DOPD) catalyzes the conversion of D‐dopachrome to 5,6‐dihydroxyindole. DOPD was found to have amino acid sequence homology with macrophage migration inhibitor factor (MIF), suggesting that DOPD functions as a proinflammatory cytokine. We here demonstrate the physicochemical properties of DOPD by nuclear magnetic resonance (NMR). The native molecular weight of rat DOPD was about 37 kDa as calculated from Sephadex G‐100 column chromatography. Since the deduced molecular weight of its cDNA (117 amino acid residues) is 12.5 kDa, it is assumed that DOPD exists as a homotrimer in native form. Since several methyl proton resonances were observed in the high magnetic field region of the one‐dimensional 1H‐NMR spectrum, DOPD appeared to have a hydrophobic core in which methyl groups and aromatic groups are located close to each other. From a simple integration of one‐dimensional 1H‐NMR spectra, the contents of the α‐helix,β‐strand, and random coil were calculated to be 16%, 68%, and 16%, respectively. Since the denaturation temperature of DOPD is exceedingly high at the range between 90‐100°C, it is considered that the high β‐strand content may contribute to its heat stability.
Involvement of an endogenous metalloprotease in the activation of protoxin in Bacillus thuringiensis subsp. kurstaki
Authors: Kumar N. Suresh , Venkateswerlu G.
Published: July 2007

Insecticidal crystal proteins harvested from sporulated cultures of Bacillus thuringiensis subsp, kurstaki contain the protoxin (Mr 132 kDa) and minor amounts of toxin (66 kDa). The proteolytic processing of 132 kDa protoxin to an active 66 kDa toxin is brought about by exogenous proteases or larval gut enzymes. Under denaturing / reducing conditions this conversion is also mediated by endogenous protease(s) of the producer organism. This endogenous protease is identified as a metalloprotease as the activation process is inhibited by ethylenediamine tetraacetic acid at 2 mM concentration.
Free radicals in the cerebrospinal fluid are associated with neurological disorders including mitochondrial encephalomyopathy
Authors: Ihara Yuetsu , Kibata Masayoshi , Hayabara Toshiyuki , Katayama Shizuo , Konishi Hiroshi , Miura Kenzo , Kohno Masahiro , Kawai Motoko , Yokoi Isao , Mori Akitane
Published: July 2007

The free radical levels in the cerebrospinal fluid of 8 patients with neurological diseases and 9 undergoing lumbar anesthesia for surgery were measured. The ascorbate free radical level 10 min after lumbar puncture showed a positive correlation with the hydroxyl radical level. In the patient with mitochondrial encephalomyopathy, the levels of hydroxyl and ascorbate free radicals increased upon discontinuation of treatment and decreased upon its resumption, and the ascorbate free radical levels without therapy fell after lumbar puncture. The free radical levels in the cerebrospinal fluid may reflect the degree of oxidative stress in the central nervous system.
Interaction of bromocresol green with different serum albumins studied by fluorescence quenching
Authors: Trivedu V. D. , Saxena I. , Siddiqui M. U. , Qasim M. A.
Published: July 2007

The binding of bromocresol green to bovine serum albumin at micromolar concentrations leads to quenching of protein fluorescence. This property has been used here to study interaction of bromocresol green with bovine serum albumin as a function of pH and ionic strength. The transformation of fluorescence quench data obtained with bromocresol green into Scatchard plots yielded an association constant of 3.06×107 1M‐1 and a binding capacity of about 1.0. The affinity of bromocresol green for bovine serum albumin remains virtually unchanged between pH 4.0 and 8.0 but decreases by about 7 fold with increase in ionic strength from 0.01 to 1.0. Six other serum albumins obtained from cat, dog, human, pig and sheep have also been studied for bromocresol green binding. Although all the albumins studied bind bromocresol green, they show considerable differences in their affinities towards the dye. It appears that despite a great degree of overall similarity in their structure and conformation, serum albumins from different species differ in their ligand binding properties.
Thapsigargin induces apoptosis in SH‐SY5Y neuroblastoma cells and cerebrocortical cultures
Authors: Nath Rathna , Raser Kadee J. , Hajimohammadreza Iradj , Wang Kevin K. W.
Published: July 2007

Thapsigargin, a specific inhibitor of the endoplasmic reticular Ca2+‐ATPase, has been used previously to mobilize calcium release from intracellular calcium stores. We now show that thapsigargin (1‐10 μM) induces apoptosis in a neuroblastoma cell line (SH‐SY5Y) and in fetal rat cerebrocortical cultures. Cell death measured by lactate dehydrogenase release was observed 24‐48 hours after treatment with thapsigargin. In both cases, DNA extracts from thapsigargin treated cells showed laddering, typical of endonuclease‐mediated internucleosomal cleavages. The presence of DNA fragments was also confirmed by an ELISA designed for detecting nucleosomes in apoptotic cells. Cycloheximide reduced the extent of DNA fragmentation and injury in thapsigargin‐treated cells. Dantrolene, an inhibitor of calcium release from intracellular stores partially abolished the effect of thapsigargin, suggesting that the initial Ca2+ rise may be the signalling event in this apoptotic cell death pathway. We propose that thapsigargin‐induced cell death in cultured neuronal cells maybe a useful system to study the molecular and genetic events involved in apoptosis.
cDNA sequence analysis of a novel neurotoxin homolog from Taiwan banded krait
Authors: Chang Long‐Sen , Lin Jordge
Published: July 2007

The cDNA encoding a novel protein was constructed from the cellular RNA isolated from the venom glands of Bungarus multicinctus (Taiwan banded krait) by reverse transcription‐polymerase chain reaction. The deduced amino acid sequence of this novel protein contains 68 amino acid residues with 10 cysteine residues. Comparative sequence analyses show that it is structurally related to α‐bungarotoxin and κ‐bungarotoxins from Bungarus multicinctus venom. Eight out of the ten cysteine residues in this protein are located at the homologous positions as those in the neurotoxins. However, instead of the fifth disulfide linkage appearing in loop II of α‐bungarotoxin and κ‐bungarotoxins, the other two cysteine residues in this novel toxin are situated at the N‐terminal region. Phylogenetic analyses suggest that it probably represents a small evolutionary divergence between the long and short neurotoxins.
Fucoidan inhibits leukocyte recruitment in a model peritonial inflammation in rat and blocks interaction of P‐selectin with its carbohydrate ligand
Authors: Preobrazhenskaya M. E. , Berman A. E. , Mikhailov V. I. , Ushakova N. A. , Mazurov A. V. , Semenov A. V. , Usov A. I. , Nifant'ev N. E. , Bovin N. V.
Published: July 2007

Neutrophil recruitment into systemic inflammatory sites in vivo is thought to be initiated by selectin‐mediated endothelial adherence. The effect of fucoidan (natural sulfated polymer of L‐fucose) on the selectin dependent PMN migration into rat peritoneum following the induction of inflammation by peptone injection was studied. Peritonitis was characterized by an increase in the total cell number (from 45.3×106 to 91.6×106'rat), and by highly elevated PMN content (from 0.2% to 58%) in the rat peritoneal cavity 3 h after peptone injection. Intravenous administration of fucoidan was found to reduce, in a dose‐dependent manner, neutrophil migration into peritoneum. Fucoidan in a dose as low as 0.8 mg per rat caused 96.8% reduction of neutrophil extravasation. The inhibitory effect of fucoidan was also dependent on the time intervals between the peptone and fucoidan injections. The maximal inhibitory effect of fucoidan was observed within the first 15 rain after the induction of peritonitis and it was maintained at a level of 80% during 1.5 h. Administration of fucoidan 2.5 h after peptone injection had practically no effect on PMN extravasation. Since P‐selectin is known to play a key role at the earlier stages of PMN extravasation, it was suggested that the inhibitory effect of fucoidan was mostly due to its interaction with P‐selectin. The in vitro experiments demonstrated the high affinity of fucoidan for both isolated P‐selectin and P‐selectin in plasma membranes of activated platelets.
Evidence for the essential histidine residues in geranylgeranyl transferase type 1 from bovine testis
Authors: Kim Hyunkyung , Yoon Bo‐Young , Yang Chul‐Hak
Published: July 2007

Geranylgeranyl transferase was purified 30,000‐fold by sequential use of 30‐50% ammonium sulfate fractionation, Q‐Sepharose anion exchange chromatography, Phenyl Superose hydrophobic interaction chromatography, Sephacryl S‐300 gel filtration chromatography, and peptide (YREKKFFCAIL) affinity chromatography. Geranylgeranyl transferase, when incubated with diethyl pyrocarbonate (DEPC), a histidine‐specific reagent, shows time‐dependent inactivation, and the activity is restored by the addition of neutral hydroxylamine. The inactivation follows pseudo‐first order kinetics with a second order rate constant of 0.319 M‐1min‐1. The overall results thus provide evidence that a histidine residue in the active site is involved in the catalytic mechaism of the geranylgeranyl transferase reaction.
Formation and characteristics of an unusual λ‐DNA species
Authors: Cao En‐hua , Sun Xue‐guang , Bai Chun‐li , Liu Ci‐quan , He Yu‐jian , Wang Xin‐wen , Fang Ye
Published: July 2007

An unusual DNA species, termed as DNA species A, has been isolated and purified from thermal‐denatured λ‐DNA Hind III by Sephadex G‐200 gel filtration. Our studies indicate that DNA species A is resistant to DNase I digestion and has a higher melting point. The new DNA species showed a lower absorbency at 260 nm, and a lower fluorescence quantum yield after interaction with ethidium bromide (EB) than native double‐stranded λ‐DNA. CD spectrum of DNA species A consists of a broad positive band centered at 245 nm and a weak negative band at 220 nm. transmission electron microscope (TEM) visualizations showed that their lengths of DNA species A fell mainly in three regions (300‐500 nm, 750‐1000 nm and 1500 nm) that corresponded to three fluorescence bands in the EB‐stained gels. Their apparent width and height were 65‐75 nm and 2.2 nm respectively as observed by images of atomic force microscope (AFM).
Thermal stabilization of carboxypeptidase a as a function of pH and ionic milieu
Authors: Li Xinhui , Solomon Beka
Published: July 2007

In this study we investigated the contribution of pH, phosphate anions and salt concentration to the catalytic and structural thermostability of the carboxypeptidase A (CPA). The concentration of 75‐100 mM phosphate as well as neutral pH values were found to be optimal for stabilizing CPA at high temperatures. Although moderate concentrations of sodium chloride had no effect on thermal stability, high concentrations of the salt destabilized the enzyme. The experimental results and theoretical analysis suggested that the main contribution to heat stabilization of CPA is related to intramolecular electrostatic interactions and Arginine and/or Lysine are the putative groups able to bind phosphate and stabilize the enzyme molecule against thermal denaturation.
Purification of recombinant human precursor acid α‐glucosidase
Authors: Van Hove Johan L. K. , Yang Helen W. , Oliver Letealia M. , Pennybacker Mark F. , Chen Yuan‐Tsong
Published: July 2007

Large quantities of recombinant acid α‐glucosidase are needed for in viva experimentation of enzyme replacement therapy in Pompe disease. We describe a new purification method for the purification of this recombinant enzyme from tissue culture medium consisting of concanavalin A affinity chromatography, hydrophobic interaction chromatography, affinity chromatography on Superdex, and anion exchange chromatography. The new method is amenable to scale up, and has increased speed, and improved reproducibility with similar high yield and purification efficiency when compared to previous methods.
Attenuation of renomedullary phospholipase C isozyme, PLC‐δ1, in spontaneously hypertensive rats
Authors: Lee Kweon‐Haeng , Cho Young‐Jin , Cha Seok Ho , Endou Hitoshi
Published: July 2007

The distributional patterns of PLC isozymes within the kidney were investigated using spontaneously hypertensive rats (SHRs) and normotensive Wistar‐Kyoto (WKY) rats at 4 and 12 weeks of age. PLC‐β1, PLC‐β3 and PLC‐δ1 quantified by Western blot analysis, were present in the highest concentrations in the inner medulla of rats at both 4 and 12 weeks of age. On the other hand, PLC‐β4, PLC‐γ1 and PLC‐γ2 were distributed almost equally among the regions for the rats of both ages. When compared with WKY rats at 12 weeks of age, the amounts of PLC‐β1, PLC‐β3, PLC‐γ1, PLC‐γ2, and PLC‐δ1 in the inner medulla of SHRs were significantly lower, and the amount of PLC‐δ1 in the inner stripe of the outer medulla was also significantly lower. Even at the prehypertensive stage at 4 weeks of age, the inner medullary concentration of PLC‐δ1 was significantly lower in SHRs than WKY rats. These results suggest that PLC‐δ1 would play an important role in the development of hypertension.
Sheep testicular and epididymal angiotensin converting enzyme: inhibitions by captopril, lisinopril and enalapril
Authors: Udupa E.G. Padmanabha , Rao N. Mallikarjuna
Published: July 2007

Inhibition of angiotensin converting enzyme(ACE) in presence of captopril(C), lisinopril(L) and enalapril(E) were investigated in testis and epididymis of sheep using Hip‐His‐Leu as substrate. Captopril, lisinopril and enalapril were competitive inhibitors of the enzyme from both tissues. Differences in the I50 and Ki values using these three inhibitors reflects the affinities of these inhibitors for the, ACE. In addition, the relative potencies of captopril, lisinopril and enalapril were different for testicular ACE(C > L > E) and epididymal ACE(L>C >E). This observation suggests differences between the active sites of the testicular and epididymal ACE which may reflect on their functions in vivo.
Vitamin D receptor in IMR‐90 human fibroblasts and antiproliferative effect of 1,25‐dihydroxyvitamin D3
Authors: Stio M. , Celli A. , Lunghi B. , Raugei G. , Modesti A. , Treves C.
Published: July 2007

This study examines the effect of 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3], serum or forskolin on the proliferation of IMR‐90 fetal lung fibroblasts and demonstrates, for the first time, the presence of 1,25(OH)2D3 receptor (VDR) in this cell line. In quiescent, subconfluent cultures neither the treatment with 100 nM 1,25(OH)2D3 nor that with 50 μM forskolin influenced proliferation, while a significant increase was observed after incubation of the cells with 10% fetal bovine serum. Either cell number, determined on growing IMR‐90 human fibroblasts after 48 or 72 h incubation with 100 nM 1,25(OH)2D3 or [3H]thymidine incorporation (24, 48 or 72 h incubation) significantly decreased, while protein content per cell increased. Northern blot analysis revealed the expression of the VDR gene, the VDR mRNA bands being prominent in 1,25(OH)2D3, serum or forskolin treated fibroblasts. VDR mRNA levels slightly decreased, when growing fibroblasts were exposed to 100 nM 1,25(OH)2D3 for 48 or 72 h.
Functional residues at the active site of bovine brain adenosine deaminase
Authors: Lupidi G. , Marmocchi F. , Venardi G. , Cristalli G.
Published: July 2007

Brain adenosine deaminase was investigated in order to identify amino acid residues essential for its catalytic activity. The pH dependence of log Vmax shows that the enzyme activity depends on two ionizing groups with pK values of 5.4, that must be unprotonated, and 8.4, that must be protonated, for the catalysis. These same groups are observed in the Vmax/Km profiles. The plausible role of histidine residues at the active site of brain adenosine deaminase was proved by chemical modification with (DEP). The histidine specific reagent inactivated the enzyme following a pseudo first‐order kinetics with a second‐order rate constant of 8.9 10‐3 (±1.8 10‐3) M‐1 min‐1. The inhibition of the enzyme with PCMBS was studied monitoring the enzyme activity after incubation with the inhibitor. Brain adenosine deaminase exhibited a characteristic intrinsic tryptophan fluorescence with an emission peak centered at 335 nm. Stern‐Volmer quenching parameters in the presence of acrylamide and iodide indicated that tryptophan residues are buried in the native molecule. Tryptophan residues also showed a high heterogeneity that was increased after binding of ground‐ and transitionstate analogs to the enzyme.