bluuebunny/crossref_metadata_2025 huggingface dataset:
D‐myo‐inositol derivatives alter liposomal membrane fluidity
Authors: Brailoiu Eugen , Margineanu Anca , Toma Catalin P. , Filipeanu Catalin M. , Rusu Valeriu , Branisteanu Dimitrie D.
Published: July 2007

We investigated the effect on membrane fluidity induced by D‐myo‐inositol derivatives (IP3, IP4, IP5, IP6). Fluidity was determined as the anisotropy of fluorescence polarisation from liposome model membranes labelled with DPH (1,6‐diphenyl‐1,3,5 hexatriene). IP3 (10‐10 to 10‐5 M) increased the membrane fluidity with a maximum effect at 10‐5 M. For IP4, IP5 and IP6, at concentrations less than 10‐6 M these derivatives increased the membrane viscosity (i.e. reduced fluidity). This effect was enhanced when the derivatives were incorporated in the vesicles, rather than added to the vesicle suspension. In this case IP5 and IP6 increased viscosity over the reference values. We conclude that inositol derivatives directly modified membrane fluidity which could play a role in their effects in biological systems, beside the one mediated by binding to specific receptors.
Lithium regulation of protein phosphorylation in rat cerebral cortex slices in vitro
Authors: Singh Tryambak Deo , Shankar Raj
Published: July 2007

The aim of the present study was to evaluate the direct effect of Li+ and Na+‐Li+ exchange on protein phosphorylation in rat cerebral cortex slices incubated in Krebs Ringer medium. When Na+ concentration was varied in the incubation medium, either by replacement with Li+ or sucrose, a variable effect on [32P] phosphate incorporation into proteins was observed. Protein phosphorylation in cerebral cortex slices was very low in the absence of Na+, and some dependence of phosphorylating system of neural tissue on extra cellular concentration of Na+ was evident. Lithium was not able to replace sodium as far as protein phosphorylation in cortical slices is concerned. Ouabain was more effective in a Li+ containing medium in inhibiting protein phosphorylation, presumably due to improper functioning of the sodium pump.
Ascorbate peroxidase and catalase cooperate for protection against hydrogen peroxide generated in potato tubers during low‐temperature storage
Authors: Mizuno Masashi , Kamei Michiyo , Tsuchida Hironobu
Published: July 2007

We investigated the behavior of the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APx), in potato tubers during storage at low temperature. SOD activity increased temporarily within 3 weeks and was higher at 1°C than at 20°C. APx activity also increased more at low (1°C) than at higher temperatures (5 and 20°C). The contents of ascorbic acid (AsA), which is the substrate of APx, decreased immediately within 3 weeks and then gradually decreased until 15 weeks. The activity of CAT, the other enzyme which can scavenge hydrogen peroxide, decreased once in the first six weeks and thereafter increased to 15 weeks. Thus, the enhancement of the active oxygen‐scavenging system that was induced by low temperature in potato tubers could result not only in a decrease of AsA but also in combined increases in APx and CAT activity whose manners were different.
Structural characterization of human glyoxalase II as probed by limited proteolysis
Authors: Aceto Antonio , Dragani Beatrice , Melino Sonia , Petruzzelli Raffaele , Gualtieri Gabriella , Principato Giovanni , Saccucci Franca
Published: July 2007

Human glyoxalase II is partially proteolyzed by trypsin, under non denaturing conditions, only at the level of the C‐terminal region. The proteolytic cleavage resulted in an inactivation of the enzyme without loss of the secondary structure. Sodium dodecyl sulphate polyacrylamide gel‐electrophoresis and microsequence analysis showed that the glyoxalase II is proteolyzed at the level of Arg 184 and Lys 230 and undergoes a third cleavage in a region located at the beginning of the supposed C‐terminal domain. The proteolysis occurs either in the presence or in the absence of specific inhibitors. Our limited proteolysis experiments and secondary structure prediction give evidence for the presence of two domains characterized by different pattern of secondary structure.
Aquaporin 3 is induced in rat alveolar epithelium after birth
Authors: Koyama Nobuyuki , Inase Naohiko , Tanaka Michiko , Ichioka Masahiko , Sasaki Sei , Marumo Fumiaki
Published: July 2007

Aquaporin (AQP) is the water channel protein associated with water‐permeability, in which AQP1, AQP3, AQP4, and AQP5, are expressed in respiratory organs. Because water filling the fetal lung has to be absorbed after birth, aquaporins are postulated to contribute to this water reabsorption. To examine the potential role of AQP3 around birth, we studied mRNA expression and localization of AQP3 in fetal and postnatal rat lung. AQP3 mRNA was most prominently expressed in rat lung on the day of birth, began to decrease on the 2nd day after birth, and was not observed on the 4th day and later. In situ hybridization demonstrated that AQP3 mRNA was expressed in alveolar epithelium. The expression of AQP3 mRNA may influence water reabsorption of the perinatal rat lung.
Identification by bacterial expression of the yeast genomic sequence encoding L‐galactono‐γ‐lactone oxidase, the homologue of L‐ascorbic acid‐synthesizing enzyme of higher animals
Authors: Nishikimi Morimitsu , Ohta Yuriko , Ishikawa Takahiro
Published: July 2007

Baker's yeast possesses L‐galactono‐γ‐lactone oxidase that is similar in enzymic properties to L‐gulono‐γ‐lactone oxidase, the terminal enzyme of L‐ascorbic acid biosynthesis in higher animals. Computer‐assisted comparison of amino acid sequences revealed the occurrence of a homologue of rat L‐gulono‐γ‐lactone oxidase in the database of the proteins encoded by the Saccharomyces genome. The gene encoding the homologue was amplified by polymerase chain reaction and expressed as a fusion protein with glutathione S‐transferase in Escherichia coli cells with expression vector pGEX‐5X‐3. The expressed protein was found to show L‐galactono‐γ‐lactone oxidase activity and also to have covalently bound flavin, as is the case with purified L‐galactono‐γ‐lactone oxidase. Thus the gene for this enzyme was unequivocally identified.
The role of counterions in the interaction of bifunctional surface active compounds with model membranes
Authors: Sarapuk J. , Kleszczynska H. , Rózycka‐Roszak B.
Published: July 2007

Interaction of two series of bifunctional surfactants (bromides and chlorides) with red blood cells and planar lipid membranes was studied. The aim of the work was to determine the role of counterions in the mechanism of interaction of bifunctional cationic surfactants with model membranes. In each case bromides influenced model membranes to a greater degree than the corresponding chlorides. The possible explanation of the obtained results is presented. It seems that the greater ability of bromides to destabilize model membranes in comparison with chlorides can be attributed to the greater mobility and the smaller radius of the hydrated bromide ion. This may underlie the greater ease that this anion can modify the surface potential of the lipid bilayer, thus enhancing the interaction of the cationic surfactant with such a modified bilayer.
In vitro transcription of c‐jun gene using fractionated nuclear extract from regenerating rat liver
Authors: Sharma Dipali , Choudhary Shailesh Kumar , Dixit Aparna
Published: July 2007

Regenerating rat liver serves as a source of proliferating cells, such a system can be used to study the regulation of genes involved in proliferation. We have established an in vitro transcription capable of supporting accurate transcription of cloned c‐jun gene using fractionated nuclear extract prepared from partially hepatectomized rat liver. EcoR I linearized plasmid ‐132/+170 jun‐CAT containing c‐jun gene promoter region (‐132 to +170 nucleotides) was transcribed in an in vitro run‐off transcription assay and a transcript of expected size i.e. 370 nucleotides was obtained. The intensity of the transcript was dependent on the concentration of fractionated nuclear extract as well as template. The transcription was α‐amanitin sensitive indicating that it is directed by RNA polymerase II.
Differential oligomerization of membrane‐bound CD38/ADP‐ribosyl cyclase in porcine heart microsomes
Authors: Chidambaram Natesavelalar , Wong Ee Tsin , Chang Chan Fong
Published: July 2007

A protein fraction displaying ADP‐ribosyl cyclase activity was purified from porcine heart microsomes which appeared as a major band of 45 kDa on Coomassie blue stained SDS‐PAGE gel under reducing condition. Protein immunoblot analysis with antiserum to recombinant rat CD38 showed a series of bands (45‐285 kDa) under non‐reducing condition, while only the 45 kDa monomer under reducing condition. The high molecular weight oligomers of CD38 were found to be stable even upon treatment with various concentrations of SDS in the sample buffer and also upon incubation at lower temperature. These oligomers of CD38 also displayed higher ADP‐ribosyl cyclase activity than that of the monomer.
Effect on colon cancer cells of human interferon‐β gene entrapped in cationic multilamellar liposomes
Authors: Shimizu Minoru , Akiyama Seiji , Ito Katsuki , Kasai Yasushi , Takagi Hiroshi , Kito Mariko , Ohishi Nobuko , Yagi Kunio
Published: July 2007

When cultured cells of human colon cancer cell line SW480 were transfected with human interferon‐β (hIFN‐β) gene by means of cationic multilamellar liposomes, the endogenously produced hIFN‐β exhibited a remarkable anti‐proliferative effect on the cells, which was more effective than that of exogenously added hIFN‐β. This effect lasted for several days, and was blocked completely by the addition of sufficient amounts of anti‐hIFN‐β antibody. From experiments using a transwell plate and an infusion pump, we found that endogenously produced hIFN‐β acted effectively on the cells around the transfectants and that the growth‐inhibitory effect was totally retained upon continuous dilution of the medium. These data indicate that hIFN‐β expressed endogenously by transfer of its gene acted on these cancer cells mainly in a paracrine manner. Although the transfection with hIFN‐γ gene also revealed a definite growth‐inhibitory effect on the same tumor cells, the extent was less than that of hIFN‐β gene.
Reductive dechlorination of DDT to DDD by rat blood
Authors: Sugihara Kazumi , Kitamura Shigeyuki , Ohta Shigeru
Published: July 2007

The present study provides evidence that the reductive dechlorination of DDT (p, p'‐dichlorodiphenyltrichloroethane) to DDD (p, p'‐dichlorodiphenyldichloroethane) mediated by rat blood proceeds in the presence of both a reduced pyridine nucleotide and aflavin. The reduction appears to proceed in two steps. The first step is reduction of a flavin such as FAD, FMN or riboflavin by NADPH or NADH, either enzymatically or nonenzymatically. The second step is nonenzymatic reductive dechlorination of DDT by the reduced flavin, catalyzed by the heme group of hemoglobin.
Hydrogen peroxide‐supported activities of semisynthetic flavocytochrome 2B4
Authors: Shumyantseva Victoria V. , Avdeenko Yuliya L. , Moskvitina Tatyana L. , Bulko Tatyana V. , Archakov Alexander I.
Published: July 2007

Semisynthetic flavocytochromes, obtained by covalent binding of riboflavin with cytochromes P450 2B4, were able to catalyze the H202‐mediated reactions of aniline p‐hydroxylation, aminopyrine N‐demethylation and p‐nitroanizole O‐dealkylation. The rates of the flavocytochrome‐catalyzed, H202‐supported reactions far exceeded those of the appropriate NADH‐dependent reactions and were comparable with the cytochrome P450 2B4‐catalyzed, peroxide‐mediated reaction rates. The kinetic parameters (kcat, Km) for the peroxide‐dependent flavocytochrome P450 2B4 reactions were obtained. Sodium cyanide and SKF‐525A, a specific P450 inhibitor, were both shown to inhibit these reactions. The generation of active oxygen species by flavocytochrome 2B4 was registered by chemiluminescence intensity.
A novel rapid‐reaction spectrophotometric method for monitoring monovalent anion exchange by human erythrocyte band 3
Authors: Salhany James M.
Published: July 2007

Thiocyanate (SCN‐) uptake into human erythrocytes and resealed ghosts was measured by monitoring the intracellular reaction of SCN‐ with methemoglobin using a dual wavelength stopped‐flow apparatus. The cellular reaction was considerably slower than the reaction of SCN with methemoglobin in solution, indicating that SCN‐ diffusion and not chemical reaction was rate limiting. This view was confirmed by showing that the uptake rate followed saturation kinetics ( Km〈9 mM), thus indicating that SCN‐ transport involves a facilitated diffusion process. Addition of DIDS ( 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonate) totally inhibited SCN‐ uptake, thus identifying band 3 as the sole facilitator. Substitution of iodide or sulfate for trans chloride, slowed SCN‐ uptake by 4‐fold and 35‐fold respectively. Reducing the trans chloride concentration from 150 to 2 mM decreased the extent of the reaction, and slowed the observed rate by about 2‐fold. These results define a new approach for the continuous monitoring of monovalent anion exchange by human erythrocyte band 3.
Subcellular distribution of the R‐subunits of cAMP‐dependent protein kinase in LS‐174T human colon carcinoma cells
Authors: Kondrashin A. A. , Nesterova M. V. , Cho‐Chung Y. S.
Published: July 2007

The analysis of the particulate preparations of LS‐174T human colon carcinoma cells in confluent stage of growth revealed different distribution for regulatory subunits (R) of cAMP‐dependent protein kinase (PKA) in the subcellular compartments. The LS‐174T cell lysates were subjected to differential and discontinuous sucrose density gradient centrifugation. The obtained fractions were assayed for marker enzymes and photoaffinity labeled with 8‐N3[32P]cAMP. The whole lysates and cytoplasmic fraction exhibited the presence of both RIα and RIIα ‐ subunits of PKA. The fractions exhibiting high activity of the marker enzymes for plasma membranes, Golgi apparatus and mitochondria contained mainly RIIα. In the fractions of lysosomes and microsomes RIα and RIIα were found in nearly equal amounts.
Effects of La3 on lipid fluidity and structural transitions in human erythrocyte membranes
Authors: Li Xin‐Min , Ni Jia‐Zuan , Chen Jian‐Wen , Hwang Fen
Published: July 2007

The effects of La3+ on the structure and function of human erythrocyte membranes were investigated by fluorescence polarization, spin‐labeled electron spin resonance (ESR) and differential scanning calorimetry (DSC). The results showed that increasing concentrations of La3+ inhibited (Na++K+)‐ATPase and Mg2+‐ATPase activities. La3+ lowered the lipid fluidity of erythrocyte membranes and induced structural transitions in erythrocyte membranes.
Induction of apoptosis by polyunsaturated fatty acids and its relationship to fatty acid inhibition of carnitine palmitoyltransferase I activity in Hep2 cells
Authors: Colquhoun Alison
Published: July 2007

The effects of fatty acids on the induction of apoptosis were studied over a 24 hour period in Hep2 human larynx tumour cells. While oleic and linoleic acids had little effect on the apoptotic index, the polyunsaturated fatty acids α‐linolenie, γ‐linolenic, arachidonic, eicosapentaenoic and docosahexaenoic acids all induced apoptosis, beginning at ∼6 hours after fatty acid exposure. By 24 hours after exposure, the apoptotic index had reached as much as 19% in the presence of docosahexaenoic or α‐iinolenic acid. The correlation between degree of unsaturation of the 18‐carbon fatty acids and the apoptotic index was r2=0.45, 0.89, 0.84 and 0.98 at 6, 8, 12 and 24 hours, respectively. The correlation between mitochondrial carnitine palmitoyltransferase I (CPT I) activity, 6 hours after exposure, and the apoptotic index was r2=0.842 and 0.798 at 12 and 24 hours, respectively. The inhibition of CPT I and subsequent fatty acid oxidation by polyunsaturated fatty acids leads to a significant increase in apoptosis, suggesting that CPT I may be involved in the processes of programmed cell death in Hep2 human turnout cells.
Captopril ameliorates myocardial and hematological toxicities induced by adriamycin
Authors: Al‐Shabanah Othman , Mansour Mahmoud , El‐Kashef Hassan , Al‐Bekairi Abdullah
Published: July 2007

Adriamycin has a wide spectrum of antitumor activity with dose related cardiotoxicity as a major side effect. The objective of this study was to investigate the influence of captopril, a sulphydryl containing angiotensin converting enzyme inhibitor, on the cardio‐and hematotoxicity of adriamycin in normal rats. A single dose of adriamycin (15 mg/kg) caused myocardial toxicity after 24 h manifested biochemically by elevation of serum enzymes:‐ Aspartate transaminase (AST, EC: 2.6.1.1), lactate dehydrogenase (LDH, EC: 1.1.1.27), creatine phosphokinase (CPK, EC:2.7.3.2) and the cardiac iso‐enzymes of LDH and CPK. The hematotoxicity was characterized by severe leukopenia and anemia that appeared after 72 h of adriamycin adminstration. Captopril (60 mg/kg i.p) 1 h before adriamycin injection ameliorated the biochemical toxicity induced by adriamycin. This was evidenced by a significant reduction in serum enzymes, after 24 and 48 h and a significant reduction of serum cardiac iso‐enzymes after 48 h. Also restoration of the white blood cell counts as well as hemoglobin concentration occured after 72 h of captopril adminstration. These results suggest that captopril may be benificial as a protective agent against cardio‐and hematotoxicity induced by adriamycin.
Enhancement by copper, zinc superoxide dismutase of DNA damage and mutagenicity with hydrogen peroxide
Authors: Kim Ryoung Hyo , Lee Su Min , Park Jeen‐Woo
Published: July 2007

Oxidative DNA damage caused by hydrogen peroxide was enhanced by copper, zinc superoxide dismutase (CuZnSOD) in a concentration‐dependent manner, as reflected by the formation of 8‐hydroxy‐2′‐deoxyguanosine (8‐OH‐dG) and strand breaks. Hydroxyl radical scavengers such as sodium azide, mannitol and 5,5‐dimethyl‐l‐pyrroline N‐oxide (DMPO), a metal chelator, diethyenetriamine‐ pentaacetic acid, and catalase decreased strand breaks and 8‐OH‐dG formation in DNA. The deoxyribose assay showed that hydroxyl free radicals were generated in the reaction of CuZnSOD with H2O2. CuZnSOD also caused enhancement of mutation in the pUC18 lacZ' gene in the presence of H2O2 when measured as a loss of a‐complementation. Based on these results, we interpret the effects of CuZnSOD on hydrogen peroxide induced DNA damage and mutation as due to reactive oxygen species, probably hydroxyl free radicals, formed predominantly by the reaction of hydrogen peroxide and free Cu2+ released from oxidatively damaged CuZnSOD.
RGD‐containing trypsin with both platelet aggregation inhibitory activity and proteolytic activity
Authors: Nie Xin , Tang Jian‐Guo
Published: July 2007

Arg‐Gly‐Asp (RGD) motif mediates cell adhesion as a major determinant in interactions of disintegrins with cell surface receptors. In order to obtain a mutant trypsin with both high affinity to integrins and retained proteolytic activity, RGDS and RGD were inserted into a rigid tuna region and a flexible loop of trypsin, respectively. Wild type trypsin and substituted mutant trypsins, 37RGDS and 77RGD, were expressed in E. coli and purified. Kinetic properties, autolytic stability as well as platelet aggregation inhibitory activity of both 37RGDS and 77RGD were determined. 37RGDS and 77RGD give retained proteolytic activities of 34% and 87%, respectively, and both become less stable to autolysis. 37RGDS shows an obvious inhibition rate of 29% for platelet aggregation and 77RGD gives a rather weak rate of 14% at the same protein concentration of 3.5 μM, while the wild type trypsin shows no inhibitory activity.
Reduced level of ATF is correlated with transcriptional repression of DNA topoisomerase IIα gene during TPA‐induced differentiation of HL‐60 cells
Authors: Lim Kyu , Lee Jong‐In , Yun Kyung‐Ah , Son Mee‐Young , Park Jong‐Il , Yoon Wan‐Hee , Hwang Byung‐Doo
Published: July 2007

DNA topoisomerase II is a marker for the proliferation state of mammalian cells in culture, and the protein levels are markedly higher in exponentially growing cells than quiescent cells and can be downregulated by growth of the cells at high density and serum starvation. Correlation between ATF and TPA‐repressed DNA topoisomerase IIα (Topo IIα) mRNA has been investigated during TPA‐induced differentiation of HL‐60 cells. Topo IIα RNA and unknotting activity were reduced at 24 hours in TPA‐treated HL‐60 cells. The level of Topo IIα mRNA and the activity were gradually decreased in proportion to the concentration of TPA. Two DNA‐protein complexes were formed by DNA mobility shift assay when ATF‐binding site was incubated with nuclear extract prepared from TPA‐free HL‐60 cells, and the amount of ATF was vanished after TPA treatment. TPA‐repressed Topo IIα mRNA and ATF levels were partially restored after pretreatment of staurosporin. These results suggest that the reduced level of ATF may be important to the transcriptional repression of Topo IIα gene during TPA‐induced differentiation in HL‐60 cells and related to protein kinase C signal pathway.
Alloxan diabetes regulates adenosine deaminase activity in mice: tissue‐ and age‐ specific correlation
Authors: Singh L. Shanjukumar , Sharma Ramesh
Published: July 2007

Effect of alloxan induced diabetes on the activity of adenosine deaminase (ADA) was studied in the liver, spleen, stomach and small intestine of mice at two different postnatal ages (preweaned, 15‐day and postweaned, 60‐day). Alloxan significantly stimulates (133%) ADA activity in the liver of 15‐day old mice, while it has no significant effect in the 60‐day old animals. In contrast, ADA activity was moderately increased (25%) in spleen of both the ages. However, no significant influence of alloxan was observed on ADA activity of stomach at either age of mice. On the other hand, alloxan treatment increases (69%) intestinal ADA activity in 15‐day old mice, with no significant change in 60‐day old animals. Thus, alloxan diabetes increases ADA activity in an age‐ and tissue‐specific manner. Stimulation of ADA activity in diabetic mice might play role in immune and other metabolic dysfunctions in diabetic conditions.
Inhibitory effects by a submandibular gland extract on luteinizing hormone‐stimulated testosterone production by testicular cells
Authors: Koshika Shinri , Izukuri Kazuhito , Kato Yasumasa , Saito Shigeru , Hosaka Masahiko
Published: July 2007

In order to evaluate the role of the submandibular gland (SMG) on testosterone (T) production by the testis, primary cultured testicular cells were prepared from rats that had the submandibular gland surgically ablated (G‐) and control (sham operated) rats (S.O.) respectively. The cells were incubated with or without 100 ng/ml luteinizing hormone (LH) and/or SMG extract. The same linear increase in T secretion was shown by both S.O.G‐ cells on multi‐stimulation with LH for up to 96 hrs. However, while an equivalent response was shown for S.O. cells after a single LH stimulation at 96 hrs, T secretion by the G‐ cells reached a plateau after 24 hrs. The level at 96 hrs was thus approximate 30% and 33% of those of S.O. cells with and without multi‐stimulus by LH for 96 hrs, respectively. When S.O. cells were cultured with SMG extract, LH‐stimulated T secretion was dose‐dependently inhibited and there was no effect on basal T secretion. The inhibitory effect was abolished by treatment at 95 °C for 5 min. Ultra‐filtration indicated that the molecular size of the inhibitory agent was greater than 30,000. It is proposed that SMG may contain a high molecular weight, heat labile soluble factor(s) which affects T secretion by inhibiting LH action in testicular cells.
Identification of a carbohydrate‐binding site in physarum haemagglutinin I
Authors: Morita Masashi , Iwado Yukiko , Okamura Shoji
Published: July 2007

Carbohydrate‐binding peptides from trypsin‐digests of Physarum lectins (haemagglutinins I and II) were isolated by affinity column chromatography. The amino acid sequence of the peptlde fragment from haemagglutinin I was determined to be 48TVHQSWY54. A similar amino acid sequence was found in the peptide fragment from haemagglutinin II, in which alignment of valine, histidine, tryptophan and tyrosine was identical. Deletion of the heptapeptide sequence (TVHQSWY) by site‐directed mutation abolished the haemagglutinating activity. The replacement of Trp53 by alanine resulted in a complete loss of the haemagglutinating activity, suggesting that the tryptophan residue in the heptapeptide sequence is essential for carbohydrate binding.
The expressed α domain of mouse metallothionein‐I from Escherichia coli displays independent structure and function
Authors: Xiong Yi , Chen Yiwu , Ru Binggen
Published: July 2007

The α domain of mouse metallothionein‐I (mMT‐I) was expressed in E. coli as a C‐terminus of the 26 KD glutathione‐S‐transferase and purified from the cell lysates. The amino acid composition and molecular weight of the expressed protein are as expected. The metal‐binding stoichimetry was determined to show that divalent metals bind to the expressed α domain at the desired ratio of 4:1. The ultraviolet absorbance, circular dichroism spectra and the atomic force microscopy indicate that it can form the proper metal‐thiolate structure as in the whole MT molecule. The apparent affinity of the expressed α domain to bind cadmium is 1.8‐fold stronger than the recombinant mMT‐I when detected by the reaction with DTNB. The ability to scavenge hydroxyl free radicals remains higher than the whole MT molecule. All the results demonstrate that the expressed α‐domain from E. coli exhibits independent biochemical and physiological structure/function without the assistance of β domain.
Quenching of chlorophyll fluorescence by quinones
Authors: Samuilov Vitaly D. , Borisov Alexander Yu. , Barsky Eugene L. , Borisova Olga F. , Kitashov Andrei V.
Published: July 2007

Quinones caused quenching of Chl a fluorescence in native and model systems. Menadione quenched twofold the fluorescence of Chl a and BChl a in pea chloroplasts, chromatophores of purple bacteria, and liposomes at concentrations of 50‐80 μM. To obtain twofold quenching in Triton X‐100 micelles and in ethanol, the addition of 1.3 mM and 11 mM menadione was required, respectively. A proportional decrease in the lifetime and yield of Chl a fluorescence in chloroplasts, observed as the menadione concentration increased, is indicative of the efficient excitation energy transfer from bulk Chl to menadione. The decrease in the lifetime and yield of fluorescence was close to proportional in liposomes, but not in detergent micelles. The insensitivity of the menadione quenching effect to DCMU in chloroplasts, and similarity of its action in chloroplasts and liposomes indicate that menadione in chloroplasts interacts with antenna Chl, i. e., nonphotochemical quenching of fluorescence occurs.
Chromium levels in patients with internal diseases
Authors: Zima T. , Mestek O. , Tesar V. , Tesarová P. , Nemecek K. , Zák A. , Zeman M.
Published: July 2007

Chromium (Cr), an essential element, mainly affects saccharide (potentiated insulin action via interaction with insulin receptor on the cell surface) and lipid metabolism (inhibition of hydroxymethylglutaryl‐CoA reductase with a hypolipidemic effect). The aim of the study was to describe Cr serum levels in different diseases (malignant, metabolic, renal) using an advanced analytical technique with correlation to other biochemical parameters. The concentration was measured using atomic absorption spectrometry with electrothermal atomization.

The Cr levels were increased in hemodialysis patients ‐ HD (3.67± 0.35 μg/L) compared to controls ‐ C (0.40±0.12 μg/L), in significantly changed in diabetic patients ‐ DM (0.29±0.08 μg/L) and patients with lymphoproliferative disease ‐ LP (0.24±0.07 μg/L), and decreased in hyperlipidemic patients ‐ HL (0.15±0.03 μg/L). There were no differences in Cr concentration between DM treated by diet or peroral antidiabetic drugs; likewise hypolipidemic drugs in HL did not change the Cr concentration. The biochemical parameters ‐ total protein, transferrin in LP group, glucose in DM group, total cholesterol, triacylglycerols, LDL‐cholesterol, apolipoprotein B and A‐I did not correlate with serum Cr concentration. However, the HDL‐cholesterol concentration marginally significantly (p < 0.07) correlated with it. The role of Cr in humans has not yet been fully characterized. To prevent some complications in patients, it may be important to monitor the Cr levels. Chromium supplementation may be indicated in some diseases with no controversy concerning the importance of decreased serum and/or tissue levels and documented positive effects of Cr supplementation on the quality of life (e.g. hyperlipidemia).
Cholesterol sulfate induces changes in human erythrocyte thermostability
Authors: Przybylska Maria , Faber Miłosz , Zaborowski Andrzej , Bryszewska Maria
Published: July 2007

The influence of cholesterol sulfate (CS) on human red blood cell thermosensitivity was studied by flow cytometry and scanning electron microscopy. It was found that the effect of this sterol on erythrocyte stability is biphasic. Exposure of red blood cells (RBC) to the elevated temperature (51°C) induced perturbation of the cell membrane and led to haemolysis. Preincubation of cells with CS at a concentration of 1 × 10‐5 M protected them, to a certain extent, against lysis. In contrast, enrichment of RBCs with CS during the incubation with lower (0.4 × 10‐5 M) or higher (4‐8 × 10‐5 M) CS concentrations substantially augmented the fragility of the cells. The fact, that at the sublytic concentrations CS stabilises the cell membrane, may be explained by the ability of this amphipathic compound to link hydrophilic and lipophilic domains of the cell membrane and to increase the degree of the lipid bilayer order. Higher CS concentrations cause cell lysis in a detergent‐like manner. Our data support the conclusion that CS can be considered to be a potent thermosensitizer, which enhances the selectivity of biological drug carriers.
Fusion expression of human pro‐urokinase with E. coli thioredoxin
Authors: Sun Ai‐Long , Hua Zi‐Chu , Yao Ju , Yang Yong‐Hua , Yin Da‐Qiang
Published: July 2007

Human pro‐urokinase (pro‐UK) was cloned into plasmid pET32b and fused to the E. coli thioredoxin (trxA). When expressed in E. coli AD494(DE3), the fusion protein Trx‐pro‐UK accumulated as insoluble inclusion bodies and amounted to 35% of total cellular proteins. When co‐expressed with molecular chaperones human protein disulfide isomerase (PDI) and E. coli GroESL, all the expressed products still existed in the form of insoluble inclusion bodies.
Changes in the activities of three diagnostic enzymes in the heart of rats following the consumption of diets deficient in zinc and essential fatty acids
Authors: Adisa A. O. , Odutuga A. A.
Published: July 2007

Changes in the activities of alkaline phosphatase (ALP), lactate dehydrogenase(LDH) and glutamate dehydrogenase (GDH) in the heart following the consumption of diets deficient in zinc and essential fatty acids (EFA) by rats were investigated. The study was a 2 X 2 factorial design in which rats were fed diets deficient in zinc (5μg/g) or EFA (4% hydrogenated coconut oil) or both. The control diet was adequate in both zinc (100μg/g) and EFA (4% soyabean oil). The experimental diets were fed for forty‐two days after which time the activities of the enzymes were terminally determined in the heart of the rats. The result showed that the activity of ALP was markedly elevated in rats consuming either zinc or EFA deficient diet and slightly in the group deficient in both EFA and zinc. LDH and GDH activities were significantly depressed by EFA only. An interactive effect of zinc and EFA was indicated only for ALP activity. The data indicate a resistance of cardiac tissue to moderate zinc deficiency. However, the effects of EFA were apparent.
Possible role of alteration in pH, but not in ionic strength, in the modulation of Ca2 ‐dependent and diacylglycerol‐induced association of protein kinase C‐α with plasma membrane
Authors: Nishino Noriaki , Sugimoto Isamu , Hashimoto Eikichi
Published: July 2007

In an attempt to elucidate the mechanism of the membrane binding of protein kinase C‐α (PKC‐α), effects of pH and ionic strength were examined on Ca2+‐dependent and diacylglycerol‐induced association of this PKC with rat liver plasma membrane. In the absence of diacylglycerol, the membrane‐bound PKC‐α increased according to the changes in pH from 6 to 8. However, these reactions were not seriously influenced by alterations in ionic strength (0‐240 mM NaCl). In the presence of diacylglycerol, this pH‐modulated binding of PKC‐α to plasma membrane was more effective under lower Ca2+ concentration at pH 8. These results suggest that alkaline pH conditions seem to be important for preferable association of PKC‐α with plasma membrane.
Sequence of electron carriers in the process of methanol oxidation by a new obligate methylotrophic bacterium
Authors: Yang Song Suk , Lee Jung Sup , Kim Young Min , Kim Si Wouk
Published: July 2007

From pink soluble fractions prepared from cells cultured in a copper‐free medium, active methanol dehydrogenase (MDH) and two soluble c‐type cytochromes (c‐I and c‐II) were purified homogeneously. The green fractions from cells grown on a medium containing 1.0 mg/ℓ of copper had inactive MDH, cytochrome c‐II, and blue copper protein. The amount of copper retained in the blue copper protein increased with cultivation time. The oxidized blue copper protein was similar to the classical type I blue copper proteins since it had the novel absorption peak at 625 nm. However, when the blue protein was reduced with MDH or dithionite, it showed the same spectrum as ferrocytochrome c‐I. The isoelectric points of cytochrome c‐I, blue copper protein and cytochrome c‐II were 9.08, 9.08 and 6.52, respectively. These results suggest that the identity of the purified blue copper protein is cytochrome c‐I, and copper ions bind to the cytochrome as methanol is depleted in the culture medium. In addition, MDH activity was not detected at all in the methanol‐depleted condition. The data suggest that blue copper protein acts as a negative regulator for MDH. The electrons were transferred as follows: MDH → cytochrome c‐II → cytochrome c‐I (blue copper protein). It was also revealed that the initial ‘docking’ of MDH and cytochrome c‐II is accompanied by electrostatic interactions between lysine or arginine residues on the α‐subunit of MDH and carboxyl groups on the cytochrome c‐II.
Interactions of photosensitized tetracycline with serum albumin
Authors: Khan Mateen A. , Muzammil Salman , Musarrat Javed
Published: July 2007

Interactions of tetracycline with bovine serum albumin (BSA) were studied by fluorescence quenching and circular dichroism (CD) analysis. The binding isotherm exhibited at least 13 tetracycline binding sites on the albumin molecule. Amongst these, four were found to be high affinity sites and the remainder were loose sites. The Scatchard analysis demonstrated the binding constant and capacity of BSA to be 4.6×106 liters/mole and 3.6, respectively. The CD data revealed a significant decrease in the mean residue ellipticity (MRE), indicating alterations in the protein helicity. A reduction of 20% in the α ‐ helical content of the albumin was noted at higher levels of tetracycline in the presence of Cu (II) ions. Thus the strong in vitro interactions of tetracycline with albumin resulted in eonformational changes in its globular structure and insinuate potential health risk due to possible macromolecular damage, under physiological conditions, from the formation of tetracycline/Cu(II) complexes.
Different sources of acidity in glucose‐elicited extracellular acidification in the yeast Saccharomyces cerevisiae
Authors: Lapathitis Georgios , Kotyk Arnost
Published: July 2007

Three wild‐type strains of Saccharomyces cerevisiae, viz. K, Y55 and Σ1278b, two mutants lacking one or both of the putative K+ transporters, trklΔ and trklΔtrk2Δ, and a mutant in the plasma membrane H+‐ATPase, viz. pmal‐105, were compared in their extracellular acidification following addition of glucose and subsequent addition of KCl; in ATPase activity in purified plasma membranes; and in respiration on glucose. The glucose‐induced acidification was the greater the higher the respiratory quotient, i.e. the higher the anaerobic metabolism. A markedly lower acidification was found in the ATPase‐deficient pmal‐105 strain but also in the TRK‐deficient double mutant. The acidification pattern after addition of KCl corresponds to expectations in the TRK mutants; however, a similarly decreased acid production was found in the ATPase‐deficient mutant pmal‐105. The highest rate of ATP hydrolysis in vitro was found with the trklΔtrk2Δ mutant where glucose‐, as well as KCl‐induced acidification were lowest. Likewise, the pmal‐105 mutant with extremely low acidification showed only a minutely lower ATP hydrolysis than did its parent Y55 strain. Apparently, several different sources of acidity are involved in the glucose‐induced acidification (including extrusion of organic acids); in fact, contrary to the general belief, the H+‐ATPase may play a minor role in this process in some strains.
Evaluation of 9‐cis retinoic acid for a new remedy of human retinoblastoma
Authors: Tsukamoto Hidetoshi , Kurokawa Tomonori , Hirata Kohji , Ishibashi Sadahiko , Mishima Hiromu K.
Published: July 2007

We investigated the effect of two isomers of retinoic acid (RA), all‐trans RA and 9‐cis RA, on the proliferation of Y79 human retinoblastoma cells. The two isomers inhibited the cell proliferation in a concentration‐dependent manner. The IC50 for this inhibition by all‐trans RA and 9‐cis RA was 1.50 and 0.15 μM, respectively. The inhibitory effect of 9‐cis RA on Y79 cell growth was observed within 24 hr, thereafter the cell number was gradually decreased. In contrast, no inhibition by all‐trans RA of Y79 cell growth was observed within 24 hr, thereafter the cell number was slightly increased. In these cases, the cell viability at 4 days after the addition of 9‐cis RA and all‐trans RA was more than 90 % and 95 %, respectively. These results indicate that the two RA inhibit the proliferation of Y79 human retinoblastoma cells without inducing the cell death and that the effect of 9‐cis RA on the inhibition of Y79 cell growth is much greater than that of all‐trans RA.
Sphingosine induces phospholipase D and mitogen activated protein kinase in vascular smooth muscle cells
Authors: Taher Mohiuddin M. , Abd‐Elfattah Anwar S. , Sholley Milton M.
Published: July 2007

The enzymes phospholipase D and diacylglycerol kinase generate phosphatidic acid which is considered to be a mitogen. Here we report that sphingosine produced a significant amount of phosphatidic acid in vascular smooth muscle cells from the rat aorta. The diacylglycerol kinase inhibitor R59 949 partially depressed sphingosine induced phosphatidic acid formation, suggesting that activation of phospholipase C and diacylglycerol kinase can not account for the bulk of phosphatidic acid produced and that additional pathways such as phospholipase D may contribute to this. Further, we have shown that phosphatidylethanol was produced by sphingosine when vascular smooth muscle cells were stimulated in the presence of ethanol. Finally, as previously shown for other cell types, sphingosine stimulated mitogen‐activated protein kinase in vascular smooth muscle cells.
Effects of prostaglandins and nitric oxide on rat macrophage lipid metabolism in culture: implications for arterial wall‐leukocyte interplay in atherosclerosis
Authors: Senna S. M. , Moraes R. B. , Bravo M. F. R. , Oliveira R. R. , Miotto G. C. , Vidor A. C. , Belló‐Klein A. , Irigoyen M. C. C. , Belló A. A. , Curi R. , de Bittencourt P.I. Homem
Published: July 2007

Macrophages/foam cells have a pivotal role in atherogenesis although little is known about the way lipid imbalance, a hallmark of atherosclerosis, leads to lipid accumulation in these cells. Modified low‐density lipoproteins are associated with macrophage lipid dysfunction in atherosclerosis, but a possible role for altered lipogenesis leading to lipid accumulation remains to be elucidated. Since endothelium‐derived nitric oxide (NO) and prostaglandins (PGs) are physiological autacoids whose production may be impaired in atherosclerosis, the effects of these mediators on de novo lipid synthesis in 24‐h cultured rat peritoneal macrophages is investigated. In resident (unstimulated) cells, 1 μM PGE2 and the stable analog of PGI2 carbaprostacyclin (cPGI2, 1 μM) deviated the overall [1‐14C]acetate from incorporation into cholesterol, free fatty acids and triacylglycerols favoring the formation of phospholipids. In inflammatory (thioglycollate‐elicited) macrophages, these eicosanoids likewise reduced tac‐incorporations into all the lipid fractions tested. Also, cPGI2 and PGE2 reduced [4‐14C]cholesterol uptake from inflammatory cells but did not interfere in 14C‐cholesterol export. The PGE2‐derivative PGA2 (10‐20 μM) reduced 14C‐incorporations into all the lipids in resident cells while it enhanced phospholipid synthesis by up to 129% at the expense of reduced incorporations into the other test lipids. The NO donor S‐nitroso‐N‐acetylpenicillamine (SNAP, 1‐10 μM), when added to macrophages in the presence of superoxide dismutase (SOD, to avoid the reaction of superoxide with NO), significantly reduced lipogenesis especially in inflammatory cells. These findings suggest that endothelium‐derived NO and PGs may be associated with macrophage lipid accumulation by modulating lipogenesis and cholesterol uptake within these ceils.
Identification and partial characterization of three calcium‐ and zinc‐independent gelatinases constitutively present in human circulation
Authors: Makowski Gregory S. , Ramsby Melinda L.
Published: July 2007

Three constitutive gelatinases in human plasma were identified and characterized relative to known matrix metalloproteinase (MMP) gelatinases: MMP‐2 (fibroblast 72‐kDa) and MMP‐9 (neutrophil 92‐, 130‐, and 225‐kDa). Substrate gel electrophoresis (gelatin zymography) revealed an apparent Mw of 78‐, 82‐, and 89‐ kDa for these gelatinases. Densitometry revealed that MMP‐9 and MMP‐2 were highly calcium sensitive requiring 50‐150 μM and 500 μM calcium for half‐maximal activity, respectively. Of the new gelatinases, only the 89‐kDa form demonstrated slight calcium activation. The three gelatinases were unaffected by known MMP inhibitors: EDTA (5 mM), 1,10‐phenanthroline (2 mM), and pepstatin (18 μM). Serine and thiol protease inhibitors (leupeptin, aprotinin, PMSF, TLCK, TPCK, antichymostatin, antipain) were also ineffective. Solution‐phase IEF revealed that the 78‐ and 82‐kDa forms focused at neutral pl 6.72‐7.95 whereas the 89‐kDa focused at an acidic pI 4.89‐5.18 (similar to neutrophil and fibroblast forms). The data indicate that these gelatinases are not MMPs or partially activated MMPs. Their role in normal and pathological conditions is not known.
Differences in ethidium bromide and 4′‐6‐diamidino‐2‐phenylindole staining profiles with regard to DNA fragmentation during apoptosis
Authors: Shono Masayuki , Shimizu Ichiro , Omoya Toshihiro , Hiasa Aya , Honda Hirohito , Tomita Yumiko , Ito Susumu
Published: July 2007

To simply and directly evaluate DNA fragmentation during apoptosis induced in mouse cultured hepatocytes by an anti‐Fas antibody, we examined the fluorescence intensity in cell nuclei stained with ethidium bromide and 4′‐6‐diamidino‐2‐phenyhndole by optiphoto fluorescence microscopy. The intensity of the former staining for the nuclear DNA of apoptotic cells was clearly decreased compared to that of non‐apoptotic cells, whereas no difference in the fluorescence intensity for the latter stain between the apoptotic and non‐apoptotic groups was observed. Thus, the use of optiphoto fluorescence microscopy, in conjunction with both stains, constitutes a useful tool for the evaluation of apoptotic DNA fragmentation.
The shortening of the N‐terminus of myosin essential light chain A1 influences the interaction of heavy meromyosin with actin
Authors: Efimova Natalya N. , Stępkowski Dariusz , Nieznanska Hanna , Borovikov Yurii S.
Published: July 2007

The effects resulting from the removal of the N‐terminus of heavy meromyosin (HMM) A1 light chain by papain digestion are investigated. The fluorometry of TRITC‐phalloidin labelled actin in ghost fibers is used as a tool for sensing conformational changes of rigor complex of phosphorylated and dephosphorylated HMM with actin filament. The experiments were performed both under conditions assuring saturation of RLC with magnesium cation (4 mM EGTA) or calcium cation (0.1 mM CaCl2), and in constant presence of 1 mM magnesium chloride. HMM native and with A1 shortened from the N‐terminus is used. As it was observed previously rigor complex of actin filament and native HMM shows sensitivity to the kind of cation saturating RLC and to the phosphorylation status of RLC. In particular, the sin2θ parameter of actin bound rhodamine‐phalloidin fluorescence polarization representing roughly the flexibility of actin filament HMM complex changes significantly with the changes of RLC phosphorylation and cation saturation. Removal of the N‐terminus of A1 reduces this sensitivity to cation and phosphorylation both in the case of dephosphorylated and phosphorylated HMM. Our results suggest that the N‐terminus of A1 plays significant role in the rigor interaction of myosin heads with actin and is involved in modulatory function of RLC in this interaction.
Complementation of the growth defect of an RNPA49 mutant of Escherichia coli by overexpression of arginine tRNACCG
Authors: Kim Myung Soon , Park Bo Hyun , Kim Semi , Lee Young Jae , Chung Jae Hoon , Lee Younghoon
Published: July 2007

We previously found that overexpression of arginine tRNACCG from Brevibacterium albidum complements the rnpA49 mutation, which is responsible for the thermosensitivity of Escherichia coli RNase P function. In this present work, we show that the E. coli homologue tRNA also complements the same mutation, but other tRNAs do not. These results suggest that the rnpA49 mutation causes a major cellular defect in an RNase P reaction to generate the mature arginine tRNACCG.
The age‐associated decrease in the amount of amplifiable full‐length mitochondrial DNA in human skeletal muscle
Authors: Kovalenko Sergey A. , Kopsidas George , Islam Mohammed M. , Heffernan Damien , Fitzpatrick Jennifer , Caragounis Aphrodite , Gingold Elliot , Linnane Anthony W.
Published: July 2007

There has been a continuous evolution in our concept [1] that mtDNA undergoes a range of mutations with age and that such alterations lead to a decline in mitochondrial bioenergy capacity. Here we report that a wide range of deletion mutations accumulate with age and the amount of full‐length mtDNA (FLmtDNA) amplifiable by extra‐long PCR (XL‐PCR) markedly decreases with age. An analysis of single human quadriceps muscle fibres reveals a close correlation between the decrease in FLmtDNA and the decline in cytochrome c oxidase activity, an exemplifier of mitochondrial bioenergy. However, Southern blotting analysis of unamplified genomic DNA shows that there is little decrease in FLmtDNA in aged quadriceps. The results are interpreted to indicate that while there is little change in the total mtDNA with age, nonetheless a significant proportion of this mtDNA is extensively damaged such that it cannot be amplified by XL‐PCR. The amplifiable FLmtDNA, which putatively represents the functional component of the mtDNA, decreases markedly with age.