bluuebunny/crossref_metadata_2025 huggingface dataset:
Altruistic DNA
Authors: Patrushev L. I.
Published: July 2007

There has been designed a model to explain the main function of abundant DNA sequences in the eukaryotic genome. The non‐coding abundant sequences are supposed to provide two levels of protection of functionally important genome regions against mutations: global and differential. At the first level rather high excess of the nucleotide sequences over the coding ones decreases probability of mutations in coding and other functionally important parts caused by intranuclear mutagens. The second level is achieved by specific chromatin condensation in individual microcompartments of the interphase nucleus and physical accessibility of functionally important sequences to mutagens. These mechanisms might control the optimum mutation rates in some functionally important loci in ontogenesis and might be of help to establish a preferable trend of evolutionary transformations of biological species in phylogenesis.
Purification of α‐mannosidase activity from Indian lablab beans
Authors: Tulasi Rajasekhar Baru , Nadimpalli Siva Kumar
Published: July 2007

Seeds of Dolichos lablab var. typicus (Indian lablab beans contain a glucose/mannose specific lectin that was affinity purified on Sepharose mannose columns in our laboratory. The unbound fraction from this matrix showed α‐mannosidase activity. In the present study this has been purified to homogeneity by a combination of ion‐exchange, hydrophobic chromatography and gel filtration. Purified α‐mannosidase had an apparent molecular weightof 195,000±5,000 with 4.5% carbohydrate. On SDS‐PAGE under reducing conditions, the enzyme dissociated into two major bands corresponding to Mr 66,000 and Mr 44,000. An antibody to the well studied jack bean α‐mannosidase cross‐reacts with the enzyme from the lablab beans suggesting antigenic similarity between these two legume mannosidases.
Dicarbanonaborates in yeast respiration and membrane transport
Authors: Kotyk Arnošt , Lapathitis Georgios
Published: July 2007

Two derivatives of carborates, sodium 5,6‐dichloro‐7,8‐dicarbanonaborate (CB‐Cl) and sodium 5‐mercapto‐7,8‐dicarbanonaborate (CB‐SH) were found to inhibit endogenous as well as glucose‐induced respiration of the yeast Saccharomyces cerevisiae. Both substances slightly increased endogenous acid production, were neutral toward H+‐ATPase‐associated acidification but pronouncedly inhibited the K+‐stimulated acidification. The same effects were observed also with an ATPase‐deficient mutant of the yeast. The ATP‐hydrotyzing activity of yeast plasma membranes in vitro was severely reduced. The membrane potential was substantially increased toward more negative values. The H+‐symporting uptake of glutamic acid was considerably decreased, that of adenine was diminished much less. The effects of the dicarbanonaborates are obviously pleiotropic but their inhibition of ATP hydrolysis and of uptake of H+‐symported substances, on the one hand, and absolute lack of effect on ATPase‐catalyzed acidification, on the other, pose an unresolved problem.
Toxicity of daunorubicin and naphthoquinones to HL‐60 cells: an involvement of oxidative stress
Authors: Dičkancaité Eglé , Čénas Narimantas , Kalvelyté Audroné , Serapiniené Nijolé
Published: July 2007

Incubation of HL‐60 cells with anthracycline daunorubicin caused an appearance of viable apoptotic, nonviable apoptotic, necrotic (nonviable nonapoptotic) and chromatin‐free (late apoptotic) cells. Both necrotic and apoptotic cell responses were partly prevented by antioxidant N,N′‐diphenyl‐p‐phenylene diamine (DPPD) and iron‐chelating agent, desferrioxamine, suggesting an involvement of activated oxygen species. The comparison of cytotoxicity of daunorubicin and of 5‐hydroxy‐ and 5,8‐dihydroxy‐1,4‐naphthoquinones revealed that at equitoxic concentrations, hydroxynaphthoquinones induced a larger number of necrotic cells in comparison to daunorubicin, a process being partly prevented by DPPD and desferrioxamine. However, we have found that cytotoxicity of daunorubicin was markedly higher in comparison with a series of naphtho‐ and benzoquinones, where an increase of cytotoxicity upon increase in single‐electron reduction potential of quinones (E17) was observed, pointing out to redox cycling as to the main factor of cytotoxicity. This discrepancy could be explained by additional factor(s) of daunorubicin cytotoxicity, e.g. DNA‐intercalation, or selective accumulation of daunorubicin in cell nucleus.
Isolation of a gene encoding nodulin‐like intrinsic protein of Escherichia coli
Authors: Fushimi Kiyohide , Bai Liqun , Marumo Fumiaki , Sasaki Sei
Published: July 2007

Members of the membrane intrinsic protein (MIP) family are expressed in various organisms including plants, insects, and vertebrates. E. coli is known to have a MIP member gene, glycerol facilitator (GlpF). Here we report the isolation of E. coli gene encoding BniP, bacterial nodulin‐like intrinsic protein. BniP encodes a 231 amino acid, 24 kDa protein with 42 % amino acid identity to Nod26, 38 % amino acid identity to AQP1, and 29 % amino acid identity to GlpF. Analysis of deduced amino acid sequence predicted a hydrophobic protein with six membrane‐spanning domains. Expression of BniP in Xenopus oocytes induced slight increase in osmotic water permeability, but not glycerol or ion permeability. Our results showed that BniP is a new member of the MIP channel‐forming proteins of E. coli.
Mutagenic and genotoxic activities of four pesticides: Captan, foltaf, phosphamidon and furadan
Authors: Saxena Sangeeta , Ashok B. T. , Musarrat Javed
Published: July 2007

The mutagenic and genotoxic potential of four pesticides viz. captan, foltaf, phosphamidon and furadan was evaluated by the Ames mutagenicity assay and their DNA damaging ability on radiation repair defective E. coli K‐12 strains respectively. The mutagenic spectrum revealed captan to be most mutagenic in the absence of metabolic activation, while the presence of S9 mix led to an attenuated mutagenic response. Foltaf, phosphamidon and furadan were detected as relatively weaker mutagens. A significant decrease in the survival of SOS defective mutants, recA, lexA and pol‐ of E. coli was observed as compared to their wild‐type counterparts in the presence of the pesticides. The role of SOS repair genes gains further support from the Salmonella strains triggering the error‐prone SOS response.
Mouse deoxyribonuclease I (DNase I): Biochemical and immunological characterization, cDNA structure and tissue distribution
Authors: Takeshita Haruo , Yasuda Toshihiro , Nakajima Tamiko , Hosomi Osamu , Nakashima Yoshimitsu , Kishi Koichiro
Published: July 2007

Mouse urinary deoxyribonuclease I (DNase I) resembles rat and human DNase Is in terms of its proteochemical and enzymological properties. Furthermore, mouse DNase I was demonstrated to be immunologically closer to the rat than to the human enzyme. A 1176 bp full length cDNA encoding mouse DNase I was constructed from RNA obtained from the kidney and parotid glands. The amino acid sequence up to the 45th residue from the N‐terminal of the mature enzyme was identical to that deduced from the cDNA sequence. This DNase I was distributed most densely in the parotid glands from the standpoint of both enzyme activity and gene transcript levels.
Effect of free fatty acids on erythrocyte morphology and membrane fluidity
Authors: Zavodnik Ilya B. , Zaborowski Andrzej , Niekurzak Andrzej , Bryszewska Maria
Published: July 2007

Changes in red blood cell shape and membrane properties in response to the interaction with free fatty acids and their derivatives were studied by light scattering at small and large angles, light microscopy and fluorescence anisotropy. The influence of these agents depended on the end groups and increased with increasing chain length. The fatty acids exerted a biphasic effect on the cell size, shape and surface properties, and induced erythrocyte aggregation. After transient size alteration with a reduction in diameter, caused by low free fatty acid concentrations (up to 5‐10 μM in the case of palmitic acid), fatty acids increased the erythrocyte diameter at higher concentrations (20‐60 μM in the case of palmitic acid). The aliphatic aldehydes and methyl esters of fatty acids significantly decreased the cell diameter at the concentrations used. Changes in erythrocyte shape and size were accompanied by changes in membrane microviscosity. Palmitic acid decreased the rotational diffusion of the fluorescence probe incorporated into the membrane whereas methyl ester of palmitic acid and laurie aldehyde increased probe mobility. Also the erythrocyte modification by malondialdehyde influenced cell morphology and highly decreased membrane fluidity.
Relation of biological activity of mutant forms of recombinant human tumor necrosis factor alpha and accumulation of sphingosine in murine liver
Authors: Alessenko A. V. , Korobko V. G. , Khrenov A. V. , Rozhnova U. A. , Soloviev A. S. , Shingarova L. N.
Published: July 2007

TNF‐α induced sphingomyelin hydrolysis by sphingomyelinase and both sphingosine and ceramidc generation have been reported to be implicated in a number of TNF‐α responses, including cytotoxicity and apoptosis. We found that sphingosine, a highly cytotoxic product of enzymatic degradation of sphingomyelin, is accumulated in liver of mice treated with TNF‐α. To determine the role of sphingosine in TNF‐α toxicity, TNF‐α mutants differing in their cytotoxicity to L929 cells as well as haemorrhagic tumor necrosis, tumor regression and lethal toxicity in mice were used in our experiments. The mutants with highest toxicity and tumor‐necrotizing activity caused accumulation of sphingosine exceeded its control level 5,5 times in murine liver cells. TNF‐α variants which caused moderate increase in sphingosine content were significantly less toxic. The observed relationship between toxicity of TNF‐α mutants, the toxicity of sphingosine, and the extent of its accumulation in murine liver provides evidence to suggest that this sphingomyelin metabolite may be mediator of TNF‐α ‐ induced cell damage and death.
Half‐life of Escherichia coli polyadenylated lipoprotein mRNA
Authors: Taljanidisz Janos , Shen Ping , Sarkar Nilima
Published: July 2007

In the light of recent evidence that the half‐life of bacterial mRNA may be modulated by polyadenylation at the 3′ end, we determined the half‐life of polyadenylated lpp mRNA, which is an abundant and comparatively stable message encoding a major lipoprotein of the outer membrane. Messenger RNAs were pulse‐labeled with [3H] adenosine and poly(A) RNA was isolated at various times after pulse‐labeling by affinity chromatography on oligo(dT) cellulose. The amount of lpp mRNA remaining was quantitated by hybridization with the antisense strand of the lpp gene cloned in M 13mp18. The observed half‐life for polyadenylated lpp mRNA was 12 min. This represents the first half‐life measurement for a specific polyadenylated mRNA in E. coli, and is slightly longer than the half‐life of total lpp RNA reported earlier. It coincides with the functional half‐life for lpp mRNA determined by Inouye and coworkers by measuring the rate of lipoprotein synthesis at various times after rifampicin addition. This suggests that polyadenylated lpp RNA is the predominant and translationally active form of lpp mRNA within the cell.
Use of an automated sequencer to determine the sequence specificity of DNA damage
Authors: Hardman Leigh C. , Murray Vincent
Published: July 2007

An automated sequencer was used to determine the sequence specificity of DNA damage caused by hedamycin in the plasmid pUC19 using a linear amplification/Taq DNA polymerase method. Previously, manual DNA sequencers have been in widespread use to investigate the sequence specificity of a DNA damaging agent. Manual DNA sequencers are restricted in the length of DNA sequence that can be read at base pair resolution for densitometry. An automated sequencer can greatly expand on the length of analysable DNA sequence. An additional important capability of the automated sequencer, is the ability to quantitate the intensity of damage at each base pair site. Thus we have used the automated sequencer to elucidate the sequence specificity of DNA damage for 300 bp. We have carried out an extended analysis of the sequence specificity of hedamycin DNA damage and found that the sequences 5′‐cGt‐3′, tGt and cGg are preferentially damaged. The sequence specificity of cisplatin was also investigated.
Nitroaromatic betulin derivatives as redox cycling agents
Authors: Miškiniene Vanda , Dičkancaite Egle , Nemeikaite Aušra , Čenas Narimantas
Published: July 2007

We have synthesized nitroaromatic derivatives of triterpenoid betulin (lup‐20(29)‐ene‐3β,28‐diol), betulin‐(28)‐5′‐(aziridin‐l‐yl)‐2′,4′‐dinitrobenzoate and betulin‐(28)‐5′‐nitro‐2′‐furoate. These compounds were reduced in single‐electron way by ferredoxin: NADP+ reductase and flavocytochrome b2 at rates comparable with their simple structure analogs. Besides, these compounds were substrates for DT‐diaphorase. Their toxicity to bovine leukemia virus‐transformed lamb fibroblast culture was partly prevented by antioxidant N,N′‐diphenyl‐p‐phenylene diamine and desferrioxamine, indicating an involvement of oxidative stress in their cytotoxicity.
Human placenta cytidine deaminase: a zinc metalloprotein
Authors: Vincenzetti Silvia , Cambi Alessandra , Balducci Enrico , Natalini Paolo , Volpini Rosaria , Vita Alberto
Published: July 2007

Cytidine deaminase, a tetrameric enzyme purified from human placenta, was shown to contain a single atom of tightly bound zinc per subunit by Inductively Coupled Plasma Optical Emission Spectrometry analysis. The metal appears to be involved in catalysis, as suggested by the inhibition exerted by 1,10‐phenanthroline and dipicolinic acid. This hypothesis is further supported by the finding that the presence of substrate protects the enzymatic activity from dipicolinic acid inhibition. Furthermore the total cysteine residues per subunit were investigated by sulphydryl groups titrating agents.
Interactions of aldolase and glyceraldehyde‐3‐phosphate dehydrogenase: molecular mass studies
Authors: Lal Ashish K. , Kayastha Arvind M. , Maihotra O. P.
Published: July 2007

A gel penetration technique, that measures the dilution undergone by protein equilibration on a short tightly packed gel column, has been employed to determine the molecular masses of aldolase (160 kDa), glyceraldehyde‐3‐phosphate dehydrogenase (GPDH; 145 kDa) in the absence and presence of each other and of other proteins. The dilution factor (concentration of protein applied/concentration of protein after equilibration) was found to be inversely related to the molecular mass of the protein. In equimolar mixtures of aldolase and GPDH, 0.5‐2.5 μM each, the two enzymes exhibited a common molecular mass value of 309‐316 kDa. These enzymes did not undergo any self association or dissociation in this concentration range. Moreover, their molecular masses were unaffected by the presence of other proteins tested. When the concentration of one of these enzymes (aldolase or GPDH) was held constant and that of the other varied, the dilution factor of the former was decreased as the concentration of the latter was increased until it corresponded to a molecular mass of ca. 310 kDa at equimolar concentrations of the two enzymes. Further increase in the concentration of the variable enzyme had no effect. It has been suggested that aldolase and GPDH form a 1:1 complex of dissociation constant equal to or less than 5×10‐8 M. The complex was found to dissociate in the presence of KCl, (NH4)2SO4, ATP and NADH whereas its formation was favoured by fructose‐1,6‐bisphosphate, glyceraldehyde‐3‐phosphate, NAD+, ADP, AMP and phosphate ions.
Helicobacter pylori lipopolysaccharide inhibition of gastric somatostatin receptor: effect of sucralfate
Authors: Piotrowski J. , Slomiany A. , Slomiany B. L.
Published: July 2007

In this study, we investigated the inhibitory effect of Helicobacter pylori lipopolysaccharide on the binding of somatostatin to gastric epithelial cell membrane receptor, and assessed the effect of antiulcer agent, sucralfate, on this process. The assays conducted with the somatostatin receptor, purified from the solubilized epithelial cell membranes by affinity chromatography on Affi‐Gel‐ [D‐ Tryp8]SRIF‐14, revealed a dose‐dependent inhibition in receptor‐somatostatin binding by the lipopolysaccharide which reached a maximum of 94.1%. This effect of H. pylori lipopolysaccharide was countered by sucralfate that produced a 92.5% restoration in receptor‐somatostatin binding at 70 μg/ml of the drug. The findings demonstrate that sucralfate is capable of reversing the interference by H. pylori lipopolysaccharide with somatostatin‐receptor binding on gastric mucosal G‐cells.
The α2ß1 integrin binds to the CB4 peptide of the α2(I) collagen chain
Authors: Staatz William D. , Walsh John J. , Santoro Samuel A.
Published: July 2007

The adhesion of platelets and other cells to type I collagen is mediated by the α2ß1integrin. A binding site for the α2ß1 integrin within the α1(I) collagen chain has previously been localized to the cyanogen bromide fragment α(I)‐CB3. We noe show by use of inhibitory monoclonal antibodies against the α2ß1 integrin, that platelets also adhere to purified α2(I) collagen chains by a mechanism mediated by the α2ß1 integrin. Moreover, following isolation of cyanogen bromide fragments of the α2(I) collagen chain by HPLC, we demonstrate that α2ß1 integrin‐mediated adhesion is restricted to the CB4 fragment of the α2(1) collagen polypeptide. These findings indicate the presence of at least two spatially distinct binding sites for the α2ß1 integrin on the native type I collagen triple helix.
Staurosporine‐induced apoptosis of immortalized mouse proximal tubule cells is modulated by bcl‐2 expression level
Authors: Takeda Michio , Kobayashi Mami , Osaki Takako , Shirato Isao , Endou Hitoshi
Published: July 2007

Mouse terminal proximal tubule (S3)cells treated with staurosporine (STS) underwent morphological and biochemical changes characteristic of apoptosis. Treatment of S3 cells with STS, H‐7, PMA, herbimycin A or genistein caused DNA fragmentation. STS inhibited the activity of protein kinase C but not of cdc‐2 kinase in S3 cells. No change in cell cycle distribution was observed in S3 cells treated with STS. However, treatment of S3 cells with STS resulted in a decrease in the level of bcl‐2 mRNA expression in the cells. Overexpression of bcl‐2 inhibited the degree of STS‐induced apoptosis of S3 cells. In conclusion, STS induces apoptosis of mouse S3 cells via inhibition of various protein kinases including protein kinase C, and the apoptosis is modulated by the bcl‐2 expression level.
Cytotoxic activity of acetylcholine receptor ligands
Authors: Blishchenko E.Yu. , Mirkina I. I. , Mernenko O. A. , Yatskin O. N. , Satpaev D. K. , Strizhkov B. N. , Karelin A. A.
Published: July 2007

Acetylcholine receptor ligands were studied for cytotoxicity in K562 human erythroid leukemia tumor cells. Cytotoxicity of carbachol, an agonist of acetylcholine receptors, atropine, an antagonist of muscarinic acetylcholine receptor, neurotoxin 11 (NT II) from Naja naja oxiana cobra venom and tubocurarine, antagonists of acetylcholine receptor of nicotinic type was exhibited in the 10‐7‐10‐5 M concentration range. Several cytolytic processes, two for carbachol and three for other ligands, corresponding to different concentrations of each ligand were detected. All acetylcholine receptor ligands induced internucleosomal DNA fragmentation.
Effect of selenium on human phospholipid hydroperoxide glutathione peroxidase expression and host cell susceptibility to lipid hydroperoxide‐mediated injury
Authors: Sun Qian , Kojima Hisanori , Komura Sadaaki , Ohishi Nobuko , Yagi Kunio
Published: July 2007

We recently isolated stable transfectants expressing human phospholipid hydroperoxide glutathione peroxidase (PHGPx) from the cells of guinea pig cell line 104C1 (Biochem. Biophys. Res. Commun. 219, 486‐491, 1996). Among them, one transfectant, designated 104C1/O4C, expressed high glutathione peroxidase activity toward dilinoleoyl phosphatidylcholine hydroperoxide (PCOOH); and another one, 104C1/O2D, moderate activity. In the present study, we investigated the effect of selenium on the PHGPx activity and on the lipid hydroperoxide‐mediated cell injury in the transfectants to clarify further the action of PHGPx in preventing oxidative injury of the cells. When transfectant 104C1/O2D cells were cultured in the medium added with 250 nM selenium, glutathione peroxidase activity toward PCOOH increased 8‐fold. Western blot analysis also revealed an increase in the amount of protein immunoreactive against anti‐rat PHGPx antibody in this transfectant. Lipid hydroperoxide‐mediated cell injury to the transfectant 104C1/O2D was significantly suppressed in accordance with the increase in the enzyme activity when the cells were cultured in the medium added with selenium. On the contrary, neither glutathione peroxidase activity toward PCOOH nor susceptibility to the injury was affected by selenium addition to the medium of the parental 104C1 cells, which have no selenium‐dependent glutathione peroxidase. These results clearly support our previous conclusion that expression of PHGPx is responsible for the protection of host cells from lipid hydroperoxide‐mediated injury.
Activity profile of placental superoxide‐superoxide dismutase system in pregnant mice and its possible relation with placental steroidogenesis
Authors: Laloraya Malini , Thomas Monzy , Jain Sudhir , Jafri Farzana , Pradeep Kumar G.
Published: July 2007

Placenta in mouse generate increasing quantities of superoxide dismutase from day 13 of pregnancy until parturition. This is associated with a concomitant reduction in the activity of superoxide radical. This findings points to the steroidogenic control of the later half of pregnancy by the placental axis. Parturition is associated with an abrupt spurt in superoxide radical. This is a novel finding and could be a consequence of the estrogen surge at labour. It is suggested that this abrupt increase in superoxide radical level at parturition may remould the placental membrane fluid at the point of its attachment with uterine membranes so as to facilitate the separation of placenta from uterus.
Endoproteases other than furin have a role in hepatic proprotein processing
Authors: Ledgerwood Elizabeth C. , Brennan Stephen O. , George Peter M.
Published: July 2007

The enzyme or enzymes responsible for dibasic‐directed proprotein processing in the liver have not yet been unequivocally identified, although there are a number of potential candidates. We have compared a Kex2‐like proalbumin convertase activity present in rat liver ER/Golgi membranes with recombinant furin, a candidate hepatic convertase. Using a series of mutant recombinant proalbumins as substrates the biochemically identified convertase and furin had very similar specificities with both preferring a substrate with an ArgXaaArgArg processing motif. Kinetic studies with normal and ‐4R proalbumin suggested however that the proalbumin convertase was not identical to furin. This was confirmed in immunoabsorption studies which demonstrated that furin only accounts for approximately half of the convertase activity. Therefore at least two proprotein convertases with overlapping specificities are involved in hepatic proprotein processing.
Effects of methylmalonate and propionate on [3H]glutamate binding, adenylate cyclase activity and lipid synthesis in rat cerebral cortex
Authors: de Mello C. F. , Rubin M. A. , Coelho J. , Wajner M. , Souza D. O. G.
Published: July 2007

The effects of methylmalonic (MMA) and propionic acid (PPA), metabolites that accumulate in methylmalonic and propionic acidemia respectively, on [3H]glutamate binding, adenylate cyclase activity and [U‐14C]acetate incorporation into lipids were investigated in rat cerebral cortex. Neither acid effected [3H]glutamate binding, regardless of the presence of sodium in the incubation medium. Also, the acids had no effect on basal or GMP‐PNP‐stimulated adenylate cyclase activity. These results suggest that MMA and PPA do not interact with glutamate binding sites and have no effect on basal or guanine nucleotide‐stimulated adenylate cyclase acitivity. In contrast, [U‐14C]acetate incorporation into brain lipids was significantly blocked by both acids, the effects being more pronounced with PPA, indicating an inhibition of brain lipid biosynthesis caused by MMA and PPA. These results may explain at least in part the hypomyelinization and/or demyelinization characteristic of patients affected by methylmalonic acidemia and propionic acidemia.
Discrimination of a novel type of rat brain δ opioid receptors by enkephalin analog containing structurally constrained cyclopropylphenylalanine (∇Phe)
Authors: Chuman Yoshiro , Yasunaga Teruo , Costa Tommaso , Shimohigashi Yasuyuki
Published: July 2007

Four different stereoisomers of cyclopropylphenylalanine (∇Phe) were incorporated into [D‐Ala2,Leu5]enkephalin at the position 4. These conformationally restricted enkephalin analogs were evaluated for their binding characteristics to μ and δ opioid receptors in rat brain. A striking finding is that the E‐(2R,3S)‐isomer binds to a novel class of δ receptors and discriminates this receptor from the ordinary δ receptor. This new type of δ receptor suspected to be a receptor which suppresses the thermal analgesia mediated through μ receptor. The Z‐(2R,3R)‐isomer was very potent with several times more enhanced affinity to δ receptors than to μ receptors, but could not differentiate the δ receptors. The Z‐(2S,3S)‐isomer was weak, and E‐(2S,3R)‐isomer was almost inactive.
Identification of protein kinase C isoforms in marine mussels
Authors: Barcia R. , Lopez‐Garcia J. M. , Girardini J. E. , Ramos‐Martinez J. I.
Published: July 2007

In this paper we have examined the distribution of some isoforms of protein kinase C in different tissues from the sea mussel Mytilus galloprovincialis Lmk. By immunoblot analysis, we have detected the presence of at least three PKC isoforms, all preferably associated with the cellular cytosolic fraction. The Ca2+‐independent form PKC δ was separated from the Ca2+‐dependent forms (PKC α and β) by means of an ionic change chromatography on DE‐52. A comparative study was carried out on the phosphorylatable non‐artificial substrates. The M.B.P. protein proved to be the best substrate, while the worst was HIIIS histone which, however, is frequently used in evaluation assays of the activity.
Rutoxyl [rutin/4‐acetamide‐1‐hydroxy‐2,2,6,6‐tetramethylpiperidinium] is a new member of the class of semi‐natural products of high pharmacological potency
Authors: Metodiewa Diana , Skolimowski Janusz , Kochman Agata , Karolczak Stefan
Published: July 2007

A novel complex, Rutoxyl [rutin/4‐acetamide‐1‐hydroxy‐2,2,6,6‐tetramethyl‐piperidinium] was synthesized and its structure and anticancer activity were investigated. The results reported here are consistent with our idea, that the formation of such a complex of two biologically active molecules: polyphenolic flavonoid antioxidant (Rutin) and nitroxylamine of nitroxide antioxidant (Tempace), stabilized by hydrogen bond(s) can result in a supra‐additive properties.
Lys‐49‐phospholipases A2 as active enzyme for β‐arachidonoyl phospholipid bilayer membranes
Authors: Yamaguchi Yoko , Shimohigashi Yasuyuki , Chiwata Tsuyoshi , Tani Ayako , Chijiwa Takahisa , Lomonte Bruno , Ohno Motonori
Published: July 2007

Phospholipases A2 containing Lys‐49 have been reported to be extremely weak or inactive as enzyme. We have recently shown that basic proteins I and II (BP‐I and BP‐II), Lys‐49‐PLA2s isolated from the venom of Trimeresurus flavoviridis (Habu snake), are potent to hydrolyze the arachidonate of 2‐arachidonoyl‐1‐stearoyl‐L‐3‐phosphatidylcholine (ASPC) in bilayer vesicles. In order to ensure such enzymatic activity of Lys‐49‐PLA2s, two other Lys‐49‐PLA2s from different snake venoms, myotoxin II (from Bothrops asper) and App‐K49 (form Agkistrodon piscivorus piscivorus), were examined. Myotoxin II was found to be very active, even more potent than BP‐II, liberating about 80% of arachidonic acid from liposomes. App‐K49 was also active (about 50%) for ASPC liposomes. They were very weak or almost inactive for ASPC micelles and monomers. All these Lys‐49‐PLA2s were inactive for ASPC liposomes in the absence of Ca2+. These results clearly demonstrated that Lys‐49‐PLA2s are the enzymes to hydrolyze the C2‐ester bond of ASPC in bilayer membranes.
Micromolar calcium prevents isolated rat liver mitochondria form anoxia‐reoxygenation injury
Authors: Schild Lorenz , Plumeyer Frank , Reinheckel Thomas , Augustin Wolfgang
Published: July 2007

Rat liver mitochondria were exposed to extramitochondrial free calcium between 0 and 5 μM and/or 5 minutes of anoxia followed by 10 minutes of reoxygenation. At concentrations higher than 4 μM, the membrane potential collapsed indicating the permeability transition of the mitochondrial membrane. Anoxia‐reoxygenation shifted this transition to lower calcium concentrations. Anoxia‐reoxygenation alone resulted in the decrease of ADP stimulated respiration down to about 40 % of its initial value. Between 1 and 2 μM, a protective effect in terms of respiration and oxidative protein modification was found. It is concluced that calcium may suppress the formation of reactive oxygen species during anoxia‐reoxygenation before permeability transition occurs.
Effect of malathion on kinetic parameters of acetylcholinesterase (EC 3.1.1.7)In vitro
Authors: Kamal Mohammad Amjad
Published: July 2007

Kinetic analysis of the interaction of malathion with camel erythrocyte acetylcholinesterase was investigated in the present study. The Michaelis‐Menten constant (Km) for the hydrolysis of acetylthiocholine iodide (ASCh) was found to be 53.15 μM and the Vmax was 0.287 μmol/min/mg protein. The Kmapp and Vmaxapp were both decreased by increased malathion concentration. Dixon as well as Lineweaver‐Burk plots and their secondary replots indicated that the nature of the inhibition was of the pure uncompetitive type with Ki value estimated as 102.1 ppm. The Kiapp decreased while Vmaxiapp increased by an increased concentration in ASCh.
Mice lacking a functional CHK gene have no apparent defects in the hematopoietic system
Authors: Samokhvalov Igor , Hendrikx Jan , Visser Jan , Belyavsky Alexander , Sotiropolous Damianos , Gu Hua
Published: July 2007

Non‐receptor tyrosine kinase Chk has been implicated in hematopoietic development. To study the function of Chk in vivo, we have generated chk‐deficient mice using gene targeting. Overall development of mice homozygous for this mutation was apparently normal. Blood counts, FACS analysis of hematopoietic cell populations, CFU‐C and CAFC assays showed no significant difference between wild type and mutant animals. Thus, the dispensability of Chk for mouse development and hematopoiesis suggests that its function may be redundant in vivo, and most likely be compensated by activity of a closely related protein tyrosine kinase Csk.
Transport of glutathione S‐conjugates in Escherichia coli
Authors: Kałuzna Agnieszka , Bartosz Grzegorz
Published: July 2007

There are no reports concerning glutathione‐S‐conjugates transport in prokaryotic cells, although many studies have been performed in eukaryotic systems. This study demonstrates that glutathione S‐conjugates (2,4‐dinitrophenyl‐S‐glutathione and bimane‐S‐glutathione) are transported also out of Escherichia coli K12 cells. This transport is inhibited by vanadate, fluoride and other inhibitors of glutathione S‐conjugate transport in mammalian cells suggesting a similarity of this process to that known in eukaryotes.
Augmentation of protein kinase C activity and liver cell proliferation in lead nitrate‐treated rats
Authors: Liu Jer‐Yuh , Liu Cheng‐Chung , Wang Chau‐Jong , Yen Cheng‐Chieh , Hsieh Yih‐Shou , Chen Wen‐Kang , Liu Chia‐Pin , Lin Jen‐Kun
Published: July 2007

It is shown that lead alters calcium mediated cellular processes in several biological systems. Calcium enhances the activity of protein kinase C (PKC) which takes part in eliciting cell mitosis. In this study, the effects of lead nitrate on the activity of PKC enzyme were investigated in rat liver. The PKC activity was determined at 12, 24, 48, 72, 120, 168 hours after treatment with a single dose of lead nitrate in male Wistar rats. The results showed that the specific PKC activity of the purified particulate fraction was increased and reached a maximum at 24 hour, and lasted for 48 hours. This augmented activity of PKC was parallel with the increase of the lead level in the purified particulate fraction, although the protein levels of PKC α, PKC δ and PKC ζ were unchanged. Moreover, the frequency of mitotic cells also exhibited a significant increase, and like PKC activity, reached its maximum at 24 hour with accompany signs of liver enlargement. The results suggest that the PKC activation may be involved in promoting liver cell proliferation in lead nitrate‐treated rats.
The role of reactive oxygen species (ROS) in the effector mechanisms of human antimycobacterial immunity
Authors: Fazal Nadeem
Published: July 2007

The aim of this study was to determine the role of reactive oxygen species (ROS) in checking the growth of intracellular mycobacteria within human phagocytes. Peripheral blood‐derived neutrophils and monocyte‐derived macrophages were isolated from Chronic Granulomatous isease (CGD) patients and normal healthy human volunteers. CGD patients are known to have a defect in the NADPH oxidase pathway, resulting in their neutrophils and monocyte‐derived macrophages being unable to generate oxygen radicals which are required to kill intracellular bacteria. The cells were then infected with Bacilte Calmette Guerin (BCG) or Mycobacterium avium, and the bacterial growth in each cell type determined by Colony Forming Units (CFU) estimate. The results obtained indicate that there was no demonstrable inhibition in the intracellular mycobacterial growth within neutrophils or macrophages derived from either Chronic Granulomatous Disease (CGD: deficient in NADPH oxidase pathway) or normal healthy volunteers. Macrophage treatment with either IFN‐γ or TNF‐α had no effect.
Multiple cellular proteins are recognized by the adeno‐associated virus Rep78 major regulatory protein and the amino‐half of Rep78 is required for many of these interactions
Authors: Hermonat Paul L. , Carter Charleata A. , Parham Groesbeck P. , Quirk J. Gerald , Santin Alessandro D.
Published: July 2007

Adeno‐associated virus (AAV) encoded Rep78 is a multi‐functional protein which is required for AAV DNA replication, is able to regulate both AAV and heterologous gene expression at the transcriptional level, and appears necessary for site specific integration of AAV DNA into human chromosome 19. By comparison with the analogous replication protein of the polyomaviruses, large T antigen, it seemed likely that Rep78 would interact with cellular proteins to carry out at least some its functions. This study demonstrates that Rep78 is able to interact with multiple cellular proteins from cellular extracts as measured by West(far)‐western, coimmunoprecipitation, and Rep78‐affinity chromatography analysis. Eight cellular proteins of approximately 180, 140, 120, 95, 75, 55, 45, and 35 kDa (+/‐10%), were observed to bind Rep78 in all three assay systems. Two others, of 30 and 24 kDa, were observed in two of three assay systems. Furthermore, using truncated Rep78 proteins, it is demonstrated that the amino‐terminus is required for most Rep78‐cellular protein interactions. However, the extreme carboxy‐terminus of Rep78 was found to be all that is required for binding to the 55 kDa cellular protein.
Differentiation of substrate‐binding sites in pyruvate kinase by selective photoaffinity labeling
Authors: Mozumdar Subho , Wang J. H.
Published: July 2007

The four substrate‐binding sites in porcine liver pyruvate kinase have been labeled with the photoaffinity reagent 8‐azido‐2′‐O‐dansyl‐[α‐32P]ATP (AD‐ATP) under different experimental conditions. In the dark, the native pyruvate kinase was reversibly and competitively inhibited by AD‐ATP, with KI=2.8 μM and KADP=0.18 mM. Under UV‐irradiation, the enzyme was covalently labeled in the presence of Mg2+ by AD‐ATP and inactivated irreversibly. Measurement of this photoinactivation process in the presence of various concentrations of ADP gave KI=4.0 μM and KADP=0.2 mM. A linear plot of the relative specific activity of the partially photolabeled enzyme after gel‐filtration vs. the number of label per pyruvate kinase molecule introduced in the presence of Mg2+ shows that each covalent label completely inactivates a tetrameric pyruvate kinase molecule. In the strict absence of Mg2+, up to three substrate‐binding sites in each pyruvate kinase molecule can be labeled by AD‐ATP without decreasing enzyme activity. Subsequent addition of Mg2+ enables AD‐ATP to label the remaining site and inactivate the enzyme. These observations show that there are one catalytic and three non‐catalytic substrate‐binding sites in each pyruvate kinase molecule. A probable structural reason for possible functional differentiation of intrinsically identical substrate‐binding sites in all tetrameric enzymes is suggested.
Competitive inhibition of mouse brain nitric oxide synthase by amiloride: a case for enzyme cross‐inhibition
Authors: Ascenzi Paolo , Federico Rodolfo , Menegatti Enea , Venturini Giorgio
Published: July 2007

Amiloride, a diuretic and antihypertensive drug, inhibits Na+ transporting systems, diamine oxidases and the human urinary plasminogen activator. Present results indicate that amiloride is a competitive inhibitor of mouse brain nitric oxide synthase (NOS; Ki= 4.5×10‐4 M, at pH 7.5 and 37.0°C), but not a nitric oxide (NO) precursor, representing a case for enzyme cross‐inhibition. The decreased levels of NO, as a consequence of NOS inhibition, might induce some clinically‐adverse amiloride reactions, such as an unexpected reduced antihypertensive effect.
Chaperone‐like function of lipocortin 1
Authors: Kim Geum‐Yi , Lee Hee‐Bong , Lee Soo Ok , Rhee Hae Jin , Na Doe Sun
Published: July 2007

Lipocortin 1 (LC1) is a 37 kDa member of the annexin family of proteins. It has been proposed to act as a mediator of some of the actions of glucocorticoids in anti‐inflammatory and immune suppressive functions. LC1 has been shown to play a role in cell proliferation, apoptosis, and differentiation. However, the exact biological functions of LC1 still remain obscure. Here it is shown that LC1 displays a chaperone‐like function. Stoichiometric amounts of LC1 suppressed thermally induced inactivation and aggregation of the test enzymes citrate synthase and glutamate dehydrogenase. LC1 was also effective in refolding guanine hydrochloride‐denatured glutamate dehydrogenase, as judged by circular dichroism spectroscopy.
Dual temperature model for the estimation of energetics parameters for acetylcholinesterase inhibition by cyclophosphamide
Authors: Kamal Mohammad Amjad
Published: July 2007

The present work addresses the “dual temperature” model for the estimation of Gibb's free energy change (ΔG), enthalpy change (ΔH), heat of activation (ΔH*) entropy change (ΔS), temperature coefficient (Q10) and activation energy (Ea) of chicken brain acetylcholinesterase (AChE) inhibited by cyclophosphaxnide monohydrate (CP). In this investigation, the PZ factor (number of sterically and energy wise favorable collisions occurring between CP and AChE) has also been studied. The inhibitor has considerably increased all energetics parameters except ΔS and Q10. These results are in general agreement with the data from the other reported studies. The significance of the use of CP in cancer therapy and various aspects of thermodynamic parameters have also been discussed.
Isolation of cDNA encoding full‐length rat (Rattus norvegicus) poly(ADP‐ribose) polymerase
Authors: Beneke Sascha , Meyer Ralph , Bürkle Alexander
Published: July 2007

Poly(ADP‐ribose) polymerase (EC 2.4.2.30) is a nuclear enzyme which binds to DNA breaks and then catalyzes the covalent modification of acceptor proteins with poly(ADP‐ribose). Poly(ADP‐ribose) polymerase activity contributes to the recovery of proliferating cells from DNA damage and to the maintenance of genomic stability, which may be mediated by effects on chromatin structure, DNA base‐excision repair and cell cycle regulation. We established the complete eDNA sequence of rat poly(ADP‐ribose) polymerase by RT‐PCR and direct sequencing of amplification products and compared it with that of other mammalian species. The amino acid sequence homology is strikingly high. The best conserved regions are the known functional modules ofpoly(ADP‐ribose) polymerase.
Characterisation of bovine structure‐specific recognition protein 1 (SSRP1) cDNA
Authors: Nielsen Peter Kresten , Haaning Jesper , Sottrup‐Jensen Lars
Published: July 2007

A partial bovine SSRP1 (structure‐specific recognition protein 1) cDNA has been isolated from a osteoblast cDNA library and sequenced. The bovine SSRP1 cDNA of 1870 bp encoding 460 amino acid residues showed 86% and 98% sequence identity with human SSRP1 at the DNA and protein level, respectively. Expression of SSRP1 mRNA was detected in many human tissues, with a particularly high level in kidney.
Identification of 14 kDa auxin‐binding proteins in a tobacco plasma membrane subfraction responsive to auxin
Authors: Szponarski Wojciech , Peltier Jean‐Benoît , Tena Guillaume , Rossignol Michel
Published: July 2007

Auxin binding by tobacco plasma membrane proteins was investigated. After photolabeling with [3H]IAA, 350 polypeptides were resolved on 2D gels and analyzed. Thirteen polypeptides were selected according to physico‐chemical criteria. The labeling of three of them was further shown to increase, after treatment of cells with auxin, specifically in that plasma membrane subfraction where the sensitivity to the hormone of the H+‐ATPase is enhanced by the treatment of cells. These polypeptides were those that exhibited the more specific labeling features according to physico‐chemical criteria. They had similar apparent molecular weight (ca 14 kDa) that distinguished them from other auxin‐binding proteins described up to now, and exhibited similar amino acid compositions. These 14 kDa polypeptides are proposed to constitute a group of new auxin‐binding proteins, potentially involved, within specialized plasma membrane domains, in the stimulation of the proton pump by the hormone.
Multiprotein complex formation on the c‐myc promoter
Authors: Logan Thomas J. , Moberg Kenneth H. , Hall David J.
Published: July 2007

The E2F element is a cis‐acting DNA sequence within the P2 promoter of the c‐myc proto‐oncogene. While it is required for optimal transcription, the multiprotein complexes formed on this site have not been well characterized. We show that in extracts of human glioblastoma cells and NIH3T3 fibroblasts, significant E2F transcription factor binding to the c‐myc E2F site occurs as a both a monomer (the active form) and as only two mutually exclusive complexes with the retinoblastoma gene product (pRb) or the cyclin A protein. The E2F protein monomer was found predominantly in the cytosolic fraction of the cellular extracts while the pRb and cyclin A complexes in the nuclear fraction, indicating that the monomer has novel physical properties. Thus, protein complex formation on the c‐myc E2F site appears to contribute in a unique way to transcriptional activation.
Identification of actin as an estradiol 17‐β‐stimulated protein in the human placenta
Authors: Sudha S. , Kumari Usha , Rao Veena S. , Rao A. Jagannadha
Published: July 2007

Addition of estradiol 17‐β to first trimester human placental minces resulted in an increased synthesis of a protein of apparent molecular weight 45 kDa. The specific involvement of estrogen in the stimulation of this protein was established by demonstrating a reduction in the level of this protein by the addition of CGS 16949 A, an inhibitor of aromatase, a key enzyme in the biosynthesis of estradiol 17‐β and ICI 182,780, an estrogen receptor antagonist. The protein was purified to homogeneity and N‐terminal sequencing of two of the internal peptides obtained by enzymatic digestion of the protein, as well as the absence of a free N‐terminal indicated that it could be actin. This was confirmed by Western blotting using commercially available actin antiserum. The role of estradiol 17‐β in the stimulation of actin synthesis in human placenta was also established by monitoring the quantitative inhibition of DNase I by actin.
Differential expression of ferritin heavy chain in THP‐1 cells infected with Mycobacterium bovis BCG
Authors: Lim Jong‐Seok , Lee Seung‐Hwan , Lee Eunsik , Kang Young , Kim Jae Wha , Kim Jin Koo , Kim Hyeon Hoe , Lee Chongwook , Kim Sang Jae , Bai Gill Han , Lee Hee Gu , Kim Kwang Dong , Chung Tai Wha , Choe Yong‐Kyung
Published: July 2007

To identify the host genes induced or suppressed by infection of mycobacteria, the reverse transcriptase polymerase chain reaction (RT‐PCR) and the differential display reverse transcriptase polymerase chain reaction (DD RT‐PCR) methods were used. In this study, cDNAs complement to mRNA extracted from human peripheral monocyte derived naive THP‐1 cells, THP‐1 cells infected with live Mycobacterium bovis BCG, THP‐1 cells treated with heat‐killed BCG, and THP‐1 cells incubated with IgG‐coated glass‐beads were compared on the sequencing gel. One (TG2‐1) of the clones selected by DD RT‐PCR is 446 bp long and is identical to human ferritin heavy (H) chain gene. Northern blot analysis confirmed that ferritin H chain gene has been markedly over‐expressed in monocytic THP‐1 cells incubated with live and dead M. bovis BCG. Differential display techniques of host genes whose expression levels were varied by infection of mycobacteria could provide information about the response of macrophages to mycobacterial infection.