bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural and functional roles of the amino‐terminal region and collagen‐like domain of human serum mannan‐binding protein
Authors: Ma Yong , Yokota Yasunori , Kozutsumi Yasunori , Kawasaki Toshisuke
Published: July 2007

The serum mannan‐binding protein (S‐MBP) is a Ca2+‐dependent C‐type animal lectin specific for mannose and N‐acetylglucosamine, which plays an important role in first‐line host defense. To study the structure and function relationship of the lectin, a full‐length human S‐MBPcDNA was expressed in Sf9 insect cells using a baculovirus expression system, and a cDNA encoding the carbohydrate recognition domain (CRD) of human S‐MBP was expressed in E. coli. The properties of the recombinant S‐MBP and recombinant S‐MBP‐CRD were compared with those of the native human S‐MBP and the CRD of the native S‐MBP. The results indicated that functional human S‐MBP can be successfully expressed in Sf9 cells and functional S‐MBP‐CRD in E. coli. In addition, the amino‐terminal region and collagen‐like domain are required for higher oligomer formation and play important roles in complement activation.
Identification and characterization of an RNA specific primer for human tumor necrosis factor receptor I (TNFR‐I)
Authors: Kuo Kou‐Wha , Leung Maria FKL , Leung Wai‐Choi
Published: July 2007

An RNA specific primer amplified TNFR‐I cDNA from cellular RNA by single‐tube (ST) RT‐PCR without interfering with the presence of genomic DNA of cell was identified. The primer detected TNFR‐I from 1 ng of cellular RNA by ST RT‐PCR, and was not cross‐reacted with TNFR‐II despite of the homologous sequence between TNFR‐I and ‐II. In addition, the mechanism of the RNA specificity of the primer was investigated. An intron of 0.43 kb was inserted in the 5′‐925 of TNFR‐I mRNA sequence. The result corresponded with the previous determination of TNFR‐I gene structure. Thus, it appears that the RNA specificity of the primer might be resulted from the antisense primer hybridizing to exon‐intron junction. Owing to its high sensitivity and specificity to TNFR‐I RNA, the RNA specific primer may be potentially utilized for the determination of human TNFR‐I gene expression by in situ RT‐PCR.
Identification and cloning of a novel type I sering / threonine kinase receptor of the TGF‐β / BMP superfamily in rat prostate
Authors: Kang Yong , Reddi A. Hari
Published: July 2007

A cDNA clone encoding a new member of the type I serine / threonine kinase receptors of the TGF‐β / BMP superfamily was isolated from a rat prostate cDNA library. The predicted sequence of the encoded protein is 493 amino acids with a molecular weight of 55 kDa. It contains a putative cysteine rich extracellular domain, a transmembrane domain, a conserved type I receptor GS domain and a cytoplasmic serine / threonine kinase domain. The kinase domain shares 80% homology with that of ALK‐4 and ALK‐5 whose ligands are activin and TGF‐β. Northern blot analysis revealed that this prostate serine / threonine kinase receptor I (PSKR‐I) is highly expressed in rat dorsolateral prostate, coagulating gland (anterior prostate), cerebellum and adipose tissue.
Is protein kinase C alpha (PKCα) involved in vasopressin‐induced effects on LLC‐PK1 pig kidney cells?
Authors: Dibas Adnan , Mia Abdul J. , Yorio Thomas
Published: July 2007

The involvement of protein kinase C (PKC) in vasopressin‐induced effects on renal water reabsorption is still unresolved. Activation of PKC can be detected by its translocation from the cytosol (C) to the plasma membrane (PM). In LLC‐PK1 cells, the redistribution of PKCα, a predominant isoform of PKC detected, was studied utilizing western blotting after stimulation with vasopressin. Vasopressin (100 mU/ml) failed to induce a translocation of PKCα from the C to the PM. By contrast, phorbol myristate acetate (PMA, 200 nM), a potent activator of PKC, induced a relocalization of PKCα from the C to the PM. After 2 hours of treatment of cells with PMA, PKCα was predominantly detected in the PM and absent from the C. These results suggest that the signal transduction pathway of vasopressin in LLC‐PK1 cells does not involve PKCα activation and translocation.
The effect of NG‐monomethyl‐L‐arginine(LNMMA), an no‐synthase blocker on the survival of intracellular BCG within human monocyte‐derived macrophages
Authors: Fazal Nadeem
Published: July 2007

Human peripheral blood monocytes are permissive for the growth of Mycobacterium bovis (BCG), but the fate of nonpathogenic Mycobacterium bovis in these cells is not clearly known. Both oxidative and nonoxidative pathways have been implicated in killing of intracellular mycobacteria. Since human monocytes are not so far confirmed to release Reaction Nitrogen Intermediates (RNI), we tried indirect approach to inhibit the production of NO by the addition of NG‐monomethyl‐L‐arginine (LNMMA)‐an NO synthase blocker, in the infected cell cultures. In our studies adherent human peripheral blood monocytes were found to be permissive for the growth of Mycobacterium bovis, as measured by [3H] uridine uptake and confirmed by Colony Forming Unit (CFU) estimate. The killing of Mycobacterium bovis was not blocked by LNMMA, suggesting that it was not due to the production of Reactive Nitrogen Intermediates. Culture of the monocyte‐derived macrophages for 1 to 14 days before infection had no effect on the fate of Mycobacterium bovis, and no nitric oxide was detected in the culture supernatants of these infected cell cultures. Graded doses of sodium nitrite at physiological concentrations however failed to affect cell free cultures of Mycobacterium bovis. These findings suggest that the addition of NG‐monomethyl‐L‐arginine (LNMMA), an antagonist of L‐arginine oxidation and inhibitor of NO production, had no effect on the fate of Mycobacterium bovis in human monocytes and macrophages.
Salinity and osmotic stress‐regulated proteins in cowpea Rhizobium 4a (peanut isolate)
Authors: Wankhade Sharad , Apte S. K. , Rao K. Koteswara
Published: July 2007

The effect of salinity and osmotic stress on protein synthesis was studied in cowpea Rhizobium 4a. Osmotic component of salinity stress has been shown to induce ten proteins in a salt tolerant/ osmosensitive cowpea Rhizobium. 4a (groundnut isolate). Of these seven polypeptides were induced only by salt/osmotic stress while two were induced by heat shock. The results demonstrate a commonality as well as stress specificity of protein synthesis regulation.
Dopaquinone hydroxylation through topaquinone cofactor in copper amine oxidases: A simplified chemical model
Authors: Rinaldi Andrea C. , Rescigno Antonio , Sollai Francesca , Soddu Giulia , Curreli Nicoletta , Rinaldi Augusto , Finazzi‐Agrò Alessandro , Sanjust Enrico
Published: July 2007

A simple model, 4‐tert‐butyl‐1,2‐benzoquinone, was chosen to study the hydroxylation step of the tyrosine‐derived Dopaquinone residue at the active site of copper amine oxidases in the self‐catalytic generation of the Topaquinone cofactor. This hydroxylation step was studied both in the presence and absence of free copper(II), and was found to be dependent on pH value but not on the presence of metal ions. It is therefore proposed that, hydroxide ion and not water should be the true reactive species in this key biosynthetic step of the Topaquinone cofactor, and that the active site Cu2+ is implied, at this point of cofactor biosynthesis, in the quinonisation of Topa rather than in the hydroxylation of Dopaquinone.
Specific mitochondrial DNA deletions in canine myocardial ischemia
Authors: Marin‐Garcia Jose , Goldenthal Michael J. , Ananthakrishnan Radha , Mirvis David
Published: July 2007

The effect of myocardial ischemia on mitochondrial DNA (mtDNA) structure and the presence of specific mtDNA deletions was determined using the model of Ameroid constriction of canine coronary arteries. The incidence of specific deletions was high in both the endocardial and epicardial tissues perfused by the occluded vessel as compared to myocardial tissues perfused by the unconstricted vessels. Our results show that specific mtDNA deletions similar to the 5 kb and 7.4 kb human mtDNA deletions occur following canine myocardial ischemia. However the presence of these deletions did not correlate with specific mitochondrial enzyme defects.
Effect of insulin on DNA synthesis and kinetic parameters of thymidine kinase during liver regeneration
Authors: Mola P. Waliaula , Sudha B. , Paulose C. S.
Published: July 2007

The effect of insulin on cell proliferation in vivo has been studied in hepatectomised streptozotocin‐ diabetic rats. The extent of cell proliferation in sham and hepatectomized‐ control, diabetic and insulin treated rats were monitored by determining DNA content and [3H]thymidine incorporation into DNA. The kinetic parameters of thymidine kinase a regulatory enzyme for DNA synthesis was also studied in these groups. The rate of DNA synthesis in liver of streptozotocin ‐diabetic rats was significantly higher 24 hrs post‐hepatectomy compared to control and insulin treated diabetic groups. Kinetic studies of thymidine kinase revealed that there was no change in the Michaelis ‐Menten constant (Km) whereas maximum velocity (Vmax) was elevated in the diabetic hepatectomized groups compared to control and insulin treated hepatectomized groups. Thus our study elucidates the role of insulin in thymidine kinase activity and DNA synthesis.
Subcellular redistribution of hsp72 protein during cisplatin‐induced apoptosis in HeLa cells
Authors: Meléndez‐Zajgla Jorge , García Claudia , Maldonado Vilma
Published: July 2007

Exposure of HeLa cells to cisplatin results in the activation of apoptotic cell death. This drug induces DNA damage and generates reactive oxygen intermediates. Since cisplatin is highly reactive and binds to diverse proteins, it could create abnormal protein structures or nonspecific aggregates. For these reasons, we analyzed the expression and subcelullar distribution of hsp72, a heat‐shock protein that enables cells under stress to cope with damaged proteins. We did not observe any changes in the expression of hsp72 protein, although, by immunofluorescence studies, we detected a dramatic redistribution of the protein. These results and its probable relevance in the drug‐induced apoptotic phenomenon are discussed.
Further evidence for the mitogenic action of urinary trypsin inhibitor
Authors: Manilal Sushila B. , Scott G. Kenneth
Published: July 2007

We have previously shown that urinary trypsin inhibitor (UTI), also known as bikunin, was mitogenic for human fibroblasts at low concentrations, and growth‐inhibitory at higher concentrations, and have identified high‐ and low‐affinity cellular binding sites for this protein. We have now investigated fibroblast proteins which interact with bikunin. Bikunin binds to proteins of about 50K and 250K. The simplest interpretation is that the 50K protein may be a proteinase which is also the low‐affinity bikunin binding site, involved in growth inhibition, and that the larger protein may be responsible for the mitogenic response to bikunin. Inhibitors of intracellular calcium mobilisation also inhibit the mitogenic response to bikunin, and, by the measurement of the efflux of pre‐loaded 45Ca2+, bikunin at mitogenic concentrations can be shown to stimulate calcium mobilisation.
Synchronized induction in bFGF and Cdk2 during gastric ulcer healing
Authors: Slomiany B. L. , Piotrowski J. , Slomiany A.
Published: July 2007

The processes of gastric mucosal repair are regulated by growth factors triggering the signals for cell cycle progression. We examined the expression of gastric mucosal basic fibroblast factor (bFGF) and cyclin dependent kinase (p34Cdk2) at different stages of chronic ulcer healing. The results of assays revealed that ulcer healing was accompanied by an increase in mucosal expression of bFGF and Cdk2. A significant induction in bFGF and Cdk2 occurred by the 2nd day of ulcer healing, reached the respective maximums of 2.3‐ and 2.2‐fold increase by the 4th day of healing, and remained still elevated (20%) on the 14th day when the ulcer was essentially healed. The results demonstrate a synchronized induction in bFGF and cell cycle regulatory kinase, Cdk2, with ulcer healing.
Amino acid sequence of catechol 1,2‐dioxygenase (pyrocatechase) isozyme αα from Pseudomonas arvilla C‐1
Authors: Nakai Chieko , Yamazaki Gyohzan , Kagamiyama Hiroyuki , Nozaki Mitsuhiro
Published: July 2007

Catechol 1,2‐dioxygenase catalyzes the oxygenative ring cleavage of catechol to form cis,cis‐muconic acid, and contains a ferric form of iron as its sole cofactor. Pseudomonas arvilla C‐1 contains three isozymes of the enzyme, αα, αβ, and ββ [C. Nakai et al. (1990) J. Biol. Chem. 265, 660‐665]. We have determined the amino acid sequence of the αα isozyme by direct analysis. The sequence shared 77% homology with isozyme ββ, and had conserved tyrosyl and histidyl residues which are thought to be involved in the binding of ferric ion.
Purification and properties of chitinase from cabbage stems with roots
Authors: Chang Chen‐Tien , Hsueh Yi‐Ling , Sung Hsien‐Yi
Published: July 2007

Chitinase has been purified from the extract of cabbage stems with roots through successive steps of ammonium sulfate fractionation, Sephadex G‐75 gel filtration, chromatofocusing and Sephacryl S‐200 HR gel filtration. By these steps, the purity of the enzyme increased by 63 fold and the recovery of the enzyme activity was 18%. The purified enzyme was homogeneous when analyzed by SDS‐PAGE#. It showed an optimal pH of 6 and optimal temperature of 60°C for hydrolysis of ethylene glycol chitin (EGC). The molecular mass of the enzyme was 41 kDa, as determined by SDS‐PAGE. Heavy metal ions (1.5 mM) Ag+, Hg2+ and Fe2+, and chemical modification agents NAI (1 mM), NBS (0.5 mM) and CHD (0.5 mM) significantly or completely inhibited the activity of the enzyme. Substrate EGC at high concentrations also inhibited the activity. BSA (0.05%), Triton X‐100 (0.5%) and glycerol (50%) provided significant protection of the enzyme from freezing inactivation.
Cloning and characterization of cDNA for human adenylate kinase 2A
Authors: Lee Younghee , Kim Jae Wha , Lee In Ae , Kang Ho bum , Choe Yong‐Kyung , Lee Hee gu , Lim Jong‐Seok , Kim Hyo Joon , Park Chankyu , Choe In Seong
Published: July 2007

We have isolated and characterized a cDNA clone encoding human adenylate kinase 2A (AK2A) from cDNA libraries of fetal liver origin. The complete nucleotide sequence of the cloned 889 nucleotide cDNA fragment indicated that the deduced gene product of human AK2A is composed of 239 amino acids with a molecular weight of 26 kD. Comparison of these nucleotide and amino acid sequences with the corresponding sequences of bovine AK2A and rat AK2 revealed a high degree of conservation. It showed 91% and 98% homologies in DNA and amino acid sequences, respectively, with bovine AK2A throughout the full open reading frame. RNA blot analysis revealed that three species of mRNA were present with approximate sizes of 3.4, 2.1, and 1.0 kb. This gene was expressed in E. coli cells and the recombinant protein was enzymatically active.
Regulation of cholesterol synthesis and esterification in primary cultures of macrophages following uptake of Chylomicron remnants
Authors: Yu Kenneth C. ‐W. , Mamo John C. L.
Published: July 2007

The effects of chylomicron remnants (CR), β‐very‐low‐density‐lipoproteins (β‐VLDL) and low‐density‐lipoproteins (LDL) on intracellular cholesterol synthesis and esterification in primary rabbit macrophages was determined by assaying for HMG‐CoA reductase activity and cholesterol esterification. At physiological cholesterol concentrations, both CR and LDL inhibited cholesterol synthesis by almost 60% while β‐VLDL was less potent achieving only 30% inhibition. Cholesterol esterification rates were increased four‐fold by CR and LDL, whereas β‐VLDL increased esterification 14 times above controls. Qualitatively, the effect of CR on cholesterol synthesis and esterification in rabbit macrophages differs from observations in transformed macrophage cells. Quantitatively, the enhanced rates of cholesterol esterification and weak inhibition of cholesterol synthesis following β‐VLDL uptake may explain why this lipoprotein rapidly induces foam cell formation in vitro.
Genetic defect of the sodium pump‐defective mutant nap‐1 from the marine Vibrio alginolyticus
Authors: Hayashi Maki , Fujii Junko , Unemoto Tsutomu
Published: July 2007

The marine bacterium, Vibrio alginolyticus, has a respiratory chain‐linked Na+‐translocating NADH‐quinone reductase (NQR). Among several mutant cells defective in Na+ pump activity, Nap1 was a very stable mutant and a spontaneous revertant could not be isolated from Nap1. Using genetic irformation from the recently sequenced nqr operon, the genetic defects in Nap1 were examined, and the sodium pump‐defective mutant Napl was found to be caused by the insertion of a 1.2 kbp DNA fragment into the C‐terminal region of nqr6 gene.
Decrease in platelet, erythrocyte and lymphocyte acetylcholinesterase activities due to the presence of protease inhibitors in the storage buffer
Authors: Martín‐Valmaseda Eva M. , Sánchez‐Yagüe Jesús , Marcos Rocío , Llanillo Marcial
Published: July 2007

The effect of the presence of protease inhibit0rs in the storage buffer on acetylcholinesterase (AChE) activity was studied in crude membrane preparations from sheep platelets before and after solubilization of the membranes with Triton X‐100. Although sensitive to the action of trypsin, the biological activity of AChE remained unchanged for as long as 6 days at 4°C in a protease‐inhibitor‐free medium. At 10‐5 M final concentration PMSF reduced AChE activity to 50% after 24 hours of storage. This reduction was abolished in mixtures in which PMSF was present together with 3 mM EGTA, 5 mM EDTA and 0.i mg/mL trypsin inhibitor. Nevertheless, under these conditions, storage periods longer than 24 hours still drastically reduced AChE activity. A mixture of EDTA, EGTA and trypsin inhibitor also produced a decrease in AChE activity after 24 hours. A more complex cocktail of inhibitors including several commonly used peptides decreased AChE activity only if PMSF or EDTA were present in the mixtures. Similar results were obtained with sheep erythrocytes or lymphocytes, and bovine erythrocytes.
Expression and reconstitution of Calcineurin A and B subunits
Authors: Wei Qun , Lee Ernest Y. C.
Published: July 2007

Calcineurin consists of two subunits, a catalytic A subunit of 60 kDa and a regulatory B subunit of 19 kDa. Both the A and B subunits of rat brain calcineurin were expressed in E. coli. The B subunit was readily overexpressed in the pET‐21 a vector with yields of >70 mg of purified B subunit per 1 culture, representing >17% of the soluble E. coli protein. About 8 mg of purified A subunit was obtained. The enzyme activities of the A‐subunit and the reconstituted AB complex were found to be comparable to that of the bovine brain enzyme. The reconstitution of the AB complex was studied and shown to be rapid.
Detection of apoptosis in the brain of the zitter rat with genetic spongiform encephalopathy
Authors: Ookohchi Tamami , Ito Hisao , Serikawa Tadao , Sato Kenzo
Published: July 2007

Zitler rat develops genetic spongilorm encephatopathy accompanied with whole‐body tremors and flaccid paresis. To elucidale the mechanism of a neuronal cell death in the brain, we determined involvement of apoptosis in this rat. By Northern blot analysis, the elevation of mRNA levels were observed in c‐jun, c‐fos, c‐myc and p53 genes which were induced by apoptotic signals; conversely, expression of bcl‐2 was shown to be decreased in the zitter rat brain in contrast to the WTC control rat. Furthermore, TUNEL staining of fragmented DNA indicated apoptotic morphology in this brain. These results strongly suggested that the spongiform encephalopathy of the zitter rat was due to apoptosis in the brain cells.
Substrate‐binding and catalytic roles of Lys194 in the C‐terminus in human adenylate kinase by site‐directed random mutagenesis
Authors: Ayabe Takanori , Takenaka Hitoshi , Takenaka Osamu , Onitsuka Toshio , Shibata Koichiro , Uesugi Seiichi , Hamada Minoru
Published: July 2007

Site‐directed random mutagenesis of Lys194 residue in the C‐terminus of human adenylate kinase (AK) was performed, and six mutants were analyzed by steady‐state kinetics. K194‐mutants variously affected the apparent Michaelis constants (Km values) for ATP and AMP, although the kcat values strikingly decreased. The Lys194 residue appears to interact not only with MgATP2‐ but also with the AMP2‐ substrates by salt bridge formation with a nucleotide and to play a functional role in stabilizing the phosphate‐transfer during catalysis. Lys194 could be essential for substrate‐holdings and in catalysis and not replaceable to the other amino acids.
Comparative binding and toxicity of saxitoxin and saxitoxinol in mice and in cultured cells
Authors: Naseem Syed M. , Creasia Donald A.
Published: July 2007

The binding characteristics of saxitoxin (STX), a known voltage‐gated sodium channel blocker, and its analog saxitoxinol (STXOL), were studied in neuroblastoma, peritoneal macrophage, hepatocytes and PC‐12 cell lines. 3H‐STXOL bound to the cell‐surface sites which appear to be the same as those occupied by 3H‐STX and which can, therefore, be identified as STX receptors. The relative agreement of respective Kd obtained by saturation, competition, association and dissociation kinetics for STX and STXOL suggest the absence of any artifact in binding measurements. Unlike STX, STXOL was non‐toxic to mice by intratracheal instillation. The major advantage of using 3H‐STXOL is that the tritium label is not exchangeable. Data from this study suggest that 3H‐STXOL can be used to identify STX receptors at 37°C.
Characterization of a cyclic compound formed after spermine oxidation by lentil amine oxidase
Authors: Padiglia Alessandra , Medda Rosaria , Paci Maurizio , Sette Marco , Lorrai Anita , Floris Giovanni
Published: July 2007

Spermine is a substrate of lentil seedling amine oxidase and is oxidized at terminal amino groups to a dialdehyde: 2 mot of hydrogen peroxide and two mol of ammonia per mol of spermine are formed. In the presence of hight amounts of spermine, the aldehydic groups formed upon oxidation of spermine by the enzyme, may react with primary amino groups of free spermine leading to the formation of aromatic pyrimidinic ring after β‐elimination at secondary amino groups.
Inhibition of camel lens ζ‐crystallin/NADPH:Quinone oxidoreductase activity by chloranilic acid
Authors: Al‐Hamidi Abdulaziz A. A. , Shehu Riskuwa A. , Duhaiman Ali S.
Published: July 2007

Camel lens ζ‐crystallin/NADPH:quinone oxidoreductase activity was inhibited by chloranilic acid (2,5‐dichloro‐3,6‐dihydroxy‐1,4‐benzoquinone) with NADPH as an electron donor and 9,10‐phenanthrenequinone (PQ) as an electron acceptor in a time‐independent but concentration dependent manner. The IC50 of chloranilic acid was 1 μM. The inhibition was noncompetitive with respect to both NADPH and PQ as deduced by Lineweaver‐Burk plots. The estimated inhibition constant (Ki) was 0.8 μM for both NADPH and PQ. Examination of other benzoquinones suggested that the presence of ‐OH and ‐Cl on benzoquinone was essential for the inhibition.
Lipids may not be involved in the recognition of apocytochrome c during its transportation
Authors: Ma Q. F. , Yang F. Y.
Published: July 2007

It was shown that apocytochrome c was less insertion into monolayers from outer mitochondrial membrane lipids than into those from microsomal membrane lipids; the (α‐helix content of apocytochrome c induced by small unilamellar vesicles prepared from outer mitochondrial membrane lipids was less than by those from microsomal membrane lipids; the import efficiency of apocytochrome c into large unilamellar vesicles from outer mitochondrial membrane lipids was also lower than into those from microsome membrane lipids. No specific affinity between apocytochrome c and outer mitochondrial membrane lipids could be found. Import of apocytochrome c across the intact mitochondria, sealed outer mitochondrial membrane and microsome membrane vesicles was compared. Results showed that apocytochrome c was accumulated only in mitochondria, but not the other two kinds of vesicles.
Upregulation of HLA class‐I gene transcription in K562 cells by analogs of splenopentin (SP‐5)
Authors: Chatterjee‐Kishore Moitreyee , Agrawal Suraksha , Singh VK , Mathur KB , Agarwal Shyam S.
Published: July 2007

We report here the capacity of three analogs of Splenopentin, to up regulate transcription of the HLA ‐B7 gene in the K562 cell line. These cells normally do not transcribe the HLA class I genes. The Splenopentin analogs used in this study are effective at very low molar concentrations and are non‐toxic to cells at these levels. Electrophoretic Mobility Shift assays indicate that this transcriptional up regulation of HLA class I genes may be related to the appearance of novel class I promoter binding factors induced in the nuclei of treated cells.
Failure of D‐fructose to stimulate protein biosynthesis in pancreatic islets
Authors: Viñambres Concepción , Villanueva‐Peñacarrillo Maria L. , Valverde Isabel , Malaisse Willy J.
Published: July 2007

Rat pancreatic islets were incubated for 90 min with L‐[4‐3H]phenylalanine in either the absence or presence of D‐glucose and D‐fructose. Relative to basal value, D‐glucose (16.7 mM) increased the incorporation of the tritiated amino acid into trichloroacetic acid‐precipitable material. However, when D‐fructose was tested in the 80 to 240 mM concentration range, it failed to stimulate islet biosynthetic activity. Since D‐fructose causes, in the same concentration range, a dose‐related stimulation of insulin release, the dissociation between the biosynthetic and secretory responses to D‐fructose supports the view that the insulinotropic action of the ketohexose does not entail the same metabolic determinants as those operative in glucose‐stimulated islets.
Gene structure of PACO1, a petal senescence‐related related gene from Phalaenopsis encoding peroxisomal acyl‐CoA oxidase homolog
Authors: Do Yi‐Yin , Huang Pung‐Ling
Published: July 2007

The putative peroxisomal acyl‐CoA oxidase gene PACO1 from Phalaenopsis was isolated from a genomic library constructed in the λEMBL3 vector by screening with PACO1 cDNA. This gene encompasses approximately 14 Kb and is divided into seven exons by six introns. The consensus GT‐AG dinucleotides situated at all the boundaries between exons and introns. According to the genomic Southern analysis with different fragments of the first intron, PACO1 gene possesses a large intron about 10 Kb interrupting the first and secondary exon.
Purification of a 63 kDa β‐d‐galactoside binding lectin from cuttlefish, Todarodes pacificus
Authors: Ozeki Yasuhiro
Published: July 2007

A β‐d‐galactoside binding lectin (TPL) was newly purified from the body walls of the cuttlefish, Todarodes pacificus with lactosyl‐agarose affinity column chromatography and reversed‐phase HPLC. Many of the biological properties of the lectin were similar to those of the representative animal β‐d‐galactoside binding lectin, “galectin”. However, with a molecular mass of 63 kDa under reducing and non‐reducing conditions on SDS‐PAGE, TPL is larger than any previously reported galectins. The native molecular mass of TPL was estimated to be about 60 kDa by gel filtration, suggesting that it exists as a monomer and that a single polypeptide containing at least two sugar binding sites. The antisera raised against TPL did not crossreact with bull frog egg galectin‐1, suggesting that TPL is immunologically distinct from the lower vertebrate galectin.
Effects of K channel inhibitors on potassium transport in bovine adrenal chromaffin granules
Authors: Lobanov Nikolai A. , Szewczyk Adam , Wójcik Grazyna , Nowotny Marcin , Nałęcz Maciej J.
Published: July 2007

The Ca2+‐independent K+ selective channel in the membrane of adrenal gland chromaffin granules has earlier been identified. In the present report we describe new properties of this potassium channel using 86Rb+, a K+ analogue, flux measurements. The studies are performed in membrane vesicles prepared from chromaffin granules. The electrogenic 86Rb+ transport is inhibited by quinacrine, barium, zinc and magnesium. The effects of other potassium channel blockers on 86Rb+ transport into chromaffin granules are also reported.
Over expression of inducible proteins in Escherichia coli by treatment with ethanol
Authors: Basu Tarakdas , Poddar Ramendra Kumar
Published: July 2007

It is reported that ethanol enhances DNA synthesis in E. coli cells [Basu, T and Poddar, R K (1994), Folia. Microbiol., 39, 3‐6]. This communication reports that during growth of E. coli in the presence of 5% v/v ethanol, the derepressed expression of the cytoplasmic enzymes β‐galactosidase and D‐serine deaminase per cell increased approximately three fold, while that of the periplasmic enzyme alkaline phosphatase decreased approximately 40% compared to control cell levels. However, in cells transformed with the plasmid pSM 456, bearing phoA‐lacZ fusion, the level of induced synthesis of the hybrid protein PhoA‐LacZ, controlled by the phoA promoter, was elevated by 25% in the presence of 5% v/v ethanol. This result suggests that the induction of the alkaline phosphatase precursor has also been enhanced by the ethanol treatment; but the inhibition in the export of the precursor across the cytoplasmic membrane, by the influence of ethanol, may represent the reason for the deficient expression of active alkaline phosphatase. It is proposed that there is an ethanol‐mediated increase in DNA synthesis, resulting in gene amplification, which may enhance the synthesis of inducible proteins in ethanol‐treated cells.
Protein involvement in thermally induced structural transitions of pig erythrocyte ghosts
Authors: Zhadan Galina G. , Cobaleda Cesar , Jones Ahmik L. , Leal Fernando , Villar Enrique , Shnyrov Valery L.
Published: July 2007

Thermal transitions in pig erythrocyte ghosts were studied by differential scanning calorimetry and thermal gel analysis (TGA). Heating of the suspension of pig erythrocyte ghosts induced at least six thermodynamically irreversible transitions. Each of these transitions is believed to be due to a localized structural transition induced by thermal stress. Using TGA and covalent attachment of the anionic transport inhibitor regions in the thermograms corresponding to the heat sorption of some proteins of the pig erythrocyte ghosts were identified.
Suppression of Helicobacter pylori urease activity by sucralfate and sulglycotide
Authors: Slomiany B. L. , Piotrowski J. , Slomiany A.
Published: July 2007

The effect of gastroprotective agents, sucralfate and sulglycotide, on the in vitro activity of H. pylori urease was investigated. The bacterium was subjected to sonication, centrifuged, and the supernatant used as an enzyme source. The assays revealed that the rate of urea degradation was proportional to enzyme protein up to 100 μg and remained constant with time for 10 min. Introduction of sucralfate or sulglycotide to the assay system led to the reduction in the rate of ammonia production. With both drugs the optimal inhibition was attained at 10 μg/ml, at which dose a 63.1% decrease in urease activity occurred with sucralfate and a 70.2% inhibition was obtained with sulglycotide. The findings demonstrate that the inhibitory action of sucralfate and sulglycotide on H. pylori urease activity may be of value in the treatment of gastric disease associated with H. pylori infection.
Further biochemical and biophysical characterisation of scyllin, Scylla serrata hemolymph lectin
Authors: Chattopadhyay Tinku , Chatterjee Bishnupada
Published: July 2007

The amino acid and carbohydrate analysis of scyllin, a low molecular weight lectin purified from Scylla serrata (edible crab) haemolymph reveal that scyllin is rich in acidic and neutral amino acids and contains high amount of mannose. UV absorption of scyllin is perturbed by DMSO at 272 nm showing the presence of tryptophan molecule in scyllin exposed and accessible to the solvent. The oxidation of tryptophan molecule by N‐bromosuccinimide results in loss of haemagglutinating activity of lectin. The study of thermodynamic parameters of scylin‐glycoproteins interaction suggests that ceruloplasmin is the most potent inhibitors of scyllin of all the glycoproteins studied.
Menadione‐dependent reduction of tertiary amine N‐oxide by rat liver cytosol
Authors: Kitamura Shigeyuki , Tatsumi Kiyoshi
Published: July 2007

This study demonstrates the menadione‐dependent reduction of imipramine N‐oxide, a tertiary amine N‐oxide, to imipramine by rat liver cytosol in the presence of NADH or NADPH. A mechanism for the cytosolic reduction of the tertiary amine N‐oxide is proposed. Menadione is converted to its reduced form by a menadione‐reducing enzyme such as DT‐diaphorase and the reduced pyridine nucleotide, followed by reduction of the tertiary amine N‐oxide to the amine by the heme group of catalytic hemoproteins in the presence of reduced menadione as an electron donor.
The histidine‐805 in motif‐C of the phage SP6 RNA polymerase is essential for its activity as revealed by random mutagenesis
Authors: Jeong Woojin , Kang Changwon
Published: July 2007

In order to identify functional residues of the bacteriophage SP6 RNA polymerase, its C‐terminal one‐twelfth region was randomly mutagenized using polymerase chain reactions of its gene under the conditions for reduced fidelity of Taq DNA polymerase. Using a two‐vector system that permits phenotypic isolation of mutants with reduced in vivo transcription activity, 3 single and 1 multiple mutants were isolated. A single substitution of Gln for His805 resulted in complete inactivation of the enzyme. A multiple mutant carrying substitutions at 808, 820, 835, 843 and 848 also abolished the activity. However, changes of Pro856→Ser and Asp862→Glu individually reduced the activity only slightly. It is noteworthy that His805 is one of the two motif‐C residues that are absolutely conserved among all the DNA polymerases and monomeric RNA polymerases.
Age‐ and Tissue‐specific regulation of chicken inorganic pyrophosphatase
Authors: Syiem Donkupar , Sharma Ramesh
Published: July 2007

The activities and hormone regulatory patterns of inorganic pyrophosphatase (PPiase) of male Rhode Island Red (RIR) chickens were studied at two different postnatal ages (5‐ and 90‐day). The endogenous activity level was found to be significantly higher in the immature (5‐day) groups for all the tissues (liver, kidney and brain) studied as compared to that of mature (90‐day). Hydrocortisone (HC) administration significantly inhibited the PPiase activity in the immature chicken liver and did not affect the enzyme in kidney and brain at either age. In contrast, insulin increased significantly the activity of PPiase in the liver of immature chicken. Kidney PPiase, however, was unaffected to insulin treatment at immature age, while it showed increased activity in mature group. On the other hand, brain PPiase activity was significantly increased at both the ages studied. These findings indicate an age‐ and tissue‐specific regulation of PPiase activity by hydrocortisone and insulin in chicken.
Cloning and characterization of a new silver‐stainable protein SSP29, a member of the LRR family
Authors: Zhu Liangjin , Perlaky Laszlo , Henning Dale , Valdez Benigno C.
Published: July 2007

Silver‐stainable proteins (SSPs) are aspartic acid‐rich nuclear proteins which are silver stained under very specific conditions. Using a degenerate oligodeoxynucleotide probe which codes for acidic amino acid residues, a cDNA for a new SSP, referred to as SSP29, has been isolated. The cDNA‐derived amino acid sequence shows SSP29 has a molecular mass of 29 kDa, leucine‐rich repeats (LRR) near the NH2‐terminal region and acidic clusters at the COOH‐terminal portion, indicating that SSP29 is also a member of the LRR subfamily of acidic proteins which have been shown to be involved in antigen‐mediated cellular responses, leukemogenesis and differentiation. SSP29 can be stained by Ag‐NOR staining. SSP29 is expressed in all human tissues and cell lines tested, localized to nucleoplasm and translocated partially to the nucleoli after heat shock. Its interaction with RNA polymerase I suggests that SSP29 may participate in signal transduction that directs nucleolar activities by regulating ribosomal RNA biosynthesis.
Evidence for antioxidant nutrients‐induced pigmentation in skin: Results of a clinical trial
Authors: Postaire Eric , Jungmann Holger , Bejot Marie , Heinrich Ulrike , Tronnier Hagen
Published: July 2007

The aim of this study was to demonstrate that modification of the cellular redox‐equilibrium occurs as a consequence of antioxidant nutrients intake (carotenoids, vitamine E and vitamine C) and that these nutrients play a role in the pigmentation of the skin without any UV exposure. We conducted a randomized, double‐blind study in 20 healthy subjects to evaluate and to compare the efficacy of two mixtures of dietary antioxidants with regard to direct determination of melanin and carotenes by chromametry at selected skin sites and multiple reflection spectrometry from a 1 cm2 region of skin of different parts of the body. Efficacy was assessed by a significant improvement of these parameters, in comparison with measurements performed on the day of randomization, before dietary supplement intake. The formulations per capsule of study dietary supplements are: 13 mg of β‐carotene, 2 mg of lycopene, 5 mg of vitamine E and 30 mg of vitamine C (B13/L2) or 3 mg of β‐carotene, 3 mg of lycopene, 5 mg of vitamine E and 30 mg of vitamine C (B3/L3).

A 8‐week B13/L2‐supplementation lead to a detectable carotenodermia whereas the B3/L3‐supplementation not.

Signicative increase of melanin concentrations in skin were found after 4, 5, 6 and 8 weeks of dietary antioxidant intake in both groups (p<0.05). These results are discussed with regard to the redox control theory of melanocytes which regulates the tyrosinase activity.
Purification and characterization of manganese superoxide dismutase from Ganoderma microsporum
Authors: Pan Shu‐Mei , Ye Jr‐Shin , Hseu Ruey‐Shyang
Published: July 2007

Manganese superoxide dismutase (Mn‐SOD) in the mycelium of Ganoderma microsporum was purified to homogeneity by heat treatment at 70°C, ammonium sulfate fractionation, DEAE‐52 anion‐exchange chromatography, and Sephacryl SH‐200 chromatography. The molecular mass of its native form was estimated to be 98 kD by size‐exclusion chromatography. This enzyme is tetrameric composed of four subunits of equal size of 25 kD. The pI of this purified Mn‐SOD was located at pH 6.34 and 5.06 by isoeleetric focusing. Comparisons of 17 amino acids from the N‐terminus ofMn‐SOD subunit with the derived amino acid sequences from the reported Mn‐SOD eDNA clones of other sources indicated a high degree of homology among the Ganoderma genus but the Mn‐SOD from G. microsporum showed a high variation when compared with other organisms.
Protection by histadine against oxidative inactivation of AMP deaminase in yeast
Authors: Murakami Keiko , Onoda Yu , Kimura Junko , Yoshino Masataka
Published: July 2007

Oxidative inactivation of AMP deaminase and its protection were analyzed under the in situ conditions of yeast cells. AMP deaminase was readily inactivated by an exposure to hydrogen peroxide plus copper in permeabilized yeast cells. Addition of ascorbic acid further enhanced the inactivation of the enzyme, suggesting the hydroxyl radical produced by the Fenton reaction is responsible for the inactivation of the enzyme. Addition of histidine caused an effective protection against the inactivation of AMP deaminase by hydrogen peroxide‐induced hydroxyl radical. The concentration of histidine required for half‐maximal effect was within physiological range. Cysteine showed less effective protection against oxidative inactivation. Other amino acids as potent copper‐chelating agents as well as trolox and taurine showed little or no effect. Histidine can act as a physiological “antioxidant” in yeast cells.
The 28S fraction of rRNA in molluscs displays electrophoretic behaviour different from that of mammal cells
Authors: Barcia Ramiro , Lopez‐García José María , Ramos‐Martínez Juan Ignacio
Published: July 2007

Electrophoresis on agarose/formaldehyde gels of rRNA in molluscs display a pattern of bands which could suggest a RNase action due to incorrect manipulation of the samples. This study shows that the disappearance of the band corresponding to the 28S fraction is due to the denaturing conditions used when electrophoresis is carried out and not to RNases action.
Glycine68 to Histidine73 has an important role in the function of human tumor necrosis factor alpha
Authors: Cen Bo , Jin Weixing , Wu Haihong , Lu Jun , Dong Xueyin , Xu Xianxiu
Published: July 2007

Mutant human tumor necrosis factor alpha(hTNF α ) genes have been constructed by in vitro mutagenesis and expressed in Escherichia coli. A deletion involving Gly68 to His73 in hTNF α remarkably decreased the solubility and biological activity of hTNF α. From the above and results of a molecular dynamics simulation it is proposed that the region of Gly68 to His73 in hTNF α has an important role in the maintenance of the 3‐D structure and modulation of the biological activity of hTNF α.
Molecular cloning of rat P38 mitogen‐activated protein kinase and it's osmotic regulation in rat kidney
Authors: Yoshida Takumi , Sone Masayoshi , Ogawa Tatsuya , Nihei Hiroshi , Ozasa Hisashi , Tsukada Kinji , Horikawa Saburo
Published: July 2007

Rat p38 mitogen‐activated protein (MAP) kinase cDNA was isolated from rat kidney cDNA library using a PCR cloning strategy. The deduced amino acid sequence consists of 360 amino acids and shares 95.3% similarity with human p38 MAP kinase. The message for rat p38 MAP kinase was about 3.4 kilobases and was highly expressed in the kidney. In water‐deprived rat kidneys, the steady‐state levels of p38 MAP kinase mRNA increased about 2.7‐fold as compared with those of control rats. This result suggests that p38 MAP kinase may play an important role in the osmoregulation in the kidney.
The influence of intersubunit cross‐linking on the conformational changes of creatine kinase during denaturation by guanidine hydrochloride
Authors: Zhu Li , Fan Ying‐xin , Zhou Jun‐mei
Published: July 2007

Intersubunit cross‐linked creatine kinase (CK) has been prepared with the cross‐linking reagent dithiobis(succinimidyl propionate) (DTSP). Unfolding of cross‐linked CK during denaturation by guanidine hydrochloride (GuHC1), as monitored by intrinsic fluorescence, circular dichroism and fluorescence of the hydrophobic probe, 1‐aniline‐naphthalene‐8‐sulfonate (ANS), occurs in two stages with increasing GuHC1 concentration. The process is similar to that of the unmodified enzyme, but in the second stage, conformational changes of the cross‐linked enzyme need higher concentration of GuHC1, suggesting that there is a stable intermediate during its unfolding transition and the intermediate is stabilized by intersubunit cross‐linkage.
Cationic and secretory effects of 6‐O‐acetyl‐D‐glucose in rat pancreatic islets
Authors: Jijakli Hassan , Kirk Ole , Malaisse Willy J.
Published: July 2007

Selected esters of D‐glucose were recently proposed as tools to supply the sugar to cells affected by a defect in the carrier‐mediated process of hexose transport across the plasma membrane. The present study extends this knowledge to 6‐O‐acetyl‐D‐glucose. At a 2.0 mM concentration, the ester was indeed found to stimulate insulin release from perifused rat pancreatic islets. This coincided with stimulation of calcium influx into the islet cells, as judged from comparison of the ester effects on 45Ca outflow from prelabelled islets perifused in either the absence or presence of extracellular Ca2+. The facilitated inflow of Ca2+ did not appear attributable to a decrease in K+ conductance, 6‐O‐acetyl‐D‐glucose augmenting 86Rb efflux from islets prelabelled with this radioactive cation. Both the latter phenomenon and the insulinotropic action of 6‐O‐acetyl‐D‐glucose failed to be antagonized by D‐marmoheptulose. These findings indicate that the cationic events coupling the recognition of D‐glucose to the stimulation of insulin release are not identical in islets exposed to the hexose or its ester.
Functional analysis of the evolutionary conserved arginine 182 residue in human glutathione S‐transferase P1‐1
Authors: Kong Kwang‐Hoon , Park Hee‐Joong , Takahashi Kenji , Inoue Hideshi
Published: July 2007

The mutational replacement of Arg182 with threonine markedly decreased the specific activities for GSH‐conjugation reaction. The kcat of R182T for GSH‐[1‐chloro‐2,4‐dinitrobenzene] conjugation reaction was about 100‐fold smaller than that of the wild type. On the other hand, the affinity for GSH of R182T was not significantly affected. The pKa of the thiol group of GSH bound in R182T was ∼0.9 pK units higher than those in the wild type, but the kcat/Km1‐chloro‐2,4‐dinitrobenzene values at high pH were not so much lower than those of the wild type. The thermostability of R182T was significantly lower than that of the wild type. Therefore, Arg182 seems to be important for the construction of the active enzyme structure.
Levels of interleukin‐6, CRP and α2 macroglobulin in cerebrospinal fluid (CSF) and serum as indicator of blood‐ CSF barrier damage
Authors: Kanoh Yuhsaku , Ohtani Hideki
Published: July 2007

We measured the levels of interleukin‐6 (IL‐6), albumin, C‐reactive protein (CRP) and α2 macroglobulin (α2M), all of which have different spectrums of molecular weight, in the cerebrospinal fluid (CSF) and serum in 121 patients to evaluate damage to the blood‐cerebrospinal fluid barrier (BCB) in meningitis. There was an extraordinary high level of IL‐6 in the CSF when patients had bacterial or viral meningitis, but the level returned to a normal range within a week in almost all of these cases. There were no significant differences in CSF albumin levels among the different disease groups. The CRP level in CSF is considered to correlate with the serum level, and CSF CRP was higher in bacterial meningitis than in viral meningitis, however, CRP in CSF was increased in some of the infectious diseases without meningitis. The α2M in CSF, which tends to be at extraordinarily high levels when there is damage to the BCB, correlated highly with CSF cell counts. CSF IL‐6 seemed to be a useful indicator to identify the acute active phase of meningitis. CRP and α2M in CSF are considered to be useful to differentiate bacterial meningitis, bacterial infection without meningitis and viral meningitis. Extraordinarily high levels of α2M, which has a high molecular weight, in CSF is indicative of BCB damage.
Integrin expression and collagenase activity of RSV‐transformed Syrian hamster fibroblasts, differing in spontaneous meatstasizing
Authors: Kozlova N. I. , Morozevich G. E. , Soloveyva N. I. , Vinokurova S. V. , Berman A. E.
Published: July 2007

The expression of extracellular matrix (ECM) specific receptors, integrins, and the activities of type IV collagenase and interstitial collagenase were investigated in two strains of oncogenically transformed fibroblasts, drastically differing in spontaneous metastasizing. Both strains were shown to express quite limited patterns of integrins. Of those, α5β1 integrin is greatly reduced on highly metastatic (HM) cells, while the expression of αvβ3 is strongly suppressed on lowly metastatic (LM) fibroblasts. No differences between the strains were found in either intracellular or secreted activities of type IV collagenase, while the activity of interstittial collagenase, secreted by HM cells, was twice as much as secreted by LM cells. The results imply the role of cooperated modifications of integrin‐directed cell‐ECM interaction and collagenase activity in establishing a metastatic phenotype.