bluuebunny/crossref_metadata_2025 huggingface dataset:
Prostaglandin E2 derivative, enprostil, reduces hepatic triacylglycerol content in Japanese quail
Authors: Murai Atsushi , Murakami Alice E. , Okumura Jun‐ichi , Furuse Mitsuhiro
Published: July 2007

This study investigated the effect of prostaglandin E2 derivative, enprostil, on the hepatic lipid metabolism of Japanese quail. In experiment 1, the birds received 0.1, 1, 10 or 100 µg enprostil/kg body weight intraperitoneally, and they were killed after 10, 30, 60 and 120 min. After 60 min, hepatic triacylglycerol content was reduced most effectively by 10 µg enprostil/kg body weight. In experiment 2, effect of enprostil (10 µg/kg body weight) on liver enzyme activities associated with lipid metabolism and plasma lipid concentrations was examined. There were no significant differences in malic enzyme and carnitine palmitoyltransferase activities. Peroxisomal β‐oxidation in birds which received enprostil was significantly lower than that in birds receiving the vehicle. Enprostil caused a significant decrease in plasma triacylglycerol and non‐esterified fatty acid concentration. We conclude that the administration of enprostil reduces hepatic triacylglycerol content through the decreased influx of non‐esterified fatty acid.
Does the reduced form of neopterin serve as an antioxidant?
Authors: Mori Hiroko , Arai Toshiyuki , Mori Kenjiro , Suzuki Toshinori , Makino Keisuke
Published: July 2007

The oxygen free radical scavenging activity of the reduced form of neopterin, 5,6,7,8‐tetrahydroneopterin (NPH‐4), was investigated in vitro using electron paramagnetic resonance spectroscopy. In the hypoxanthinexanthine oxidase (HPX‐XOD) reaction system, NPH‐4 suppressed the production of the superoxide spin adduct, but enhanced that of the hydroxyl radical spin adduct. The hydroxyl radical spin adduct was also observed in the solution containing NPH‐4 and XOD but excluding HPX. These results indicate that NPH‐4 is readily oxidized to some intermediates before the reaction with oxygen free radicals and that the substance which actually serves as an antioxidant in vivo is not NPH‐4 but its metabolites.
Acylphosphatase stimulates Ca2 transport and Ca2 ‐dependent ATPase activity in cardiac sarcoplasmic reticulum
Authors: Fiorillo Claudia , Nediani Chiara , Marchetti Elena , Pacini Alessandra , Liguri Gianfranco , Nassi Paolo
Published: July 2007

Acylphosphatase purified from heart muscle actively hydrolyzes the phosphoenzyme intermediate of cardiac sarcoplasmic reticulum Ca2+‐ATPase. This effect was evident with acylphosphatase concentrations (up to 100 units/mg sarcoplasmic reticulum protein) that fall within the physiological range, and the low value of the apparent Km, on the order of 10‐7M, suggests a high affinity towards this special substrate. Moreover, acylphosphatase addition to sarcoplasmic reticulum vesicles significantly enhanced the rate of Ca2+‐dependent ATP hydrolysis. Maximal stimulation, observed with 100 units/mg vesicular protein, resulted in an ATPase activity which was about two folds over basal value. The same acylphosphatase concentration increased at a similar extent the rate of ATP driven Ca2+ influx into sarcoplasmic reticulum vesicles. Taken together these findings lead to suppose that acylphosphatase, owing to its hydrolytic activity, induces an accelerated turnover of the phosphoenzyme intermediate, whence an overall stimulation of heart sarcoplasmic reticulum Ca2+ pump, affecting both ATP hydrolysis and Ca2+ influx.
In vitro amplification of human basic fibroblast growth factor mRNA by RNA‐specific exon‐junction primers
Authors: Sheu Hamm‐Ming , Leung Maria FKL , Leung Wai‐Choi , Kuo Kou‐Wha
Published: July 2007

Highly sensitive and RNA‐specific primers for the determination of human basic fibroblast growth factor (bFGF) gene expression by RT‐PCR were identified. The RNA‐specific primers could amplify bFGF mRNA from 10 pg to 1 ng of total cellular RNA without interfering with the presence of genomic DNA of the cell. The feasible temperatures of the primers annealed to the template were 5t°C, 60°C and 65°C. In addition, different locations of primers on the bFGF mRNA molecule yielded distinct amounts of RT‐PCR products from the same concentration of RNA, suggesting that the mRNA secondary structure of bFGF affected the RT‐PCR. Owing to high sensitivity and specificity of the primers to bFGF RNA, the RNA‐specific primers may be potentially utilized for the determination of human bFGF gene expression by in situ RT‐PCR.
Isolation and characterization of Saccharomyces cerevisiae SAB2, a suppressor gene for temperature‐sensitive phenotype of ars‐binding factor 1 mutant
Authors: Shin Yong‐Hyun , Goo Duck‐Mo , So In‐Seop , Rhode Peter R. , Campbell Judith L. , Kim Jiyoung
Published: July 2007

A high‐copy number suppressor of yeast abf1‐5 mutant, a temperature‐sensitive lethal mutant, was isolated and named SAB2 (suppressor of ABF1). Hybridization to a yeast chromoblot and to prime clone grid filters revealed that the SAB2 gene was located near the yeast SUP3 on chromosome XV. The suppressor activity was contained in a 2.5 Kbp DNA region of the SAB2 plasmid. The nucleotide sequence of the DNA region contained a long open reading frame, which turned out to encode for yeast tryptophan permease. Four putative ABF1 binding sites were found in the promoter and the structural regions of the tryptophan permease gene. Binding of ABF1 to two of the sites tested in this study was detected. Our results indicate that ABF1 may be involved in the transcriptional control of the yeast tryptophan permease gene.
Characterization of a novel nucleolytic activity of acylphosphatases
Authors: Chiarugi Paola , Raugei Giovanni , Fiaschi Tania , Taddei Letizia , Camici Guido , Ramponi Giampietro
Published: July 2007

A novel enzymatic activity on nucleic acids was discovered in both muscle type (MT) and erythrocyte or common type (CT) isoforms of acylphosphatase, an enzyme that was previously known as a hydrolase (E.C.3.6.1.7). Both deoxyribonucleic and ribonucleic hydrolitic activity were assayed on a variety of substrates. Our results demonstrate that acylphosphatase possesses both Mg++ dependent deoxyribonuclease and ribonuclease acivities, at pH ranging from 5.0 to 6.8. Furthermore, we present evidences, for both isoenzymatic forms, of the coexistence of exonucleolytic and endonucleolytic activities on DNA.
Characterization of high‐affinity protein D binding sites on the surface of rat epididymal spermatozoa
Authors: Hall Joseph C. , Tubbs Christopher E. , Li Yuehua , Ashraf Salman
Published: July 2007

125I‐labeled protein D was shown to bind to whole rat epididymal sperm in a time‐dependent, saturable, and reversible manner. Scatchard analysis indicates that there are 3.4 × 105 binding sites/sperm cell with a binding efficiency (Kd) of 2.3 × 10‐10 M. Binding of protein D to sperm reaches equilibrium by 95 min. and is reversible by 30 min. Specific binding of protein D to sperm pre‐treated with a dilute trypsin solution is reduced significantly when compared with controls. Our findings establish that protein D‐specific binding proteins displaying characteristics of a true receptor are present on the surface of rat epididymal sperm.
Hydroxycitrate causes altered pyruvate metabolism by tumorigenic cells
Authors: Board Mary , Newsholme Eric
Published: July 2007

Metabolic fates of pyruvate (CO2, lactate, citrate) in normal and neoplastic cells have been assessed. Pyruvate consumption by tumour cells falls (by 72‐85%) and mean percentage oxidation rises from 75% to 91% with hydroxycitrate. Ratios of rates of oxidation of (3‐14C‐pyruvate): (1‐14C‐pyruvate), indicating CO2 produced from TCA cycle activity: that from PDH activity, are higher for tumorigenic (0.17‐0.24) than for non‐tumorigenic (0.005‐0.04) cells and increase (0.27‐0.65 and 0.13‐0.29, respectively) with hydroxycitrate. Although maximal ATP‐citrate lyase activities do not correlate with malignancy, citrate may be a major fate of glutaminolytic pyruvate in tumour cells. Citrate accounts for 14‐37% of consumed glutamine compared with 11‐13% being recovered as CO2. By contrast, ≈︁100% of glycolytic pyruvate is converted to lactate.
Tempace and troxyl‐novel synthesized 2,2,6,6‐tetramethylpiperidine derivatives as antioxidants and radioprotectors
Authors: Metodiewa Diana , Skolimowski Janusz , Karolczak Stefan
Published: July 2007

Two novel 2,2,6,6‐tetramethylpiperidine derivatives (Tempace and Troxyl) were synthesized and their capacity to act as scavengers of superoxide, inhibitors of iron and ascorbate‐driven Fenton reaction and radioprotectors was tested. The possibility for one‐electron oxidation of novel compounds by heme‐ferryl species was also examined. The results clearly indicate that Tempace and Troxyl are acting as promising antioxidants and radioprotectors thus providing a base for further investigations and pharmacological applications.
Modulation of muscarinic receptor‐stimulated phosphoinositide breakdown by sulfhydryl group modification is a general response in different rat brain regions and depends on the stage of brain development
Authors: Balduini Walter , Cattabeni Flaminio , Cantoni Orazio
Published: July 2007

In a previous study we reported that muscarinic receptor‐stimulated phosphoinositide breakdown in rat cortical slices is significantly reduced by modification of critical thiols located at the post‐receptor level [1]. We have now extended this observation by investigating whether this effect is a general response in different brain areas and is differentially regulated, within the cortex, in the neonatal and adult brain. Experimental results have demonstrated that indeed the effect of sulfhydryl group modification on muscarinic receptor‐stimulated phosphoinositide breakdown appears to be mediated by a general mechanism operative in different brain regions (cortex, hippocampus and striatum). In addition, this response is more pronounced in the early stages of brain development, when muscarinic receptor agonists lead to the highest inositol phosphate accumulation.
Inhibition of 5′DI and 5′DII L‐tiroxine (T4) monodeiodinases. Effect on the hypothalamo‐pituitary ovarian axis in adult hypothyroid rats treated with T4
Authors: Ortega E. , Osorio A. , Ruiz E.
Published: July 2007

Hypothyroid female rats were treated with T4 and their 5′DI and 5′DII deiodinases were inhibited by PTU and IOP administration to determine whether the effect of T3 on reproductive function is a primary event at hypothalamo‐pituitary levels or ovarian levels. Hypothyroid adult female rats were divided into four groups : Hypothyroid without treatment (H); hypothyroid treated with T4 (H‐T4); hypothyroid treated with T4 plus propylthiouracil (H‐T4‐PTU), and hypothyroid treated with T4 plus iopanoic acid (H‐T4‐TOP). A group of euthyroid rats (E) was included as control. Estrous cycle, ovarian histological changes and serum estradiol and gonadotropin levels (basal and after GnRH) were searched in all groups.

In view of our results and since sexual cycles and puberal pattern in gonadotropin secretion were restored after all treatments we can suggest: That T4 could have an intrinsic effect on reproductive function in adult hypothyroid female rats or that another compensatory T3 mechanism unaffected by IOP could exist. The present report points out that the effect of T3 on reproductive function could be a primary event at hypothalamo‐pituitary levels although an effect at ovarian levels could not be excluded.
A mutant metallothionein which has inverse fragment composition exhibits high cadmium‐binding ability
Authors: Yamaguchi Rie , Kurasaki Masaaki , Kojima Yutaka
Published: July 2007

In order to investigate the role of the α‐fragment of metallothionein (MT) in metalbinding, two mutant MTs, βAlaαCys‐ and αCysβCys‐mutant MTs, expressed in Escherichia coli were analyzed for their metal‐binding ability. A mutant MT where all of the cysteine residues in the β‐fragment of MT were substituted by alanine residues and another mutant MT that had the inverse fragment composition (α‐β, i. e., β‐α in wild‐type MT) were designated as the βAlaαCys and the αCysβCys‐mutant MT's, respectively. Both expressed Cd‐binding mutant MTs were identified by amino acid analyses. From their metal‐binding capacities, the two mutant MTs exhibited higher Cd‐binding abilities than wild‐type MT. The results suggested that the α‐fragment plays a key role in the Cd‐binding of MT, and that the metal‐binding tightness of MT is dependent on the metal‐ binding ability of a prior fragment in MT.
Detection of polymorphic FGF receptor 1 mRNA in bovine lens epithelium by PCR
Authors: Marunouchi Tohru , Hosoya Hiromi , Kousaka Masashi , Lee Xiao Rong , Majima Yoshinao
Published: July 2007

The expression of FGFR 1, 2 and 3 but not FGFR 4 was detected in bovine lens epithelial cells. FGFR 1 expression of both the 2‐Ig and 3‐Ig forms was detected by a difference in the lengths of the PCR products. Furthermore, mRNA which included or excluded six nucleotides corresponding to two amino acids in the acid box region of FGFR 1 was detected by a difference in mobility of the homo‐ and hetero‐duplex strands formed in the PCR reaction.
Bovine kidney low molecular weight acid phosphatase: FMN‐dependent kinetics
Authors: Granjeiro José M. , Ferreira Carmen V. , Jucá Marilena B. , Taga Eulázio M. , Aoyama Hiroshi
Published: July 2007

A low molecular weight bovine kidney acid phosphatase, electrophoretically homogeneous and with a relative molecular mass of 17.8 kDa, was used in this work. Among the various substrates tested, FMN was found to be the most effective, at pH 7.0. Distinct activation energy values were obtained for p‐nitrophenyl phosphate‐ (45.44 kJ mol‐1) and flavin mononucleotide‐ (28.60 kJ mol‐1) hydrolysis reactions. The FMN hydrolysis was strongly inhibited by Cu2+ and pCMB, but activated by guanosine. Pyridoxal‐phosphate and vanadate were competitive inhibitors for the FMN‐dependent reaction.
Influence of mycobacterium tuberculosis catalase gene (KatG) expression on nitric oxide production and the intracellular growth of transfected mycobacterium smegmatis strains within murine macrophages
Authors: Fazal Nadeem
Published: July 2007

Expression or possession of catalase gene may interfere with the iNOS/NO pathway in mycobacteria, hence altering their capacity to survive within macrophages. Therefore, strains of M. smegmatis with an inactive catalase‐peroxidase gene (KatG), or into which the KatG gene of Mycobacterium tuberculosis had been transfected, were used to study the influence of catalase on nitric oxide (NO) production and mycobacterial survival within infected murine J774 macrophages. High levels of nitrite (40‐70 nM) were detected in IFN‐γ and LPS activated, infected murine cell culture supernatants, however, NO2‐ titres produced by infected murine cells did not diffcr between various catalase phenotype strains. Similarly, no significant difference in mycobacterial killing was also observed among the five strains of M. smegmatis tested over a 3 day infection period.
Amino acid metabolism in the kidneys of genetic and nutritionally obese rats
Authors: Herrero M. Carmen , Remesar Xavier , Bladé Cinta , Arola Lluís
Published: July 2007

The ability of the kidney to take up and/or release amino acids has been determined in two models of obesity in Zucker rats, one genetic and the other nutritional (diet‐obese). There was a noticeable increase in gluconeogenic amino acids in the arterial blood of diet‐obese animals whereas the genetically obese rats showed small variations in the levels of these amino acids. There were significant decreases in renal Gly and Ser, only in the genetically obese rats. Genetically obese animals showed an increase in Glutamine synthetase activity. The uptake and/or release of amino acids showed important variations between the groups. The diet‐obese group exhibited greater variation, since this group took up Glu, Ala, Gy, Phe and Citrulline and released Gln, Ser, Arg and Tyr. Genetically obese rats took up Gln, His and Taurine and released Ser. These different patterns may be related to variations in the whole body metabolic rate, since the diet‐obese group was more active than the genetically obese group.
Ageing‐associated large‐scale deletions of mitochondrial DNA in human hair follicles
Authors: Kao Shu‐Huei , Liuz Ching‐San , Wang Shuo‐Yue , Wei Yau‐Huei
Published: July 2007

Hair follicles plucked from the bi‐temporal region of the scalp of 433 Chinese subjects of different ages were used for the examination of ageing‐associated mutations of human mitochondrial DNA (mtDNA). By use of PCR techniques, we detected the 4,977 bp and 7,436 bp deletions of mtDNA in hair follicles from aged individuals. The frequencies of occurrence of both mtDNA deletions were found to increase with age of the subject. Moreover, we employed a semi‐quantitative PCR method to determine the proportion of the 4,977 bp deleted mtDNA (dmtDNA) in hair follicles. The results showed that the average proportion of the 4,977 bp dmtDNA in hair follicles were 0.05%±0.01%, 0.00%, 0.55%±0.05%, 0.52%±0.24%, 0.65%±0.17%, 1.33%±0.25%, and 1.89%±0.81% for the subjects in the age groups of 21‐30, 31‐40, 41‐50, 51‐60, 61‐70, 71‐80, and 81‐99, respectively. Furthermore, we screened all the subjects harboring the 4,977 bp and/or 7,436 bp deletions for tandem duplications in the D‐loop region of mtDNA by PCR with back‐to‐back primers. The results showed that none of the previously reported tandem duplications were present in all the hair follicles examined. This indicates that tandem duplications do not predispose to large‐scale deletions of mtDNA. However, the data suggest that mtDNA deletions occur and accumulate in hair follicles during human ageing. As hair follicles can be easily and non‐invasively obtained from the human, we suggest that the age‐dependent accumulation of dmtDNAs in hair follicles may be used for the monitoring of human ageing process.
The control of oxygen affinity in the three human embryonic haemoglobins by respiration linked metabolites
Authors: Hofmann Oliver M. , Brittain Thomas , Wells Rufus M. G.
Published: July 2007

The effect of CO2, ATP and lactate ions on the oxygen affinity of the three human embryonic haemoglobins have been studied. CO2 lowers the affinity of both the adult and embryonic haemoglobins for oxygen, as does ATP. The ATP effect follows a simple binding process with an equilibrium constant in the mM range. Lactate ions have no effect on the oxygen equilibrium process, over the concentration range studied. These findings are discussed in terms of the likely physiological conditions experienced by human embryonic red blood cells.
Patterns of expression of Hoxa‐11 in micromass cultures of chick limb mesenchyme from various stages suggest a role for Hoxa‐11 in the specification of the zeugopod
Authors: Rogina Blanka
Published: July 2007

During chick limb development the expression of Hoxa‐11 is specifically associated with the prospective zeugopod (radius+ulna) region. A series of micromass cultures were performed using mesenchyme from whole stage 18/19 or 20/21 limbs or mesenchyme from the tips of stage 22/23, 23/24 or 25 limbs. In such cultures, limb mesenchyme undergoes chondrogenesis paralleling its fate in vivo. Hoxa‐11 and collagen type II mRNA were examined by dot blot and in situ analysis at different time points. The highest level of Hoxa‐11 mRNA was found in micromass cultures of the tips of stage 22/23 and 23/24 limbs at 48 hours. Between stages 22 and 24, the determining events occur which subsequently lead to the formation of the zeugopod by the mesenchyme present in the tips at these stages. This correlation further supports a possible role of Hoxa‐11 in the specification of the zeugopod.
Autolytic activation mechanism of Bombyx acid cysteine protease (BCP)
Authors: Takahashi Susumu Y. , Yamamoto Yoshimi , Watabe Shoji , Kageyama Takashi
Published: July 2007

Acid cysteine proteinase in the eggs of the silkmoth, Bombyx mori, exists as an inactive proenzyme. This 47‐kDa pro‐BCP1 zymogen molecule can be processed in vitro into an enzymatically active 39‐kDa BCP molecule (11, 12). In this current study, the maximum rate of processing in vitro was achieved at approximately pH 4.0, at a temperature of 37 °C under reducing conditions. The rate of conversion was not affected by increasing concentrations of pro‐BCP. We prepared immobilized BCP bound to AH‐Sepharose and examined the activation. Immobilized pro‐BCP was autolysed, although the rate of processing was slow, indicating that the reaction might be an intramolecular one. Kinetic experiments suggest that the mechanism is likely to involve a stepwise reaction, in which pro‐BCP is converted to an active enzyme through intermediate forms releasing small peptides stepwise.

The results suggest that autocatalytic cleavage (intramolecular) is a major processing step in the early stage of pro‐BCP activation.
High level expression of recombinant plasminogen activator inhibitor‐1 in escherichia coli and generation of its mutants involving ASP125, GLU128 and GLU130
Authors: Sui Guang‐Chao , Sun Hong , Zhang Man , Hu Mei‐Hao
Published: July 2007

Plasminogen activator inhibitor‐1 (PAI‐1) cDNA was expressed in Escherichia coli (E. coli) with high efficiency using a heat‐inducible vector. About 100 mg of recombinant PAI‐1 (rPAI‐1) could be obtained from 1 liter of bacteria culture, rPAI‐1 in inclusion bodies was purified by pI precipitation and Sephadex G‐75 chromatography. After treatment with 4 mol/L guanidinium chloride and dialysis, the largely inactive PAI‐1 gained considerably in activity as judged by its reaction with low molecular weight u‐PA (LMW‐u‐PA). Degenerated oligonucleotides containing ApaI site and mutations at Asp125, Glu128, Glu130 in PAI‐1 cDNA were synthesized. To facilitate the introduction of mutations, an ApaI site was first generated in PAI‐1 cDNA using one of the oligonucteotides. Taking advantage of the ApaI site, thirteen PAI‐1 mutants involving Asp125, Glu128 and Glu130 were produced with these oligonucleotides using PCR. Most of the PAI‐1 mutants had a similar activity as compared to wild type PAI‐1, while some of the triple‐site mutants had completely lost their activity.
Interactions between heparinoids and alcohol dehydrogenase
Authors: Paulíková Helena , Valusová Eva , Antalík Marián
Published: July 2007

The interaction between polysulfated polysaecharides (low‐molecular‐weight heparin LMWH, dextran sulfate DS and pentosan sulfate PS) and yeast alcohol dehydrogenase (YADH) was investigated. The fluorescence and UV spectra of YADH after adding the tested polysaccharides have confirmed the interaction between the enzyme and these compounds. Kinetic studies have shown that LMWH, DS and PS are inhibitors of YADH (mixed type with respect to NAD). The most potent inhibitor is PS (ID50=37.5 ng/ml, Ki=0.6 μM). The inhibition effect depends on the ionic strength (the inhibition decreased by about 50% in the presence of 100 mM Na2SO4) and pH value (the inhibition decreased at pH>7). The results indicate that the inhibition effect of these polyanions is caused by their electrostatic interactions with the NAD‐binding region of YADH.
Study of peptide‐protein interaction by high‐performance liquid chromatography. Effect of hydrophobic and steric parameters
Authors: Szikszay Zoltán , Forgács Esther
Published: July 2007

The interaction of 16 peptides was studied on zein‐coated silica column by high‐performance liquid chromatography. The separation factor, theoretical plate number and the asymmetry factors of the peaks were calculated. Stepwise regression analysis was applied to study the peptide‐protein interactions and to find the molecular substructures involved in the interactions and to define their character. Zein‐coated silica column was suitable for the determination of peptide‐protein interaction and the investigated peptide‐protein interactions were found to be mixed (hydrophobic and steric) character.
Studies on the catalytic activity of human hepatic porphobilinogen deaminase
Authors: Mazzetti Marta B. , Tomio J. María
Published: July 2007

Porphobilinogen deaminase was purified from human hepatocytes. A variety of group specific reagents have been used to achieve site‐specific modifications to evaluate the potential role of such groups in the whole catalytic cycle. Treatment with dicarbonyl reagents caused a rapid loss in activity that was time and concentration, dependent. Protection experiments revealed that arginine residues are involved in the binding of the substrate. Treatment with Woodward's reagent K showed the fastest inactivation of deaminase (85% in 30 sec at 30mM) which was pH dependent and could be prevented by the presence of substrate, suggesting that deprotonated carboxylated groups from Asp/Glu are essential for catalytic activity. Kinetic analysis gave values of 0.3 sec‐1 for the k3 rate constant and 8×10‐2 M for the KI of the non covalent complex between deaminase‐Woodward's Reagent K
Reversible cell cycle arrest with concomitant p21/WAF1 overexpression and mitochondrial destruction by nitric oxide
Authors: Esumi Hiroyasu , Chin Keishou , Kurashima Yukiko , Hasegawa Fumio , Ogura Tsutomu , Yoneda Makoto , Ozawa Takayuki
Published: July 2007

When human hepatocellular carcinoma Hep‐G2 cells were treated with the NO‐generating compounds, S‐nitroso‐N‐acetyl‐DL‐penicillamine(SNAP) or (±)‐(E)‐4‐ethyl‐2‐(E)‐hydroxyimino]‐5‐nitroso‐3‐hexenamide, cells stopped growing. Most cells were found to be in either G1 or G2/M phase and the dye‐exclusion test revealed that the cells were alive. Electron microscopic examination confirmed the integrity of cells and nuclei. Nuclear staining with the DNA‐binding dye H33258 revealed that cells did not undergo apoptosis although dramatic changes in mitochondrial morphology were noticed within 6 hr of treatment with SNAP by electron microscopy. Western and northern blot analysesrevealed that cells overexpressed p21/WAF1. The growth arrest was released by withdrawal of the NO‐generating compound and cells started to divide within 24 hr after withdrawal of the compounds.
Expression or possession of catalase gene does not alter the capacity of M. smegmatis to survive within human macrophages
Authors: Fazal Nadeem
Published: July 2007

Mycobacterium tuberculosis genes are expressed and screened in Mycobacterium smegmatis as a host, hence their survival in human monocytes needs to be determined. Strains of Mycobacterium smegmatis with an inactive catalase‐peroxidase gene (KatG), or into which the KatG gene of Mycobacterium tuberculosis had been transfected, were used to study the influence of catalase on intracellular mycobacterial survival within human monocyte‐derived macrophages. The five strains of Mycobacterium smegmatis were observed to grow at similar rates, irrespective of their catalase phenotype, within IFNγ‐treated or untreated human macrophage cultures, over a 3 day infection period. Moreover, there was no differential pattern of growth or inhibition among the strains of Mycobacterium smegmatis studied.
Characterization of Ca2 transport activity associated with a non‐mitochondrial calcium pool in the rodent malaria parasite P. chabaudi
Authors: Passos Ana P. D. , Garcia Célia R. S.
Published: July 2007

Non‐mitochondrial calcium deposits were investigated in the intraerythrocytic malaria parasite Plasmodium chabaudi at the trophozoite stage by means of arsenazo III in the presence of ATP and the mitochondrial poisons, antimycin and ollgomycin. Addition of vanadate and 2,5‐di‐(t‐butyl)‐1,4‐hydroquinone (BHQ), both known to interact with SERCA pump, induced calcium release by permeabilized parasites when the medium free calcium concentration was kept at 3.5 μM. The tumor promoter thapsigargin also caused elevation of the free calcium concentration in permeabilized parasites. Our results support the view that P.chabaudi sequesters calcium in an exchangeable form and maintains its calcium homeostasis by way of an endoplasmic reticulum Ca2+ pump.
Oxidation of feric‐cytochrome c by N, N′‐bis (2‐hydroperoxy‐2‐methoxyethyl)‐1,4,5,8‐naphthalene tetracarboxylic diimide, NP III ‐ site specific modification of cytochrome c
Authors: Matsugo Seiichi , Yasui Fumihiko , Konishi Tetsuya
Published: July 2007

When the synthetic hydroxyl radical generator, N, N′‐bis (2‐Hydroperoxy‐2‐methoxyethyl)‐1,4,5,8‐naphthalene‐tetracarboxylic diimide, NP III, was photoirradiated in the presence of ferric‐cytochrome c (Fe3 +), the fragmentation peak corresponding to the specific oxidative modification at the histidine site of cytochrome c was observed in the Matrix‐Assisted Laser Desorption Ionization‐Time of Flight Mass Spectroscopy (MALDI‐TOF) measurement. Without photoirradiation, no such protein oxidation was observed in the experimental conditions employed. This result suggests the possible usage of NP‐III as an oxidative protein‐modifying reagent.
Angiotensin II type I Receptor polymorphism in African Americans lower frequency of the C1166 variant
Authors: Gainer James V. , Hunley Tracy E. , Kon Valentina , Nadeau John H. , Muldowney James A. S. , Brown Nancy J.
Published: July 2007

The C1166 variant, an A to C substitution polymorphism at the 1166 position of the angiotensin II type I (AT1) receptor, has been previously associated with hypertension in Caucasians. This study determines the frequency of the C1166 variant in an African American population.

Normotensive African American (n=99) and Caucasian (n=100) subjects were genotyped to determine the frequency of the C1166 variant. This study establishes the frequency of the C1166 variant in African Americans (0.05±0.01) and demonstrates a significantly lower frequency in African Americans compared with Caucasians (0.05 vs. 0.25, respectively, χ2=30.7, p<<0.001, 1 df).
Antagonistic regulation of protein kinase C‐induced stimulation of fibronectin synthesis by cyclic AMP in human lung fibroblasts
Authors: Lee Byung‐Heon , Park Rang‐Woon , Sohn Kun‐Young , Kim In‐San
Published: July 2007

We studied the interaction between cAMP and protein kinase C (PKC) signaling pathways in the regulation of fibronectin synthesis in human lung fibroblasts. Phorbol myristate acetate (PMA), a PKC activator, stimulated fibronectin synthesis and its mRNA expression in both normal and transformed human lung fibroblasts (WI‐38 and WI‐38 VA13, respectively). On the other hand, dibutyryl cAMP (Bt2cAMP), a cAMP analogue, did not alter fibronectin synthesis in both cell lines. The combined treatment of Bt2cAMP with PMA, however, suppressed the PMA‐induced stimulation of fibronectin synthesis and mRNA expression in these cells. This study shows that cAMP pathway antagonizes PKC pathway in regulating fibronectin synthesis in human lung fibroblasts and provides an example of antagonistic interaction between cAMP and PKC signaling pathways.
Single‐strand breaks and repair in nuclear DNA in the presence of hydralazine assayed by the nucleoid technique
Authors: Węglarz Ludmiła , Koceva‐Chyła Aneta , Drózdz Marian , Józwiak Zoifia
Published: July 2007

The nucleoid sedimentation assay was used to study hydralazine‐induced DNA structural changes and repair in the fibroblasts cultured in vitro. The drug induced a dose dependent loss in negative DNA supercoiling due to the physical breakage of the DNA. Relaxation of supercoiled DNA resulted in the nucleoids sedimenting with lower velocities than those of undamaged control cultures. Repair incubation of the cells did not cause the restoration of DNA supercoiling to control level. Unsuccessful repair of DNA damaged by hydralazine may result in maintaining the damaged DNA in the cell which could have immunologic consequences.
Development of a competitive enzyme linked immuno sorbent assay to measure α2‐macroglobulin in irradiated rat serum
Authors: Kim In Gyu , Park Seon Young
Published: July 2007

A competitive enzyme linked immunosorbent assay with antigen immobilized on the solid phase was developed to measure α2‐macroglobulin in rat serum. The cross reactivity with albumin and hemoglobin was eliminated by use of IgG fractions that were isolated after chromatography on Cibacron Blue F3‐GA Sepharose immobilized rat whole serum and hemoglobin Sepharose. Blocking materials and pH of the coating buffer had no effect on the amount of α2‐macroglobulin that binds to the plate. When the coating amount of α2‐macroglobulin was 100 ng/well, at pH 7.4, 10 mM Tris‐HCl containing 150 mM sodium chloride and the IgG amount added was 60 ng/well, then albumin and hemoglobin did not affect the assay at concentrations of 150 μg/ml or 200 μg/ml. This assay is useful for measuring the concentration of α2‐macroglobulin in normal and irradiated rat serum.
Two short peptide sequences repeated three times in carrot /gb‐tubulin
Authors: Okamura Shoji , Sonehara Kazuhiko , Naito Keiko , Okahara Koichi
Published: July 2007

There are two repeated peptides, [S,T,Y]QQ[Y,M] and MFR[R,X][V,K], which appear three times each in the region corresponding to the third exon of carrot β‐tubulins. The former peptide is flanked on both sides by a pair of identically charged groups and the latter includes two or three basic amino acids in it. The sequences are generally conserved well among the eukaryotes except for fungi, in which one of the peptides, SQQY, does not exist.
Repetitive elements in the third intron of murine apolipoprotein A‐I gene
Authors: Chiang An‐Na , Fan Kong‐Chi , Shaw Gwo‐Chyuan , Yang Ueng‐Cheng
Published: July 2007

Genomic DNAs containing the gene encoding apolipoprotein A‐I (apoA‐I) from four strains of mice and three strains of rats have been sequenced. Some peculiar repetitive sequences were found in the third intron of apoA‐I of the murine species. The striking features include regular tandem repeats of C(A)4 and C(A)6 in mice and long A‐tracts in rats. Not completely identical but very similar motifs were found in mice or rats belonging to the same species while repetitive elements from different species show some variation from their species‐specific consensus sequences. These repetitive motifs are very similar to the sequences flanking human Alu and rodent B 1 repetitive elements, although no evidence for the existence of Alu or B 1 was found near the peculiar repetitive DNA sequences in apoA‐I gene.
Role of transglutaminase in keratinization of vaginal epithelial cells in oestrous cycling rats
Authors: Vijayalakshmi V. , Gupta P. D.
Published: July 2007

Aspects of the regulation of calcium dependent transglutaminase (TGase) enzyme in the terminally differentiating vaginal epithelial cells (VEC) from cycling and oestradiol primed ovariectomized (OVX) rats are described. There is a significant increase in the TGase activity and a quantitative rise in its cross‐linked Σ(τ‐glutamyl)lysine covalent product in the VEC of oestrus rats. A similar phenomena was also evident in the oestradiol primed OVX rats. However, in the VEC of the diestrus/unprimed OVX rats, the enzyme activity and its cross‐linked product tend to be at its basal level. An increase of TGase activity in the keratinized layers of the oestrus VEC was observed. Immunohistochemical localization data parallel the biochemical measurements. These findings suggest that TGase activity may be associated with the differential status of the cell and primarily aids in the conferring structural stability and integrity to the stratified VEC. Aggregates of the keratin tonofilament bundles which are covalently cross‐linked by the formation of Σ(τ‐glutamyl)lysine in the oestrus vagina renders them resistant to denaturant and proteolytic treatments.
Antitumor activity of conjugates of the oncofetal protein alpha‐fetoprotein and phthalocyanines in vitro
Authors: Severin S. E. , Posypanova G. A. , Katukov V.Yu. , Shmyrev I. I. , Luzhkov Yu.M. , Gerasimova G. K. , Zhukova O. S. , Vorozhtsov G. N. , Kaliya O. L. , Lukyanets E. A. , Severin E. S.
Published: July 2007

The several conjugates of aluminium and cobalt complexes of phthalocyanines with human alpha‐fetoprotein have been synthesized. Their cytotoxic activity against tumor cells and human peripheral blood lymphocytes was studied. The experimental data demonstrate that the cytotoxic activity of alpha‐fetoprotein‐phthalocyanine conjugates against three types of tumor cells of various origin is much higher (for aluminium and cobalt complexes more than 1000 and 50 times, respectively) in comparison with phthalocyanines themselves. The application of phthalocyanines as conjugates with alpha‐fetoprotein makes it possible to markedly enhance the selective toxicity of phthalocyanines against human tumor cells.
Effect of calcium ion channel antagonists on chorionic gonadotropin secretion
Authors: Sharma Srikantaradhya Chidananda , Rao A. Jagannadha
Published: July 2007

By using calcium ion channel antagonists such as Verapamil and Nifedipine, we demonstrated that calcium ion channels are involved in human chorionie gonadotropin (hCG) secretion stimulated by gonadotropin releasing hormone (GnRH). Addition of 1 μM Verapamil (Class II inhibitor) resulted in an inhibition of GnRH‐stimulated hCG secretion by placenta without affecting basal release. However, while addition of 10 μM Niffedipine (Class I inhibitor) resulted only in partial inhibition, significant inhibition was observed at 100 μM concentration and above. These results suggest that calcium ion channels in placenta appear to be similar although not identical with the channels reported in the pituitary.
Cytoplasmic creatine kinases from giant pandas
Authors: Wang Xi‐Cheng , Ye Ju‐Qun , Wang Hong‐Rui , Zhou Hai‐Meng
Published: July 2007

The muscle and brain creatine kinases of giant panda have been isolated and purified. The purified muscle and brain enzymes (MM and BB) are homogeneous on both the polyacrylamide gel electrophoresis in the presence and absence of SDS. Both enzymes are dimers, consisting of two identical subunits each with a molecular weight of 42,000 daltons. The characteristics of muscle and brain enzymes have been studied, respectively. The hybridized enzyme, MB, was prepared by hybridization of MM and BB. The kinetic parameters of MM, BB and MB were determined, respectively. The results from modification of SH groups show that the SH groups of panda creatine kinases are essential for their activity and among the all SH groups in the enzyme only one per subunit is essential for enzymatic activity.
Identification of histidine residues in the sugar binding site of snake gourd (Trichosanthes anguina) seed lectin
Authors: Komath Sneha Sudha , Nadimpalli Siva Kumar , Swamy Musti J.
Published: July 2007

Chemical modification studies have been carried out on the galactose‐specific lectin (SGSL) purified from snake gourd (Trichosanthes anguina) seeds. Modification of the imidazole side chains of histidine residues with ethoxyformic anhydride resulted in a complete loss of activity of the lectin. A total of 9.5 (±0.7) histidine residues were modified per dimer of Mr 55,000 when the reaction was carried out for 2 hours. A partial protection was observed when the modification was done in the presence of 0.1M galactose, indicating that histidine residues are directly involved in the sugar‐binding activity of the lectin. Complete recovery of the lectin activity was observed when the modification was reversed by treatment with hydroxylamine. In immunodiffusion experiments, the histidine‐modified lectin reacted with rabbit antiserum raised against the native SGSL forming a precipitin line, indicating that the loss of activity upon modification was not due to changes in the overall conformation of the lectin. Modification of the side chains of lysine, cysteine and tyrosine residues did not result in any change in the activity of SGSL.
Angiotensin II and related peptides alter liposomal membrane fluidity
Authors: Brailoiu Eugen , Margineanu Anca , Miyamoto Michael D.
Published: July 2007

We investigated the effects of angiotensinogen (Ang), angiotensin I (Ang I), and angiotensin II (Ang II) on the fluidity of phosphatidylcholine vesicles. Changes in fluidity were assessed by changes in anisotopy values calculated from fluorescence polarization measurements. All three compounds produced an increase in membrane fluidity when localized inside the phosphatidylcholine vesicles. When placed outside the vesicles, Ang II increased bilayer rigidity (decreased fluidity), whereas Ang and Ang I produced no effect. These results suggest the possibility that these peptides may alter the fluidity of cell membranes by a direct action on the phospholipid bilayer, which may in turn interfere with receptor‐mediated effects.
Inductions of immediate early genes (IEGs) and ref‐1 by human chorionic gonadotropin in murine Leydig cell line (MA‐10)
Authors: Suzuki Syoji , Nagaya Takashi , Suganuma Nobuhiko , Tomoda Yutaka , Seo Hisao
Published: July 2007

The effect of human chorionic gonadotropin (hCG) on the expression of immediate early genes (IEGs) including all members of fos and jun family, and c‐myc was studied using mouse Leydig cell line (MA‐10 cells) by Northern blot analyses. In addition, the induction of ref‐1 which enhances DNA binding of fos/jun proteins was also analyzed. HCG induced a rapid and transient expression of c‐fos, fosB, c‐jun, junB, junD and c‐myc with a peak at 30 min to 1h. In contrast, induction of fra‐1 mRNA was delayed with a peak at 3 hr. However, fra‐2 mRNA was immediately increased by hCG with a peak at 1 h. The ref‐1 mRNA was expressed before the stimulation and its level was not altered by hCG at least for 8 hr. The differential induction of IEGs and continuous expression of ref‐1 mRNA suggest an important role of their gene products on the regulation of Leydig cell function by hCG.
Protein kinase C from rainbow trout brain: Identification and characterization of three isozymes
Authors: Brooks S. P. J. , Storey K. B.
Published: July 2007

Free and membrane‐associated fractions of protein kinase C (PKC) from rainbow trout (Onchorynchus mykiss) brain tissue were separated by hydroxylapatite chromatography and characterized kinetically. In both resting fish and in fish swum to exhaustion, approximately 40% of the total PKC activity was bound to membranes. Quantification of the three distinct hydroxylapatite chromatography peaks (PKC types γ, β and α) in cytosolic and membrane fractions revealed different isozyme distributions. The cytosolic fraction contained 21% PKC type γ, 52% PKC type β and 27% PKC type α. The membrane‐associated fraction contained 23% PKC type γ, 28% PKC type β and 49% PKC type α. Kinetic characterization of the three isozymes showed that PKC type γ was almost completely activated by Ca2+ alone whereas PKC type β and PKC type α were 40% and 60% activated by Ca2+. Full activity for all enzymes was observed only in the presence of phosphatidylserine and diacylglycerol. Differences in the kinetic constants for the three isozymes were also apparent. PKC type γ, had a much lower affinity for Histone III‐S when compared with PKC types β and α (100 μg/ml as compared with 1.7 and 5.7 μg/ml). PKC type γ, also had a lower affinity for calcium (0.22 μM) when compared with PKC type β (0.08 μM) and PKC type α (0.05 μM). PKC type α had a lower affinity for phosphatidylserine (8.6 μg/ml) when compared with PKC type γ (0.37 μg/ml) and PKC type β (0.89 μg/ml).
Electrostatic inhibition of hemolysis induced by organotin compounds
Authors: Kleszczynska H. , Pruchnik H. , Przestalski S.
Published: July 2007

The effect of cations on the kinetics of hemolysis caused by organotin compounds was studied. The ions used in the investigation diminish or totally inhibit hemolysis of red cells induced by organotin compounds. The degree of inhibition depends both on the kind of ion and the compounds that induce hemolysis. The ions Zn2+, Co2+, and Cd2+ present in the medium at 50 μM concentration totally protect the erythrocytes against hemolysis induced by the compound (C3H7)3SnCl. The study has also shown the monovalent ions K+ and trimethyldodecylammonium bromide are less potent inhibitors of hemolysis than divalent ions, which is not the case for two non‐ionic organotin compounds only. The studies performed indicate that hemolysis induced by organotin compounds is inhibited due to electrostatic interaction between the cations selected and erythrocyte membrane.
Differential expression of multidrug resistance (mdr) and canalicular multispecific organic anion transporter (cMOAT) genes following extrahepatic biliary obstruction in rats
Authors: Kagawa Tatehiro , Watanabe Norihito , Sato Masahiro , Nakano Atsushi , Nishizaki Yasuhiro , Hosoi Katsumi , Takashimizu Shinji , Uchiyama Junzo , Kimura Minoru , Matsuzaki Shohei
Published: July 2007

The hepatic canalicular membrane has transporters that play an important role as efflux pumps in the excretion of endogenous bile constituents or xenobiotics into bile canaliculi. To elucidate functional significance of canalicular transporters in the mechanism of cholestasis, mRNA expression levels of multidrug resistance (mdr) lb, mdr2 and canalicular multispecific organic anion transporter (cMOAT) genes were analyzed by Southern blotting of reverse‐transcribed PCR products of liver mRNA obtained from cholestatic rats that had been subjected to bile duct ligation. Both mdr1b and mdr2 mRNA expression increased after ligation. Immunohistochemical study revealed that the products of both mdr1b and mdr2 were present on the canaliculi, and that their levels increased after ligation. In contrast, cMOAT mRNA expression was not increased, but rather attenuated by ligation. The expression of canalicular transporters was differentially regulated in extrahepatic biliary obstruction, indicating the different roles are played by mdr and cMOAT in the pathogenesis of cholestasis.
A sequence highly similar to PNG is located on chromosome 22q12 in intron 15 of the LIMK‐2 gene
Authors: Carson Darek Kedra Emma , Weber Günther , Lagercrantz Jacob
Published: July 2007

In this report we describe a sequence (PNG22) highly similar to the Phospholipase C β3 Neighboring Gene (PNG). We also report that PNG22 is located in the q12 region on chromosome 22 between markers D22S1144 and D22S280. This finding explains that PNG probes cross hybridize to sequences on chromosome 22. Fine mapping using our sequence data and the complete sequence of a PAC clone (DJ515N1), located in this region, determined that PNG22 is located in intron 15 of the LIMK‐2 gene. PNG22 is 93% homologous to PNG, however it do not have the introns described for the PNG gene, instead matching the cDNA sequence. This leads us to suggest that PNG22 probably represents a PNG pseudogene. In this report we also list the exon intron borders and the genomic structure of LIMK‐2 and place it on the Sanger Center chromosome 22 Physical map. It also explains the finding that PNG probes cross hybridize to sequences on chromosome 22.
Protective role of superoxide dismutase in iron toxicity in yeast
Authors: Wisnicka Renata , Krzepiłko Anna , Wawryn Jarosław , Krawiec Zdzisława , Bilinski Tomasz
Published: July 2007

It has been found that yeast mutants deficient in cytosolic superoxide dismutase CuZnSOD are hypersensitive to ferrous iron. In contrast mutants that are deficient in catalases and cytochrome c peroxidase do not differ from the standard strain in this respect. These findings suggest that iron toxicity may depend on the redox status of the cell. They also shed light on the role of superoxide dismutases in preventing the toxic effects of oxygen.