bluuebunny/crossref_metadata_2025 huggingface dataset:
Thyroid hormone, all‐trans retinoic acid, and 9‐cis retinoic acid functioned as negative modulators of the effect of glucocorticoid on induction of α1‐acid glycoprotein mRNA in RLN‐10 cells
Authors: Li Xiaoman , Sumi Tomoyuki , Matsukawa Toru , Nakanishi Yoshinobu , Ohba Yoshiki
Published: July 2007

The expression of acute‐phase protein genes is controlled by many factors, such as IL‐1, IL‐6, glucocorticoids, thyroid hormone (T3), and retinoic acids. We studied the interaction of T3, glucocorticoids, all‐trans retinoic acid (RA), and 9‐cis retinoic acid (9cRA) on the expression of the rat α1‐acid glycoprotein (AGP) gene in vitro. Dexamethasonc (Dex) activated AGP gene expression in a rat liver derived cell line, RLN‐10. Although T3, RA, and 9cRA by themselves had no effect on AGP production, they reduced the response to Dex of the AGP gene.
Influence of phenoxyherbicides and their metabolities on the form of oxy‐and deoxyhemoglobin of vertebrates
Authors: Bukowska Bozena , Reszka Edyta , Duda Wirgiliusz
Published: July 2007

The effect of phenoxyherbicides and their metabolites on the structure of oxy‐ and deoxyhemoglobin was studied by using different doses and times of incubation of hemoglobin with the herbicide. It was ascertained that among the investigated hemoglobins the most sensitive was carp oxyhemoglobin incubated with 2,4‐D (2,4‐dichlorophenoxyacetic acid) and the least sensitive was human hemoglobin. Comparing the toxicity of 2,4‐D, MCPA (2‐methyl‐4‐chlorophenoxyacetic acid), 2,4‐DCP (2,4‐dichlorophenol), 2,4‐DMP (2,4‐dimethylphenol) it was found that the highest decrease occurred in bovine hemoglobin incubated with 2,4‐DMP. The phenoxyherbicides caused stabilization of the structure of T‐deoxyhemoglobin in vitro, in that they decreased the oxygen affinity with a simultaneous increase in methemoglobin concentration.
Reactivity of the horseradish peroxidase compounds I and II toward organometallic substrates. A stopped‐flow kinetic study of oxidation of ferrocenes
Authors: Goral Vasily N. , Ryabov Alexander D.
Published: July 2007

Reactivity of horseradish peroxidase compounds I and II (HRP‐I and HRP‐II) toward organometallicic substrates, viz water‐soluble ferrocenes RFc (R = COOH and CH2NMe2), has been studied at 25 °C, pH 6.0 and ionic strength 0.1 M. The second‐order rate constants k2 for the reaction of HRP‐I with FcCOOH and FcCH2NMe2 equal (1.00±0.04)×106 and (0.27±0.01)×106 M‐1 s‐1, respectively. The values of k3 for the reaction of HRP‐II with FcCOOH and FcCH2NMe2 equal (1.1±0.1)×l04 and (0.25±0.01)×104 M‐1 s‐1, respectively. The steady‐state kinetic study of the HRP‐catalyzed oxidation of the ferroeenes by H202 under the same conditions gave the second‐order rate constants of (0.94±0.03)×104 and (0.24±0.06)×104 M‐1 s‐1 for FcCOOH and FcCH2NMe2, respectively, which are in a good agreement with k3. The results reported here confirm the proposal that the rate‐limiting step of the steady‐state oxidation of ferrocenes is the electron transfer from the substrate to HRP‐II.
Lucigenin reduction by nadph‐cytochrome P450 reductase and the effect of phospholipids and albumin on chemiluminescence
Authors: Tsukamoto Maki , Tampo Yoshiko , Yonaha Masanori
Published: July 2007

To assess lucigenin, a chemilumigenic probe, as a detector of superoxide anion in microsomes, NADPH oxidation, lucigenin disappearance, and chemiluminescence in a system including purified NADPH‐cytochrome P450 reductase were exmnined. NADPH oxidation was increased by adding lucigenin, and concurrently, its disappearance and oxygen consumption were also stimulated. Chemiluminescence, which is negligibly emitted in the presence of the reductase alone, was remarkably amplified with phospholipids and albumin. Menadione inhibited lucigenin disappearance resulting in suppression of chemiluminescence. Lucigenin chemiluminescence measured in microsomes appears not to reflect direct superoxide anion production from microsomal components and from quinones, such as menadione.
DNA methylation by wheat cytosine DNA methyltransferase: Modulation by protease inhibitor E‐64
Authors: Vlasova Tatyana I. , Vanyushin Boris F.
Published: July 2007

Cytosine DNA methyltransferase isolated from wheat seedlings and purified in the presence of metalloprotease and serine protease inhibitors has molecular mass and specific activity equal to about 85 kDa and 250 units/mg protein, respectively. Apparent Km for AdoMet& and [I]50 for AdoHcy values are about 6 μM and 12 μM, respectively. The enzyme is active in wide pH range (pH 5.5‐8.5) and is inhibited by NaCl. The enzyme rapidly loses its methyltransferase activity in the absence of substrates. Using the cysteine protease inhibitor E‐64 it has been shown that rapid enzyme inactivation is caused by disappearance of essential enzyme SH‐groups but is not due to proteolytic enzyme cleavage.
Cloning and sequencing of the cDNA coding for pig preuteroglobin/clara cell 10 kDA protein
Authors: Sagal Rubén Gutiérrez , Nieto Antonio
Published: July 2007

Using reverse transcription‐polymerase chain reaction (RT‐PCR) on pig lung mRNAs, we have cloned and sequenced an almost full‐length complementary DNA (cDNA) coding for pig pre‐uteroglobin/Clara cell 10 kDa protein (UG/CC10), a major secretory protein of tung Clara cells. The deduced amino acid sequence indicated a preprotein of 91 residues, 21 of which corresponded to the signal peptide. Comparison of the sequence with those of known pre‐UG/CC10 from other species indicated that the pig protein resembles the structure shared by human and Lagomorpha pre‐UG/CC10 but differ from the proteins from Rodentia that are composed of 96 aminoacids and contain signal peptides of 19 residues. Some amino acids, that form part of a hydrophobic pocket inside the mature protein, are well conserved in all UG/CC10 suggesting an important function of this cavity. Northern analysis indicated that pig UG/CC10 mRNA is abundant in lung but is not detectable in liver, uterus or epididymis. The results are discussed in relation to a possible physiological function of UG/CC10.
Effect of some physiological factors on nitrogenase activity and nitrogenase mediated hydrogen evolution by mixed microbial culture
Authors: Kumar Ashok , Jain Seema R. , Kalia Vipin C. , Joshi Arvind P.
Published: July 2007

Fermentative H2 evolution, nitrogenase activity (acetylene reduction) and nitrogenase mediated H2 evolution was studied in free cells of mixed microbial population of H2 producers. At 3% glucose level, the cells produced 8.35 1 H2/mol glucose utilized. The role of nitrogenase system in H2 generation was evident by derepressed nitrogenase activity (0.46 nmoles C2H4 produced/mg protein/h) under defined in vitro conditions. For maximum expression of the activity, the cells required preactivation under anaerobic conditions by incubating at 40 °C for 20‐24h with 0.2% glucose in the culture medium. At an O2 level of more than 0.25%, the acetylene reduction activity decreased significantly and could not be detected at a level of 20%. Nitrogenase activity development was higher at acetylene: inoculum ratio between 4.2‐6.25. H2 evolution was lower when the mixed cells were incubated under an atmosphere of 10% C2H2 and 5%CO gas. This decrease in H2 evolution was also evident at 2.5‐6.5 mM NaNO3 and KNO3 concentrations in the liquid culture medium thus establishing more than 50% H2 evolution through nitrogenase.
Identification of sequences involved in both basal expression and phenobarbital induction of a CYP2B2 gene using in vitro transcription system
Authors: Shervington Amal
Published: July 2007

Cytochrome P450 2B2 and 2B1 are induced in mammalian liver in response to barbiturates such as phenobarbital. The induction of the proteins is mediated at the transcriptional level. Owing to the lack of a suitable phenobarbital‐responsive cell line to investigate the molecular mechanism responsible for the induction, we adopted an alternative approach involving the use of in vitro transcription system, DNA constructs containing a G‐free cassette under the control of various section of the 5′ flanking sequence of rat CYP2B2 gene were transcribed in nuclear protein extracts isolated from the livers of phenobarbital‐treated or untreated rats. The results of these experiments indicated the presence within the CYP2B2 promoter of two positive DNA elements, one located between ‐178 and ‐368 and the other between ‐368 and ‐984, that are involved in regulating basal transcription of the gene. In addition, a negative regulatory element is present between ‐2880 and ‐5600. A DNA sequence involved in regulating the induction of expression of the gene in response to phenobarbital is located between ‐178 and ‐368. The use of synthetic oligonucleotides as competitor in vitro transcription assays indicated the importance for transcriptional control of the CYP2B2 gene of sequences located between ‐183 and ‐199 and ‐31 and ‐72 that were identified previously [1] as binding rat liver nuclear proteins that are enriched ox activated in vivo by phenobarbital.
Detection of infectious bursal disease virus of poultry in clinical samples by RT‐PCR
Authors: Kataria R. S. , Tiwari A. K. , Bandyopadhyay S. K. , Kataria J. M. , Butchaiah G.
Published: July 2007

The RT‐PCR technique was adopted to amplify variable region of VP2 gene of infectious bursal disease virus using three different sets of primers. These primers could generate products of 643, 474 and 552 bp sizes. The authenticity of the amplicons was confirmed by their size in agarose gel, restriction enzyme digestion and by nested PCR. Out of total five clinical samples tested, IBD viral genomic RNA could be detected in four by RT‐PCR. Restriction enzyme digestion of PCR products with StuI could differentiate clinical samples from an intermediate vaccine strain currently being used in India.
Crystals of ribosomal protein L1 from a hyperthermophilic archaeon Methanococcus jannaschii
Authors: Tishchenko Svetlana , Nikonov Stanislav , Garber Maria , Kraft Alex , Köhrer Caroline , Piendl Wolfgang
Published: July 2007

Crystallographic studies of ribosomal proteins from bacteria progressed rapidly during the last decade, though the structures of ribosomal proteins from other kingdoms have not yet been published. Here we describe crystalls of archaeal ribosomal protein L1 from Methanococcus jannaschii. The protein crystals were grown in 10% PEG 10 K, 50 mM Hepes‐HCl (pH 7.5) in hanging drops equilibrated against 33% PEG 10 K, 100 mM Hepes‐HCl (pH 7.5). The crystals diffract to at least 2.5 Å resolution and belong to the space group P1 with cell parameters a=34.09 Å, b=39.39 Å, c=55.84 Å, α=83.65°, β=80.38°, γ=75.37°.
Effects of amidinopiperidine‐4‐Carboxylic acid 4‐tert‐butylphenyl ester, a specific trypsin inhibitor, on the growth of HL‐60 Cells
Authors: Zhang Minyue , Zhu Jianqin , Zhu Dexu , Muramatu Mutumi
Published: July 2007

Previous results showed that the synthetic compound amidinopiperidine‐4‐carboxylic acid 4‐tert‐butylphenyl ester (APCA‐OPhBut), a trypsin inhibitor, could specifically inhibit the activity of proteinase In and lead to growth arrest of Hela cells in early S phase. In this study, APCA‐OPhBut exhibited inhibitory effects on the growth of HL‐60 cells. Apoptotic cells were observed when the cells were cultured with APCA‐OPhBut above 50μM. Time course studies demonstrated that apoptotic cells were increased in a dose‐ and time‐dependent manner. Flowcytometric assays demonstrated that HL‐60 cells underwent slight G1 growth arrest after treatment with APCA‐OPhBut. No change of Bcl‐2 protein level was detected. The findings suggest that the intracellular trypsin‐like protease inhibited by APCA‐OPhBut not only plays a key role in DNA synthesis initiation but is also necessary for survival of certain cell lines.
Cloning and sequencing of cDNA encoding ribonuclease F1 from Fusarium moniliforme
Authors: Yoshida Hiroshi , Iizuka Marl , Norioka Naoko , Norioka Shigemi , Sakiyama Fumio
Published: July 2007

Two cDNA clones encoding ribonuclease F1 (EC 3. 1. 27. 3) have been isolated using a probe prepared by polymerase chain reaction with primers designed on the basis of amino acid sequence of the enzyme. They derived probably from the same gene and contained 393‐base pair open reading frame encoding 131 amino acid residues (Mr 13,606) including a putative 25‐residue signal peptide. The sequences of 43‐base pair 5′‐untranslated region and 125‐base pair 3′‐untranslated region including a poly(A) tail of 25 nucleotides were also elucidated. Homology analyses showed that cDNA for ribonuclease F1 has 65% homology to that for ribonuclease T1 in the coding region. At the preprotein level, they share 53% homology.
Light is associated with a 34 kDa ribonucleoprotein in spinach chloroplasts
Authors: Kanekatsu Motoki , Sawada Hiroshi
Published: July 2007

Chloroplast ribonucleoproteins (RNPs) encoded by the nuclear genome may play an important role in the post‐transcriptional steps of light‐induced chloroplast gene expression. To study the relationship between the synthesis of a 34 kDa ribonucleoprotein (p34) and light in spinach chloroplasts, a polyclonal antibody was raised against p34. Immunoblotting revealed that p34 synthesis was induced in a light dependent manner. In addition, levels of the large subunit of ribulose‐1,5‐bis phosphate carboxylase/oxygenase (Rubisco) encoded by the chloroplast genome fluctuated in relation to those of p34. These results suggest that the synthesis of p34 in response to light induces of chloroplast gene expression.
Characterization of acidic ribosomal proteins from three developmental stages of the medfly Ceratitis capitata
Authors: Kouyanou Sophia , Gagou Mary‐Elisabeth , Fragoulis Emmanuel G.
Published: July 2007

Acidic proteins extracted with 0.5 M NH4Cl and 50% ethanol from ribosomes of the medfly Ceratitis capitata showed two major bands of MW 15 and 17 kD after SDS electrophoresis. Isoelectrofocusing of acidic proteins resolved two groups of bands at pH 4.5 and 3.5. Similar patterns were observed both from the acidic ribosomal protein fraction and from total ribosomes, treated with RNase. Treatment with alkaline phosphatase reduced the number of bands with a shift to a higher pI, indicating dephosphorylation. The phosphorylation pattern of the acidic proteins changed at three different stages of development, six day larvae, white pupae and 0‐2 days old embryos. The two protein groups correspond to multi‐phosphorylated forms of eucaryotic acidic ribosomal proteins P1 and P2. This was shown by immunoblotting with specific monoclonal antibodies.
Activation of cAMP synthesis in rat brain cortical membranes by rubidium and cesium ions
Authors: Rosenthal Katri , Lember Jaanus , Karelson Ello , Järv Jaak
Published: July 2007

Rubidium and cesium chlorides accelerated cAMP synthesis in rat brain cortex membranes, while other alkali metal chlorides had no influence on the rate of this process. The effect was dose‐dependent and yielded above 2‐fold activation of adenylate cyclase. It has been shown that Rb+ and Cs+ influenced directly the catalytic subunit of the enzyme and did not substitute Mg2+ in formation of the metal‐ATP complex in this reaction. The stimulatory effect of Rb+ was additive to the activation of adenylate cyclase by the half‐maximal (0.31‐μM) as well as by the saturating (10 μM) forskolin concentrations, pointing to the fact that these effectors activate different isoforms of the enzyme in rat brain cortex.
C‐Jun N‐terminal kinase‐mediated AP‐1 activation in experimental glomerulonephritis in rats
Authors: Sakurai Hiroaki , Sugita Takahisa
Published: July 2007

We demonstrated activation of transcription factor AP‐1 in rat nephrotoxic serum (NTS)‐induced glomerulonephritis in a previous report. Here, we evaluate c‐Jun N‐terminal kinases (JNKs) activity to clarify the molecular mechanisms of AP‐1 activation in nephritic glomeruli. Increased JNKs activity was detected in glomeruli isolated from NTS‐treated rats. The kinetics of JNKs activation was similar to that of AP‐1 activation. Phosphorylated c‐Jun at Ser63, one of the target residues for JNK, was also detected in nephritic glomeruli. This is the first report demonstrating JNKs‐mediated c‐Jun/AP‐1 activation in nephritic glomeruli. These results suggest an important role of the JNK‐AP‐1 signaling pathway in the pathogenesis of glomerulonephritis.
The potential role for Cdc42 protein from rat brain cytosol in phospholipase D activation
Authors: Han Joong‐Soo , Kim Hyung‐Chul , Chung Joon‐Ki , Kang Heun‐Soo , Donaldson Jason , Koh Jai‐Kyung
Published: July 2007

Phospholipase D (PLD) has been extracted from rat brain membranes and chromatographically enriched 70‐fold. From the rat brain cytosol, Cdc42 with a Mr of about 24,000 and ADP‐ribosylation Factor (Arf) with a Mr of about 18,000 have been purified to near homogeneity. PLD was activated better by purified cytosolic Arf than by the other small G proteins tested. Cdc42 purified from rat brain cytosol showed 70% of PLD activation activity exerted by cytosolic Arf, suggesting that Cdc42 may be one of the major G proteins involved in the activation of membrane‐associated PLD. While Cdc42 or RhoA exhibited synergistic activation of PLD when administered in conjunction with Arf, Cdc42 and RhoA showed an additive effect when used together. It is possible that Arf and Rho family proteins may have different interaction sites on PLD. These findings support a role for GTP‐binding proteins of the Rho family as well as Arf in the activation of membrane‐associated PLD and further suggest that Cdc42 may be a major G protein involved in the PLD activation in rat brain.
Spin trapping for nitric oxide produced in LPS‐treated mouse using various new dithiocarbamate iron complexes having substituted proline and serine moiety
Authors: Nakagawa Hidehiko , Ikota Nobuo , Ozawa Toshihiko , Masumizu Toshiki , Kohno Masahiro
Published: July 2007

Four dithiocarbamate derivatives of 4‐substituted L‐proline and N‐methyl‐L‐serine were synthesized, and their iron complexes were prepared in Tris‐HCl buffer solution. These complexes were used as spin trapping reagents for nitric oxide in ESR spectrometry, and compared with each other in regard to their spin trapping properties in vivo. When the synthesized complexes were injected to lipopolysaccharide‐treated mice intravenously, the nitric oxide adducts were detected both in the liver and in the blood except N‐dithiocarboxy‐4‐(methoxymethyl)oxy‐L‐proline iron complex, whose nitric oxide adduct was detected mostly in the blood. When the exogenous nitric oxide adduct of this complex was injected, it was not detected in the liver, too. It is considered that this complex can trap nitric oxide in the blood by excluding the accumulation of the nitric oxide adduct in the liver.
TNFα‐induced aerobic synthesis of ATP on plasma membranes of target cells. The relation to the expression of the nuclear oncogene c‐myc
Authors: Globa A. G. , Solovyev A. S. , Terentyev A. A. , Demidova V. S. , Shulgina M. F. , Alesenko A. V. , Karelin A. A.
Published: July 2007

The accumulation of ATP by preparations of plasma membranes enriched particles (PMEP) isolated from rat hepatocytes, murine splenocytes and human T‐lymphocytes has been investigated after the binding of human and murine tumour necrosis factors (TNFα) to their specific receptors. The TNFα‐induced expression of the nuclear oncogene c‐myc in intact hepatocytes has been also studied. TNFα induced the marked biosynthesis of ATP on PMEP of hepatocytes and splenocytes within the first minute of incubation. The biosynthesis of ATP was independent of the activity of adenylate kinase and only occured in the presence of all the components of aerobic phosphorylation and the electron acceptor, cytochrome C or diferric transferrin. The level of ATP on PM correlated with the degree of expression of the nuclear oncogene c‐myc in the same target cells. Adriamycin totally suppressed the biosynthesis of ATP on PM and simultaneously inhibited the expression of c‐myc. The ATP synthesized on PM is suggested to be involved in transduction of the proliferative or growth signal to the cell nucleus.
Activation of apoptotic caspase‐3 and nitric oxide synthase‐2 in buccal mucosa with chronic alcohol ingestion
Authors: Slomiany B. L. , Piotrowski J. , Piotrowski E. , Slomiany A.
Published: July 2007

Apoptosis, the process of programmed cell death, involves activation of caspase proteases cascade that remains under the regulatory control of nitric oxide. In this study, we investigated the activity of a key apoptotic protease, caspase‐3, and the expression of nitric oxide synthase‐2 (NOS‐2) associated with buccal epithelial cells apoptosis induced by chronic ethanol diet. The assays revealed that a 7.9‐fold enhancement in buccal epithelial cells apoptosis, observed in the alcohol diet group, was accompanied by a 37.6‐fold increase in caspase‐3 activity and a 10.1‐fold increase in NOS‐2. Furthermore, the expression of NOS‐2 showed a positive correlation (r = 0.92) with the extent of changes induced in caspase‐3 activity. These results implicate caspase‐3 in the process of alcohol‐induced epithelial cells apoptosis, and point towards participation of NOS‐2 in the amplification of the cell death signaling cascade.
A new PCR‐based approach to a fast search of a wide spectrum of cry genes from Bacillus thuringiensis strains
Authors: Shevelev A. B. , Lewitin E. , Novikova S. I. , Wojciechowska Ya.A. , Usacheva E. A. , Chestukhina G. G. , Stepanov V. M.
Published: July 2007

A pair of highly degenerated primers was adapted to carry out a single‐step PCR‐detection of any known and probably unknown cry genes of classes cry1, cry4 and cry9 encoding for 130 kDa protein δ‐endotoxins in the natural Bacillus thuringiensis i(BT) strains. The Southern hybridization of the product has demonstrated that essentially remote cry‐genes like cry1Aa and cry9A (cryIG) could be represented in the single amplificate if they are simultaneously present in the genome of the analyzed strain.

Four genes were detected by the proposed scheme in the iBT ssp. galleriae 11‐67. One of them, gene cry1Ga1 was originally found and cloned using the PCR‐amplification product obtained from the genomic DNA of this strain as a probe. The new gene was completely identical to one cloned by B. Lambert (unpublished, EMBL accession number Z22510) and essentially related to cryIM (EMBL accession number Y09326), renamed according to the new nomenclature as cry1Ga2.
Fluorimetric studies on saccharide binding to the basic lectin from Artocarpus hirsuta
Authors: Gaikwad Sushama M. , Gurjar Madhura M. , Khan M. Islam
Published: July 2007

The binding of Artocarpus hirsuta lectin to galactose and its derivatives was examined by fluorescence spectroscopy. The intrinsic fluorescence intensity of the lectin was enhanced by 55 % upon binding to methyl α‐galactose without any change in the emission maximum (333nm). 4‐Methyl umbellifery α‐galactopyranoside showed 100% quenching of its fluorescence intensity upon binding to the lectin without any shift in the emission maximum (373nm). The association constant for the binding of the above sugars to the lectin decreases with increasing temperature. Methyl group in the α anomeric position of galactose enhanced the binding while that in the β position reduced the binding to the lectin. Solute quenching studies of the lectin using acrylamide, potassium iodide and cesium chloride indicated that the tryptophan residues were fully accessible to the neutral quencher, while only partly accessible to the ionic quenchers.
Purification of heparin‐binding protein HBp17 and identification of HBp17 heparin binding site
Authors: Wang Xin‐Chang , Chen Jun‐Hui , Crab J. W. , Sato J. D.
Published: July 2007

HBp17 was purified by Heparin‐Copper biaffinity chromatography and HPLC from conditioned medium of A431 cell. The purified HBp17 was digested by staphylococcus urcus V8 protease or chymotrypsin and the heparin‐binding fragments were isolated by Heparin‐Sepharose. One binding site of peptide mapping is HBp17 residues 110‐145 produced by V8. Another one is HBp17 residues 82‐143 which were produced by chymotrypsin digestion. Two binding sites of peptide mapping are overlap. Therefore the residues 110‐143 of HBp17 are the principle heparin binding site. The basic amino acid cluster in this region may be contribute to the binding of HBp17 to heparin or heparan sulfate proteoglycan on the cell surface and extracellular matrix.
Effects of eicosapentaenoic acid and its 15‐hydroperoxy and 15‐hydroxy derivatives on glucosamine synthetase activity in rabbit gastric mucosa
Authors: Fujita Tadashi , Sakuma Satoru , Yamamoto Naho , Fujimoto Yohko
Published: July 2007

The effects of eicosapentaenoic acid and its 15‐hydroperoxy and 15‐hydroxy adducts on the activity of glucosamine synthetase, the rate‐limiting enzyme of mucus synthesis, in rabbit gastric antral mucosa were examined. 15‐Hydroperoxy‐eicosapentaenoic acid inhibited the glucosamine synthetase activity at concentrations of 10, 20 and 50 μM. The effect was concentration‐dependent, and the concentration required for 50% inhibition was approximately 20 μM. Eicosapentaenoic acid and its 15‐hydroxy adduct had no significant effect on the enzyme activity at the same concentration range. The experiment utilizing Fe2+ revealed that the inhibitory effect of 15‐hydroperoxyeicosapentaenoic acid on the glucosamine synthetase activity is not due to hydroxy radical which is expected to be formed from the hydroperoxy adduct. These results suggest that 15‐hydroperoxyeicosapentaenoic acid has the potential to reduce the synthesis of gastric mucus by inhibiting the glucosamine synthetase activity.
Phosphatidylinositol 3‐kinase binds to profilin through the p85α subunit and regulates cytoskeletal assembly
Authors: Bhargavi V. , Chari Veerendra B. , Singh Surya S.
Published: July 2007

Phosphatidylinositol 3‐kinase (PI3‐K), endowed with catalytic (110kDa) and regulatory (85kDa) subunits co‐precipitates with anti‐tyrosine antibodies in mitogen‐activated cells. Association of PI3‐K with cytoskeleton activates its catalytic activity through undeciphered mechanisms. Recently Singh et al., (Biochemistry, 35, 16544‐16549, 1996) have shown that profilin activates PI3‐K activity in a concentration‐ dependent manner. Consequently, we investigated the interaction between the PI3‐K and profilin employing the GSTp85α fusion protein and the results indicate a specific interaction between pofilin and p85α. The effect of p85α/profilin complex on polymerization of actin monomers was monitored fluorimetrically employing pyrene‐labelled actin monomers. It was noted that p85α/profilin complex inhibits actin polymerization suggesting that profilin can simultaneously bind to actin as well as to p85α. The affinity of p85α/profilin complex to actin increases in the presence of p85α subunit of PI3‐K as compared to profilin itself.
The effect of pH on the unfolding pathway and stability of ribosome‐inactivating protein abrin‐II
Authors: Krupakar Jayarapu , Das Puspendu K. , Podder Sunil K.
Published: July 2007

The effect of pH on the unfolding pathway and the stability of the toxic protein abrin‐II have been studied by increasing denaturant concentrations of guanidine hydrochloride and by monitoring the change in 8,1‐anilino naphthalene sulfonic acid (ANS) fluorescence upon binding to the hydrophobic sites of the protein. Intrinsic protein fluorescence, far and near UV‐circular dichroism (CD) spectroscopy and ANS binding studies reveal that the unfolding of abrin‐II occurs through two intermediates at pH 7.2 and one intermediate at pH 4.5. At pH 7.2, the two subunits A and B of abrin‐II unfold sequentially. The native protein is more stable at pH 4.5 than at pH 7.2. However, the stability of the abrin‐II A‐subunit is not affected by a change in pH. These observations may assist in an understanding of the physiologically relevant transmembrane translocation of the toxin.
Enzymes (Isoenzyme System) as homeostatic mechanisms the isoenzyme (ADA2) of adenosine deaminase of human monocytes‐macrophages as a regulator of the 2′deoxyadenosine
Authors: Gakis Caritaos , Cappio‐Borlino Aldo , Pulina Giuseppe
Published: July 2007

Regarding homeostatic mechanisms in the enzyme (isoenzymes) and substrate system we show through a simulation model obtained by STELLA II software that: (i) a pair of isoenzymes (Is1 and Is2) that have different affinity for the substrate (Is2 affinity < ls1 affinity), can constitute an efficient homeostatic mechanism: by varying the relative concentration of the isoenzymes in the system, the levels of the substrate can be controlled; (ii) the isoenzymes ADA1 and ADA2 of adenosine deaminase (ADA) that have different affinity for the substrate 2′deoxyadenosine (ADA2 has very weak affinity for 2′deoxyadenosine) constitute, inside human Monocytes‐Macrophages, a homeostatic mechanism that assures an up‐regulation of 2′deoxyadenosine and a down‐regulation of a second substrate (adenosine) for which the affinity of the two isoenzymes is similar.
Asergillus fumigatus strains recovered from immunocompromised patients (ICP): Suptyping of strains by RAPD analysis
Authors: Khan Zafar K. , Chowdhary Latika , Gyanchandani Anita , Goswami Mamta , Farooqui Nuzhat , Ranade Shirish A.
Published: July 2007

Invasive pulmonary aspergillosis in immunocompromised patients (ICP) is the second most frequent opportunistic fungal infection. The causative organism includes 16 species of Aspergillus, of which A.fumigatus dominates the ubiquitous incidence of invasive or allergic broncho‐pulmonary aspergillosis (ABPA). The definitive diagnosis of invasive aspergillosis is difficult. We have analyzed 24 strains of A.fumigatus recovered from ICP using the RAPD technique. The profiles generated with the 20 primers tested were mostly unique. These results may have a profound impact on the management of aspergillosis, especially in the ICP.
Dexamethasone induced expression of phosphatase inhibits generation of reactive oxygen species in ehrlich ascietes tumor cells
Authors: Salimath Bharathi P. , Sharma Ranjana , Tabassum Arshia
Published: July 2007

Both pre‐activated and phorbol ester tetradecanoyl phorbol myristate acetate (TPA) activated reactive oxygen species (ROS) generation were inhibited by dexamethasone in viva. Time kinetics on influence of dexamethasone on cytosolic phosphoprotein phosphatase activity revealed that, when compared to phosphatase activity in cytosol of control Ehrlich ascites tumor (EAT) cells, a 5‐fold increase in specific activity is seen in the cytosol of EAT cells treated (in viva, 0‐90 min. 1 mg/kg body weight) with dexamethasone. Dexamethasone induced phosphatase was partially purified by conventional ion‐exchange and gel filtration column chromatographic techniques. Purified phosphatase had a molecular weight of 70 KDa by SDS‐PAGE. A dose‐dependent inhibition of TPA activated ROS generation by partially purified phosphatase in permeabilized EAT cells suggested that dephosphorylation is a major regulatory mechanism in “switching off” of the respiratory burst. Anti‐phosphatase antibodies were raised, purified and were used to quantitate cytosolic phosphatase by ELISA, which revealed that dexamethasone induces 6‐fold increase in expression of phosphatase in EAT cells by 120 min. The expression of phosphatase in EAT cell cytosol gas further confirmed by immunostaining using anti‐phosphatase antibodies, the results of which showed intense blue staining on development with BCIP/NBT.
Purification and partial characterization of thioredoxin reductase from Streptomyces aureofaciens
Authors: Horecká Tatiana , Perecko Dusan , Kutejová Eva , Mikulásová Darina , Kollárová Marta
Published: July 2007

Thioredoxin reductase (TrxR) is one of a number of flavoproteins that catalyze the transfer of electrons between pyridine nucleotides and a specific disulfide‐containing substrate. Thioredoxin reductase from Streptomyces aureofaciens 3239 has been purified to homogeneity by a two‐step chromatographic procedure including anion‐exchange chromatography and affinity chromatography on 2′5′‐ADP‐Sepharose 4B. Molar mass determined by chromatography on Superose 12 HR 10/30 and sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed 69 kDa for the native protein and 34.8 kDa for the enzyme subunit. The isoelectric point determined by isoelectric focusing gel electrophoresis was 4.3. TrxR effectively catalyzed the reduction of DTNB in the presence of S. aureofaciens thioredoxin‐1. TrxR activity in the presence of S. aureofaciens thioredoxin‐2 was only 1/4 of the activity with thioredoxin‐1 (1). The activity of pure TrxR decreased drastically in the presence of NADPH, while NADP+ as well as Streptomyces aureofaciens thioredoxin‐1 protected the enzyme from inactivation. These results indicate that thioredoxin reductase activity in bacteria could be modulated by the redox status of NADP+/NADPH and thioredoxin pools.
Site‐directed mutagenesis and steady‐state kinetic analysis of mutant enzymes of human adenylate kinase
Authors: Ayabe Takanori , Park Seung Kyu , Nagahama Hiroyuki , Maruyama Hideharu , Sumida Michihiro , Takenaka Hitoshi , Takenaka Osamu , Onitsuka Toshio , Hamada Minoru
Published: July 2007

Site‐directed mutagenesis of human adenylate kinase (AK) was carried out on residues His36, Lys55, and the C‐terminal segment (Val182, V186, and Leu193). Five mutants (H36T, K55G, V182G, V186S, and L193Stop (deletion of residues 193‐194) were generated and analyzed by steady‐state kinetics. H36T, K55G, and L193Stop mutants showed an increase of Km values (19.8‐, 19.7‐, and 11.3‐fold) for AMP2‐ compared to that for the wild‐type enzyme, and these residues appeared to interact with AMP2‐. V182G showed an increased Km value (7.4‐fold) for MgATP2‐. Therefore, V182 may be essential for interaction with MgATP2‐. V186S increased the Km value (7.0‐ and 7.5‐fold) for MgATP2‐ and AMP2‐. V186 may thus interact with both substrates. The C‐terminal domain of AK appears to be essential for MgATP2‐ and AMP2‐ binding.
Degradation of transferrin and albumin by radical reactions in human plasma evaluated by immunoblot
Authors: Yamada Yasuko , Okamoto Eiko , Tanaka Kyoko , Kojo Shosuke
Published: July 2007

Immunoblot analysis showed that transferrin and human serum albumin were degraded in a similar rate on the basis of the protein weight by radical reactions using a solution containing each protein initiated by Cu2+ and hydrogen peroxide. When plasma was treated with Cu2+ and hydrogen peroxide, the rate profile of the degradation of transferrin was similar to that of albumin based on immunoblot analysis in spite that the content of these proteins in plasma was considerably different. These results suggest that highly reactive hydroxyl radicals attack these proteins indiscriminately. Present observations demonstrate that immunoblot analysis is an effective method to analyze radical reactions of each protein in the presence of many other proteins like plasma.
Structural studies of cucumber mosaic virus: Fourier transform infrared spectroscopic studies
Authors: Renugopalakrishnan V. , Piazzolla P. , Tamburro A. M. , Lamba O. P.
Published: July 2007

The secondary structure of cucumber mosaic virus (CMV) was investigated in solution using Fourier transform infrared (FT‐IR) spectroscopy. The amide I region of intact CMV revealed a doublet at 1671 cm‐1 and 1653 cm‐1, respectively. In order to isolate the IR bands arising from the protein backbone of CMV, the FT‐IR spectra of the RNA component, isolated by phenol‐SDS treatment of purified CMV and subsequent precipation by ethanol, was obtained separately and digitally subtracted from the intact CMV spectra. After digital subtraction, the amide I region contained two bands at 1682 cm‐1 and 1644 cm‐1. The former band was ascribed to β‐sheet structures, while the later band occurs in the region between α‐helix and “unordered” structures. Resolution enhancement of the finger print amide I region was accomplished using Fourier self‐deconvolution of the digitally subtracted FT‐IR spectrum of CMV which further confirmed the presence of anti‐parallel β‐sheet structure in the protein coat of CMV. Chou‐Fasman predictions on the the coat protein also revealed the presence of β‐sheet structure in agreement with FT‐IR studies.
cDNA cloning and sequence analysis of six neurotoxin‐like proteins from Chinese continental banded krait
Authors: Qian You‐cun , Fan Chun‐yang , Gong Yi , Yang Sheng‐li
Published: July 2007

The cDNAs encoding six neurotoxin‐like proteins were cloned from the total RNA isolated from one venom gland of Bungarus multicinctus multicinctus by reverse transcription‐polymerase chain reaction. Although they shared a high degree of nucleotide sequence homology, the deduced proteins displayed a rather low extent of identical residues with the exception that the 21 residue signal peptides were highly conserved. Each mature protein consisted of 65, 66 or 68 amino acids with four or five disulfide bonds respectively. Comparative sequence analyses showed that they were structurally related to neurotoxin homologues. Phylogenetic analyses implied that they might represent two different evolutionary branches.
Antioxidant activity of aminoethylcysteine ketimine decarboxylated dimer on copper‐induced LDL oxidation
Authors: Matarese Rosa Marina , Macone Alberto , Fontana Mario , Duprè Silvestro , Cavallini Doriano
Published: July 2007

Aminoethylcysteine ketimine decarboxylated dimer (AECK‐DD), a member of a family of natural cyclic sulfur‐containing aminoacids, recently detected in biological samples, protects low density lipoprotein (LDL) against copper‐mediated oxidation, as assessed by monitoring the kinetics of conjugated diene formation, thiobarbiturie acid‐reactive substances production and LDL‐tryptophan fluorescence quenching. Moreover, AECK‐DD exerts a protective effect also against metal‐independent, peroxyl radical‐induced lipoprotein oxidation. It is of note that the concentrations exerting a protective effect against LDL oxidation are similar to those found in biological samples.
HMG CoA reductase inhibitor accelarates aging effect on diaphragm mitochondrial respiratory function in rats
Authors: Sugiyama Satoru
Published: July 2007

We examined effects of pravastatin on age‐related changes in mitochondrial function in rats. Decline in the activity of complex I of the mitochondrial electron transport chain was observed in diaphragm and psoai major in rats aged 35 and 55 weeks, and that of complex IV in rats aged 55 weeks. Pravastatin accelerated significantly age‐related decline in the activity of complex I of diaphragm mitochondria, though pravastatin did not show significant effect on normally observed age‐associated decline in the activities of complex IV of psoal major and diaphragm mitochondria. Aginng effect on mitochondrial respiratory function was not observed on heart muscle and liver in rats up to 55 weeks old, and pravastatin did not effect significantly heart and liver mitochondrial respiratory function. From these results, careful clinical examination on respiratory muscle function should be necessary in patients treated with pravastatin particularly in elderly patients.
High‐level expression of human interleukin‐17 in the yeast Pichia pastoris
Authors: Zhou Limei , Wang Jiaxi , Peng Shangyun , Duan Jubao , Cai Xin , Zou Minji , SU Qing
Published: July 2007

Human interleukin‐17 (hIL‐17) gene without the signal sequence was isolated from activated peripheral blood lymphocytes by RT‐PCR, then highly expressed in the yeast Pichia pastoris in the form of the glycosylated monomer. The monomer of rhIL‐17 stimulated mouse fibroblast 3T3 cells to secrete IL‐6 and was specifically bound to its receptors on 3T3 cells.
Influence of age on enzyme activities of pyrimidine metabolism in the chicken heart
Authors: Wegelin I. , Pane G. , Orlandini G. , Clò C.
Published: July 2007

The effect of the aging on the activities of enzymes involved in UMP‐CMP metabolism were evalued in the heart of newborn (1‐day‐old), young (20‐day‐old), adult (12‐month‐old), and aged (30‐month‐old) chickens.

In newborn animals, UMP metabolism proceeds preferentially towards cytidine compounds rather than to breakdown. In addition, two pathways different from those involved in de novo synthesis may contribute to the synthesis of UMP : one, through cytosine deaminase that shows its maximal activity; the other, by uridine kinase, the main “salvage” enzyme of pyrimidine nucleotides.

In young chickens, pyrimidine metabolism regards especially UMP. In fact, the lower activities of cytidylate phosphatase and cytosine deaminase, together with the remarkable increase of uridine kinase indicate that the metabolic flux converges on the main salvage pathway.

In adult chickens, pyrimidine catabolism is enhanced, as supported by the maximal activity of the enzymes involved in UMP‐CMP breakdown. On the contrary, the remarkable reduction of the anabolic enzymes suggests a limited resort to the salvage pathways.

Finally, in aged chickens a reduced pyrimidine catabolism and a greater utilization of the salvage pathways appear to take place, thus contributing to the maintenance of pyrimidine nucleotide pool.
The free radical‐generating function of a familial amyotrophic lateral sclerosis‐associated D90A Cu,Zn‐superoxide dismutase mutant
Authors: Kim Sung Moon , Eum Won Sik , Kwon Oh Bin , Kang Jung Hoon
Published: July 2007

The free radical‐generating functions of the D90A Cu,Zn‐superoxide dismutase (SOD) associated with Swedish familial amyotrophic lateral sclerosis (FALS) patients are investigated. The results show that both the wild‐type and mutant enzymes have identical dismutase activity, while the free radical‐generating activity of the D90A mutant is enhanced relative to that of the wild‐type enzyme. The studies suggest that the active channel of the D90A mutant is larger than that of the wild‐type enzyme. A higher free radical‐generating activity of the mutant enzyme led to the release of copper ions from the damaged protein. The generation of strand breaks in plasmid DNA was enhanced more effectively by the D90A mutant Cu,Zn‐SOD than by the wild‐type enzyme. The results suggest that the pathology of FALS may be attributed to oxidative damage caused by the gain‐of‐function of FALS Cu,Zn‐SOD mutant.
Characterization and cloning of long neurotoxin homolog from Naja naja atra
Authors: Lin Shinne‐ren , Huang Hsien‐bin , Wu Bin‐nan , Chang Long‐sen
Published: July 2007

The cDNA encoding a long neurotoxin homolog was constructed from the cellular RNA isolated fom the venom glands of Naja naja atra (Taiwan cobra) by reverse transcription‐polymerase chain reaction. BLAST searches for sequence similarity in the GeneBank databases reveal that the cDNA sequence of the long neurotoxin homolog is not highly homologous with long and short neurotoxins. Although the long neurotoxin homolog exhibited an activity to inhibit acetylcholine‐induced muscle contractions as Naja naja atra cobrotoxin, the degree of inhibition caused by the addition of long neurotoxin homolog was only approximately 35% of that observed with the addition of cobrotoxin. Moreover, the primary structure of the long neurotoxin homolog did not fulfill the characteristic features of long or short neurotoxins. Together with long neurotoxin homologs from other snake species, they probably represent an evolutionary divergence between long and short neurotoxins.
Genomic structure of the human ribosomal protein L41 gene
Authors: Go Hiroki , Miyado Kenji , Taniguchi ShuN′ichiro
Published: July 2007

Using PCR technique, genomic DNA encoding the gene of the human ribosomal protein L41 was amplified. Subsequent sequencing showed that the gene was constituted with 4 exons and 3 introns. Because the amplified gene contained introns and whole the open reading frame, we concluded the fragment corresponded to the functional gene.
Novel substrates of yeast alcohol dehydrogenase‐4. Allyl alcohol and ethylene glycol
Authors: Trivic Svetlana , Leskovac Vladimir
Published: July 2007

In the present work, we have determined the steady‐state kinetic constants for yeast alcohol dehydrogenase‐catalyzed oxidation of allyl alcohol (H2C=CH.CH2OH) and ethylene glycol (HOCH2.CH2OH) with NAD+, at pH 8.0; also, a kinetic mechanism for the former reaction was determined at the same pH. In addition, it was found that acrolein is a potent inhibitor of yeast alcohol de‐hydrogenase.
Characterization and partial purification of CD34 progenitor cell ecto‐phosphatidic acid phosphohydrolase
Authors: Harvey Kevin A. , Siddiqui Rafat A. , Reeves Margaret , Kovala Thomas , Dugan Michael , Akard Luke P. , English Denis
Published: July 2007

Phosphatidic acid and its hydrolysis product, diacylglycerol, play potentially vital roles as extracellular messengers in numerous cellular systems and may play a key role in regulating hematopoiesis. In this study, we describe an ecto‐phosphatidic acid phosphohydrolase that potentially regulates cellular responses to phosphatidic acid on bone marrow derived human hematopoietic progenitors. We partially purified hematopoietic progenitor ecto‐PAPase using a novel in‐gel phosphatase assay and then characterized the enzyme on phenotypically defined subpopulations of hematopoietic CD34+ progenitors isolated by flow cytometry. The most pronounced PAPase activity was confined to uncommitted CD34+/CD38+ hematopoietic progenitors, which lacked the expression of other lineage‐associated antigens. We conclude that hematopoietic progenitor cells at various stages of maturation possess a potent ecto‐PAPase, an enzyme well positioned to regulate progenitor cell growth and differentiation induced by phosphatidic acid and related lipids.
Pressure effects on proteolysis catalysed by calpain
Authors: Bessiere Philippe , Bancel Frédéric , Cottin Patrick , Ducastaing André
Published: July 2007

The effects of pressure on μ and m‐calpain stability and specific activity have been examined. Activity and stability of these neutral calcium‐dependent heterodimeric proteinases were studied using an in‐house built bioreactor allowing on‐line spectrophotometric monitoring with retention of pressure. Both isozymes were founded to be rather baro‐sensitive with t1/2 at 1500 bar of 6 min and 11 min for μ and m‐calpain respectively. Activity measurements under pressure showed a biphasic behavior for both proteinases with a slight activation for pressure up to 500 bar and 750 bar for m and μ‐calpain respectively. Activation volume changes indicated that the proteolytic reaction was alternatively favored and disfavored by pressure due to catalytic step activation associated with enzyme‐substrate binding step being continuously inhibited by pressure. Furthermore, autoproteolysis of calpain, a calcium dependent phenomenon was inhibited by application of pressure indicating that pressure inhibition of proteolytic activity could also be due to Ca2+‐binding decrease under pressure. Implication of these results with catalytic mechanism of these heterodimeric proteinases is also discussed.
Effects of ethanol ingestion on sperm monosaccharides and fertility
Authors: Srikanth V. , Aruldhas M. M. , Srinivasan N. , Govindarajulu P. , Balasubramanian K.
Published: July 2007

Chronic alcohol abuse is often associated with reproductive disorders. Sperm monosaccharides play an indispensable role in sperm‐egg interactions and fertilization. Ethanol (3 g/kg body weight as 25%, v/v) was given by gastric intubation twice daily for 30 days while in another group, rats which had been treated with ethanol were withdrawn from treatment for a further period of 30 days, in order to assess the reversibility of the ethanol‐induced effects. Epididymal ethanol content, sperm monosaccharides and the fertility of ethanol treated and ethanol withdrawn rats were assessed. Ethanol ingestion caused a significant decrease in sperm monosaccharides suggesting defective glycosylation of sperm surface proteins. Sperm monosaccharides and fertility were returned to normal following the withdrawal of ethanol. Ethanol‐induced changes in sperm monosaccharides may be one of the reasons for the reduced fertility of ethanol treated rats.