bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cloning and sequencing of the homologues of both the bacterial and eukaryotic initiation factor genes (hIF‐2 and heIF‐2γ from archaeal Halobacterium halobium
Authors: Hasegawa Yoshiko , Sawaoka Nozomi , Kado Nozomi , Ochi Minako , Itoh Takuzi 
Published: July 2007

AbstractThe cloning and sequencing of the genes encoding the translational initiation factors (hIF‐2 and heIF‐2 γ) was performed by screening the halophilic archaeon Halobacterium halobium genomic library with a probe constructed from the peptide IGHVDHGK that is conserved in archaeal GTP‐binding elongation factors. The codon usage by the hIF‐2 and heIF‐2 γ genes showed a preference for triplets ending in G or C. This characteristic is almost identical to that of other H. halobium genes. The translated protein of hIF‐2 and heIF‐2 γ genes is made of 414 and 583 amino acid residues, respectively, and contains the sequence motif for the binding of GTP. The sequence of hIF‐2 shows a strong similarity to the initiation factor IF‐2 from Bacteria whereas heIF‐2 γ shows a strong similarity to the initiation factor eIF‐2 γ from Eucarya.
Transcription of the rat p53 gene is induced by a 39kDa protein binding to the p53 promoter region during the liver regeneration
Authors: Lee Minhyung , Park Sunhee , Song Haisun , Park Jong‐sang 
Published: July 2007

AbstractThe adult rat liver is normally in a state of growth arrest. However, cell loss such as partial hepatectomy can induce the proliferation of the hepatocytes. Early after partial hepatectomy, the concentration of p53 mRNA increases during the prereplicative phase. In this study, we identified the cis‐regulatory element involved in the induced transcription of the rat p53 gene by DNase I footprinting assay. This element had a partial homolgy to the AP 1 recognition motif, but the competition study with AP1 oligonucleotide showed that this element was not the AP1 recognition motif. The molecular weight of the binding protein to this motif was determined as 39kDa by southwestern blotting analysis. In vitro transcription assay with the competitor containing the binding motif showed that the 39kDa protein binding to the element was requried for the induced transcription of the rat p53 gene during the liver regeneration.
Thioredoxin fusion/HIV‐1 protease coexpression system for production of soluble human IL6 in E. coli cytoplasm
Authors: Han Bao‐Guang , Song Xiao‐Guo , Peng Shan‐Yun , Wang Jia‐Xi , Ling Shi‐Gan 
Published: July 2007

AbstractIn this paper, thioredoxin (TRX) fusion expression system has been modified to produce soluble human IL6 (hIL6) without TRX moiety in E. coli cytoplasm. A novel TRX gene fusion vector was developed that contained at the 3′‐end of TRX gene a short DNA sequences encoding a linker peptide ‘‐GSGSGVSQNYPIVQHHHHHH‐’, serving not only as a specific HIV‐1 protease site but also providing six contiguous histidine (His) residues to foreign proteins. The cDNA for hIL6 was cloned into this vector resulting in plasmid pTRX@HISIL6. The cDNA for the HIV‐1 protease has been cloned into another compatible plasmid pHMM2, resulting in plasmid pHMM2‐PR. Both plasmids were transformed into E. coli strain GI724, and when induced for expression of both proteins, the correct processing of TRX@HISIL6 was obtained, producing hlL6 with His6‐tag at the N terminus named HISIL6. A fraction of HISIL6 was found in soluble form and could be purified to homogeneity by Ni‐NTA Superflow and ion‐exchange chromatography. The biological activity of purified HISIL6 was measured by MTT method in an IL‐6‐dependent cell line 7TD1 to be 2.1×108 unit/mg.
The effect of heparin and pentosan polysulfate on the thermal stability of yeast alcohol dehydrogenase
Authors: Paulíková Helena , Molnárová Milota , Podhradský Dusan 
Published: July 2007

AbstractHeparin and pentosan polysulfate as organic polyanions inhibit yeast alcohol dehydrogenase (YADH). The aim of this study was to determine the effect of heparin and pentosan polysulfate on the thermostability of alcohol dehydrogenase. Spectral and kinetic analyses showed that these compounds increase the thermal stability of the enzyme and eliminate entirely thermal aggregation. The thermostabilizing effect of unfractionated heparin and pentosan polysulfate was accelerated in the presence of NAD+. The addition of NAD+ (11 μM) to the incubation medium decreased the inhibition of the YADH activity in the presence of pentosan polysulfate (1.32 μM). Moreover, 38 % of the residual activity of YADH was found after a 5‐rain incubation at 70 °C. These findings indicate that heparinoids not only modulate the enzyme activity but also can prevent the protein's thermal denaturation.
Sequence‐specific DNA binding activity in the RAE28 protein, a mouse homologue of the Drosophila polyhomeotic protein
Authors: Nomura Midori , Takihara Yoshihiro , Motaleb Abdul , Horie Kyoji , Higashinakagawa Toru , Shimada Kazunori 
Published: July 2007

AbstractThe rae28 gene, a mouse homologue of the Drosophila polyhomeotic gene, is involved in the maintenance of the transcriptional repression states of Hox genes. In this study we synthesized the glutathione S transferase‐RAE28 (GST‐RAE28) fusion protein and examined sequence‐specific DNA binding activity in the RAE28 protein by using the selected and amplified binding site method. After five rounds of enrichment, the eluted DNAs were amplified, cloned and sequenced. The sequences of individual oligonucleotides included the following consensus sequences; 5′‐ACCA‐3′, 5′‐ACCCA‐3′, 5′‐CTATCA‐3′ and 5′‐TGCC‐3′. The oligonucleotides including these consensus sequences were show to have significant affinity with the GST‐RAE28 fusion protein. The RAE28 protein was recently shown to form multimeric protein complexes with other members of mouse Pc‐G proteins in the nucleus. These findings strongly suggest that the RAE28 protein constitutes a sequence‐specific DNA binding domain in multimeric Pc‐G protein complexes.
Use of prokaryotically expressed nucleocapsid protein as positive antigen in ELISA
Authors: Kumar S. S. , Renji R. , Saini Mohini , Goel A. C. , Sharma Bhaskar 
Published: July 2007

AbstractA cDNA library of Rinderpest vaccine virus was prepared in Zap Express vector (Stratagene). The Rinderpest ‘N′ gene specific clones were selected, characterized and thereafter expressed in E. coli XLOLR strain. The expressed protein was found to be immunogenic in western blot with hyperimmune sera. It reacted with rinderpest and ‘N′ protein specific monoclonal antibodies in Enzyme Linked Immunosorbent Assay (ELISA). Prokaryotically expressed ‘N′ protein also gave precipitin band in counter immunoelectrophoresis test (CIE). The expression of N protein was sufficient for its utility as positive antigen in CIE and ELISA used for rinderpest diagnosis.
Role of reducing co‐factor in cerulenin‐insensitivity of 6‐hydroxymellein synthase in carrot cell extract
Authors: Kurosaki Fumiya , Togashi Kousuke , Arisawa Munehisa 
Published: July 2007

AbstractThe activity of 6‐hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme of carrot, was not inhibited by cerulenin in the presence of NADPH. However, cerulenin showed a marked inhibitory activity to the synthase if the reducing co‐factor was omitted from the assay mixture. The synthase was also sensitive to the antibiotic even in the presence of NADPH when the acyl condensation site and the reducing domain at the reaction center of the enzyme were dissociated under the high ionic strength condition, In addition, the synthase activity was appreciably inhibited when NADH was employed instead of NADPH. These observations strongly suggest that a phosphate group attached to 2′‐position of adenosyl moiety of NADPH molecule plays an important role in the apparent insensitivity of 6‐hydroxymellein synthase toward cerulenin.
Presence and comparison of angiotensin converting enzyme in commercial cell culture sera
Authors: Bramucci Massimo , Miano Antonino , Quassinti Luana , Maccari Ennio , Murri Oretta , Amici Domenico 
Published: July 2007

AbstractThis study was conducted to determine the presence of the angiotensin converting enzyme in commercial sera used in cell culture medium. The aim of the research was to bring the presence of proteinases (angiotensin converting enzyme) to cell culture users' knowledge and to give some data for solving problems about the development of peptides as useful drugs. The enzymes, purified from foetal bovine, adult bovine, foetal equine, adult equine, and human sera, showed molecular weights of about 170 kDa. Captopril and lisinopril inhibited enzyme activities at nanomolar concentrations. The enzymes were able to hydrolyze, with different efficiency, angiotensin I, bradykinin and epidermal mitosis inhibiting pentapeptide. The heat inactivation of commercial sera at 56°C for 30 min showed a reduction of ACE activity of about 35‐80%. Therefore, the presence of ACE activity in commercial sera can influence the activity of biological peptides tested on cell lines cultured “in vitro”.
Total reactive antioxidant potential in human saliva of smokers and non‐smokers
Authors: Kondakova Irina , Lissi E. A. , Pizarro M. 
Published: July 2007

AbstractUric acid is the most important non‐enzymatic antioxidant present in human saliva. There is a great variability among individuals, both in salivary uric acid content and saliva total reactive antioxidant potential (TRAP). The uric acid present in saliva correlates with plasma uric acid, suggesting that the former is imported from plasma. There are not statistical differences between uric acid or TRAP values in saliva of smokers and non‐smokers. Also, smoking a cigarette does not modify the levels of antioxidants present in saliva.
Direct Transcriptional Control of the Chloroplast Genes psbA and psaAB Adjusts Photosynthesis to Light Energy Distribution in Plants
Authors: Pfannschmidt Thomas , Nilsson Anders , Tullberg Anna , Link Gerhard , Allen John F. 
Published: July 2007

AbstractTwo photosystems, I and II, absorb and convert light energy in photosynthesis in chloroplasts of green plants. The genes psbA and psaAB of the cytoplasmic chloroplast genome encode core components of photosystem II and photosystem I, respectively. Here we show that the absolute amounts of photosystem I and photosystem II respond, in a complementary manner, to changes in light quality that preferentially excite each photosystem in mustard seedlings. We also show that the initial response to altered energy distribution is a change in the rates of transcription of psbA and psaAB. Changes in chlorophyll fluorescence emission in vivo suggest that the signal initiating this change is the oxidation‐reduction state of plastoquinone, a component of the photosynthetic electron transport chain that connects photosystem I and photosystem II. The results are consistent with transcriptional effects observed previously with chloroplasts isolated in vitro and demonstrate that redox control of chloroplast transcription initiates long‐term adjustments that compensate for imbalance in energy distribution and adapt the whole plant to altered light environments.
Hypoxia, Ischemic Stroke, and Memory Deficits: Prospects for Therapy
Authors: Sun Miao‐Kun 
Published: July 2007

AbstractOxygenation of cells and tissues in mammals according to metabolic needs is always a life‐or‐death necessity. Over the last decade, life scientists have gained an impressive understanding of cellular and molecular mechanisms in hypoxic‐ischemic injury and memory deficits, survival, and death. Functional recovery from hypoxia‐ischemia depends on how the individual responds to the insult, and whether appropriate responses are initiated and maintained. Unraveling the biochemical and molecular mechanisms and responses of hypoxia‐ischemia is likely to dramatically shape and improve medical practice in the treatments of hypoxia‐ischemia and memory deficits.
Geldanamycin Induces Heat Shock Protein Expression Through Activation of HSF1 in K562 Erythroleukemic Cells
Authors: Kim Hong Ro , Kang Ho Sung , Kim Han Do 
Published: July 2007

AbstractHSP90 is one of the most abundant heat shock proteins (HSPs) in eukaryotic cells and is found in complex with several regulatory proteins such as kinases and transcription factors. Geldanamycin (GA), a benzoquinone ansamycin, specifically binds to HSP90 and disrupts the interaction of HSP90 and target proteins. Thus, GA has been used as a specific inhibitor of HSP90. In this study, we examined whether GA could affect protein synthesis and gene expression in the human erythroleukemic cell line K562. Treatment with GA, but not herbimycin A (another benzoquinone ansamycin), highly induced a 70‐kDa protein, which was revealed to be HSP70 by immunoblotting and immunoprecipitation with anti‐HSP70 antibody. The expression of HSP28 was also enhanced by GA. Furthermore, GA induced the activation of heat shock factor 1 (HSF1), but not HSF2, as determined by electromobility shift and electromobility supershift assay. In addition, similar to heat shock treatment, GA induced the phosphorylation of HSF1. Heat shock element‐binding activity and phosphorylation of HSF1 were attenuated 3 h after GA treatment. These results indicate that the functional inactivation of HSP90 by GA potentially stimulates the expression of heat shock proteins through activation of HSF1.
Investigation of the Orientation of Purple Membrane Sheets in Langmuir‐Blodgett Films by a Quartz Crystal Microbalance
Authors: Lu Tao , Rothe Ulrich , Liebau Maik , Schaffer Heike , Hufnagel Peter , Bakowsky Udo 
Published: July 2007

AbstractBy means of the quartz crystal microbalance (QCM), a convenient method was developed to determine the degree of orientation of purple membrane (PM) sheets on the air/water interface. Langmuir‐Blodgett films from both wild‐type and SH‐mutant PM (bR D36C) were vertically deposited on the surface of goldsputtered AT‐cut quartz crystals. The shift of resonance frequency of the QCM during a special washing protocol allowed us to differentiate between physically adsorbed PM fragments and any PM attached to the gold surface via chemical bonds. By washing with organic solvents, complete desorption of the wild‐type PM was achieved, whereas for the SH‐mutant, 60% of the PM fragments could not be detached from the surface. These PM sheets should be oriented with the cytoplasmic side facing the water subphase so that their SH‐groups can chemically bind to the gold surface of the quartz plate.
Peptide Binding Inhibits Aggregation of Soluble MHC Class II in Solution
Authors: Arimilli Subhashini , Astafieva Irina , Mukku Prabha V. , Cardoso Cristina , Deshpande Shrikant , Nag Bishwajit 
Published: July 2007

AbstractAffinity‐purified major histocompatability complex (MHC) class II molecules are known to bind antigenic peptide in vitro. This peptide‐bound MHC class II is known to undergo a change in structure upon stable binding of antigenic peptide. Previous results from our, and other laboratories, have suggested a relationship between MHC class II structure and peptide association that enables class II to enter into a stable conformation upon peptide binding. In this report we describe that stable binding of high‐affinity antigenic peptide to MHC class II molecule results in transition of aggregated purified MHC class II proteins to a stable heterodimeric state. Such transition was demonstrated by using purified human HLA‐DR2 class II molecule and high‐affinity myelin basic protein (MBP) 83‐102)Y83 peptide. Highly aggregated purified DR2 (high molecular weight; HMW) was first separated from heterodimer (low molecular weight; LMW) in the presence of 50‐fold molar excess of MBP(83‐102)Y83 peptide. We then show that the aggregated HMW preparation can be successfully converted into a stable dimer by further incubation with MBP(83‐102)Y83 and changing various binding parameters such as pH, temperature, reducing agent, and peptide concentrations. Under optimized conditions, the highly aggregated inactive DR2 molecules can be completely loaded with the antigenic peptide. The transformed heterodimers with bound peptide prepared by this method are biologically active, as shown by their ability to induce the production of gamma‐interferon by SS8T transformed human T cells. These results suggest that in solution, MHC class II molecules may be aggregated in the absence of bound peptide. Such aggregated MHC class II molecules can be converted to stable and biologically active heterodimers in the presence of high‐affinity antigenic peptide.
Lucigenin Luminescence Elicited by Microsomes and Its Modulation by Nitroazole Compounds
Authors: Schepetkin I. A. 
Published: July 2007

AbstractThe lucigenin luminescence elicited by rat liver microsomes and its modulation by the nitroazole compounds metronidazole and sanazole (drug AK‐2123), as well as the rates of lucigenindependent NADPH consumption and cytochrome c reduction, were studied. The obtained data suggest that the luminescence can be the result of univalent lucigenin reduction by microsomal NAD(P)Hreductases, generation of superoxide anion radical in the redox cycle of lucigenin radicals, dioxetane formation by (di)oxygenases, and catalytic action of cytochrome P450 heme on dioxetane decomposition, followed by light emission.
Effect of 13‐Hydroperoxyoctadecadienoic Acid on the Supply of Arachidonic Acid for Prostaglandin Synthesis from Arachidonoyl‐CoA Mediated by the Cytosolic or Microsomal acyl‐CoA Hydrolase in Rabbit Kidney Medulla
Authors: Sakuma Satoru , Fujimoto Yohko , Yoshimura Mari , Saeki Kazuki , Mukai Akiko , Akimoto Manabu , Fujita Tadashi 
Published: July 2007

AbstractThe effects of 13‐hydroperoxyoctadecadienoic acid (13‐HPODE) on the cytosolic or microsomal acyl‐CoA hydrolase (ACH) activity in rabbit kidney medulla and on the ACH‐mediated prostaglandin (PG) formation fromarachidonoyl‐CoA (AA‐CoA) were examined. 13‐HPODE (10, 20, and 50 mu M) had no effect on the cytosolic ACH activity but significantly inhibited the activity of the microsomal enzyme (43‐57% inhibition). PG formation was measured as follows: AA‐CoA (20 nmol) was preincubated with the cytosolic or microsomal fraction (as the source of ACH) in the presence or absence of 13‐HPODE for 5 min at 37° C, followed by incubation with the microsomal fraction (as the source of PG‐synthesizing enzymes), hydroquinone and reduced glutathione for 5 min at 37° C, and the PGs formed were measured by HPLC, with use of 9‐anthryldiazomethane for derivatization. 13‐HPODE reduced the PG formation when the microsomal fraction, but not the cytosolic fraction, was used as the source of ACH (10,20, and 50 mu M; 28‐55% inhibition). These results suggest that 13‐HPODE may modulate PG levels in rabbit kidney medulla by inhibiting the microsomal ACH activity.
Fine Structure of the Human Translocation Protein 1 (HTP1/TLOC1) Gene
Authors: Daimon Makoto , Susa Shinji , Kato Takeo 
Published: July 2007

AbstractWe characterized the genomic region corresponding to the human translocation protein 1 (HTP1/TLOC1) cDNA previously reported. An experiment using rapid amplification of cDNA ends revealed that the transcription initiation site was at 12 bp upstream from the translation initiation codon ATG. Using direct‐sequencing PCR, we determined precise intron/exon boundaries and intron‐exon composition of the gene. The gene region spanned 28 kb and was composed of eight exons and seven introns. The lengths of exons and introns range from 48 to > 1707 bp and from 0.25 to 8.2 kb, respectively. The translation initiation codon and the termination codon were located in exons 1 and 8, respectively. The nucleotide sequences of the introns were also determined in the region around the intron/exon boundaries for 63 to 442 bp. All of the sequences around the intron/exon boundaries were consistent with the 5′ and 3′ consensus sequences for splice junctions of transcribed genes. Putative lariat sequences were identified between 28 and 64 nucleotides from the 3′ splice junction for all seven introns. DNA walking experiments revealed a promoter region of 600 bp. The promoter region did not contain an apparent TATA box or a CAT box but did contain Evi‐1, GATA, v‐Myb, MZF1, and AP‐1 binding sites‐factors known as regulatory factors on expression of the gene in blood cells. Therefore, this gene may be one such gene.
Variation of Hepatic Methotrexate 7‐Hydroxylase Activity in Animals and Humans
Authors: Kitamura Shigeyuki , Sugihara Kazumi , Nakatani Keiko , Ohta Shigeru , Oh‐Hara Toshinari , Ninomiya Shin‐Ichi , Green Carol E. , Tyson Charles A. 
Published: July 2007

AbstractThis study deals with individual and species variations in the converting activity of methotrexate (MTX) to 7‐hydroxymethotrexate in animals and humans. When MTX 7‐hydroxylase was assayed in six human liver cytosols, a 48‐fold range of intersubject variation of the activity was observed. The variations were correlated to the concentrations of aldehyde oxidase activity in human subjects assayed with benzaldehyde as a substrate. Species differences of liver MTX 7‐hydroxylase activity were also observed. The activity was highest in rabbits, followed by rats, hamsters, and monkeys but was undetectable in dogs. Strain differences of MTX 7‐hydroxylase activity based on aldehyde oxidase activity were also observed in rats and mice. The results suggest that aldehyde oxidase functions as MTX 7‐hydroxylase in livers of animals and humans, and the observed differences of MTX 7‐hydroxylase activity are due to variations in the amount of aldehyde oxidase present.
Oxidative Stress, Antioxidant Defenses, and Damage Removal, Repair, and Replacement Systems
Authors: Davies Kelvin J. A. 
Published: July 2007

AbstractOxidative stress is an unavoidable consequence of life in an oxygen‐rich atmosphere. Oxygen radicals and other activated oxygen species are generated as by‐products of aerobic metabolism and exposure to various natural and synthetic toxicants. The “Oxygen Paradox” is that oxygen is dangerous to the very life‐forms for which it has become an essential component of energy production. The first defense against oxygen toxicity is the sharp gradient of oxygen tension, seen in all mammals, from the environmental level of 20% to a tissue concentration of only 3‐4% oxygen. These relatively low tissue levels of oxygen prevent most oxidative damage from ever occurring. Cells, tissues, organs, and organisms utilize multiple layers of antioxidant defenses and damage removal, and replacement or repair systems in order to cope with the remaining stress and damage that oxygen engenders. The enzymes comprising many of these protective systems are inducible under conditions of oxidative stress adaptation, in which the expression of over 40 mammalian genes is upregulated. Mitotic cells have the additional defensive ability of entering a transient growth‐arrested state (in the first stages of adaptation) in which DNA is protected by histone proteins, energy is conserved by diminished expression of nonessential genes, and the expression of shock and stress proteins is greatly increased. Failure to fully cope with an oxidative stress can switch mitotic cells into a permanent growth‐arrested, senescence‐like state in which they may survive for long periods. Faced with even more severe oxidative stress, or the declining protective enzymes and adaptive capacity associated with aging, cells may “sacrifice themselves” by apoptosis, which protects surrounding healthy tissue from further damage. Only under the most severe oxidative stress conditions will cells undergo a necrotic death, which exposes surrounding tissues to the further vicissitudes of an inflammatory immune response. This remarkable array of systems for defense; damage removal, replacement, and repair; adaptation; growth modulation; and apoptosis make it possible for us to enjoy life in an oxygen‐rich environment.