bluuebunny/crossref_metadata_2025 huggingface dataset:
Crystal and molecular structures of trans-[PdCl2(SMePh)2] ·C6H6, trans-[Pd2Cl2(μ-SPh)2(SPh2)2], and trans-[Pd2Cl2(μ-SPh)2(PPh3)2] · 4CH2Cl2
Authors: Oilunkaniemi Raija , Laitinen Risto S. , Ahlgrén Markku
Published: July 2007

The reaction of neat methylphenylsulfide with PdCl2 affords trans-[PdCl2(SMePh)2]. Upon recrystallization from dilute benzene/diethyl ether solution, crystals of trans, syn-[PdCl2(SMePh)2] · C6H6 (1 · C6H6) were isolated. [PdCl2(NCPh)2] in diphenylsulfide affords crystals of trans-[PdCl2(SPh2)2] and trans-[Pd2Cl2(μ-SPh)2(SPh2)2] (2). The latter species is a novel complex containing both thioether and thiolato ligands. A small amount of crystals of [Pd2Cl2(μ-SPh)2(PPh3)2] · 4CH2Cl2(3 · 4CH2Cl2) were isolated upon prolonged reflux a mixture of [PdCl2(PPh3)2] and Ph2S in CH2Cl2. Each Pd center shows a slightly distorted square-planar coordination. The Pd-S bond lengths involving Ph2S are 2.319(1) – 2.320(1)Å and 2.341(2)Å in 1 · C6H6 and 2, respectively and those involving bridging phenyl thiolato ligands are 2.301(2) – 2.312(2)Aring; and 2.296(1) – 2.375(1)Aring; in 2 and 3 · 4CH2Cl2, respectively. The lengths of the Pd–S bonds in 1 – 3 expectedly show a increasing trend consistent with the increasing trans influence Cl < S < P of the ligands trans to the thiolato or thioether ligands. All lattices are composed of discrete complexes that are linked together into three-dimensional networks by Cl···S and H···Cl close contacts.
Carboxylate Bridged Octaphenyltetra-antimony(V) ‘Cage’ Compounds; [(SbPh2)4(µ-O)4(µ-OH)2(µ-O2CR)2·HO2CR], where R = Me or t-Bu and the Crystal Structure of [(SbPh2)4(µ-O)4(µ-OH)(µ-OEt)(µ-O2CMe)2]
Authors: Gibbons Martin N. , Abdul Malik K.M. , Hursthouse Michael B. , Bryan Sowerby D.
Published: July 2007

Reactions of [SbPh2BrO]2 with carboxylates did not give [SbPh2(O2CR)O]2, the expected substitution products, which are highly sensitive to hydrolysis by traces of moisture, and cage compounds containing an Sb4O6 unit were obtained instead. With silver acetate and sodium trimethylacetate the products were [(SbPh2)4(µ-O)4(µ-OH)2(µ-O2CR)2]·HO2CR, R = Me 1 and R = t-Bu 2, respectively. An unusual compound, [(SbPh2)4(µ-O)4(µ-OH)2 (µ-O2CMe)2] 3, in which one of the bridging hydroxo groups in 1 is replaced by an ethoxo group, was also isolated and its crystal structure determined.
Synthesis and Characterisation of Amine Adducts of Tri(4-bromophenyl)boroxine, Tri(3-nitrophenyl)boroxine, and Tri(3-aminopheny1)boroxine; Molecular Structure of 3-Picoline·tri(4-bromopheny1)boroxine
Authors: Beckett Michael A. , Hibbs David E. , Hursthouse M.B. , Owen Paul , Abdul Malik K.M. , Sukumar Varmaar K.
Published: July 2007

Twenty five 1:l adducts of the triarylboroxines (4-BrC6H4)3B3O3(PBB) (3-NO2C6H4)3- B3O3 (MNB), and (3-NH2C6H4)3B3O3 (MAB) with N donor ligands (cyclohexylamine, 4- picoline, 3-picoline, piperidine, morpholine, isobutylamine, triethylamine, pyridine, isoquinoline, benzylamine) have been prepared by reaction of stoichiometric quantities of ligand and triarylboroxine in Et2O at room temperature. All 25 adducts have been characterised by elemental analysis, Mpt, IR and by 1H and 13C NMR. MNB and 4 of its adducts have been characterised by 1lB MAS NMR. In solution variable temperature 1H NMR of selected PBB derivatives and of MNB·cyclohexylamine indicate that a ligand dissociation-recombination process is occuring with ΔG+ of ca. 39–54 kJ·mol−1. The crystal and molecular structure of 3-picoline·PBB has been determined by a single-crystal X-ray diffraction study. The colourless compound crystallises in the orthorhombic space group (Pbca) with a = 14.1710(10), b = 15.446(5), and c = 22.418(7) A, with Z = 8, calculated density = 1.737 Mg/m3, and final R values [I > 2σ (I)] of R1 = 0.0376, and ωR2 = 0.0691 from 3786 independent reflections.
Sodium and Potassium Derivatives of 2-(Diphenylphosphinoyl)-2-Phenyl-Ethenol: Synthesis, Structure and Reactivity
Authors: Petrova Jordanka , Momchilova Snezhana , Haupt Erhard T.K.
Published: July 2007

The sodium and potassium derivatives of 2-(diphenylphosphinoyl)-2-phenylethenol (2 and 3, respectively) are obtained and their (E)-enolate structure is proved by spectroscopic methods. The study of their reactivity shows that regiospecific O-acylation of 2 and O-alkylation of 3 takes place, the corresponding 1-acetoxy-2-(diphenylphosphinoy1)-2-phenyl-ethene 4 and 1-benzyloxy-2-(diphenylphosphinoyl)-2-phenyl-ethene 5 is isolated. The reaction of carbonyl olefination is not observed.
Oxygen Transport and Exchange in the Microcirculation
Authors: PITTMAN ROLAND N.
Published: July 2007

The cardiovascular system is responsible for maintaining an adequate convective delivery of oxygen to the smallest branches of the network of blood vessels—the microcirculation—from which oxygen passes to the parenchymal cells by passive diffusion. The aim of this brief review is to trace the development of the study of oxygen transport from the point of view of the microcirculation. August Krogh performed measurements that allowed him to use his keen insight to draw conclusions about oxygen transport that remained the foundations of this field for decades. After an extended period of neglect, Duling rekindled interest in the field of oxygen transport by discovering that substantial amounts of oxygen diffused from the arteriolar network. Subsequent investigations confirmed this finding in various vascular beds and extended these studies to capillaries and venules. The important contributions of computational modeling and new techniques in intravital microscopy continue to led to more advances in our understanding of the role of the microcirculation in the supply of oxygen to tissues. Current work is applying the concepts and principles learned in normal tissues to pathophysiological situations, as well as increasing our understanding of artificial oxygen carriers, oxygen sensing, and the connections between nitric oxide and oxygen transport.
Therapeutic Intervention in Inflammatory Diseases: A Time and Place for Anti‐Adhesion Therapy
Authors: NORMAN M. URSULA , KUBES PAUL
Published: July 2007

The recruitment of leukocytes from the blood into tissue is central to the development and maintenance of the majority of inflammatory diseases. This multistep process requires a series of leukocyte–endothelial adhesive interactions, involving several families of adhesion molecules. Molecules that block these interactions have been targeted as potential therapeutic treatments for acute and chronic inflammatory diseases. However, many of the anti‐adhesion therapy clinical trials have yielded disappointing outcomes. This review discusses some of the animal models that raise questions about the suitability of anti‐adhesion therapy to treat certain inflammatory diseases. The authors suggest that it is crucial to understand the underlying mechanisms and timelines of leukocyte recruitment in each affected tissue and inflammatory disease to develop more effective anti‐adhesion therapy.
Irreversible inactivation of Caldariomyces fumago chloroperoxidase by hydrogen peroxide
Authors: Shevelkova Angelina N. , Ryabov Alexander D.
Published: July 2007

The rate constants for the H2O2‐induced irreversible inactivation (kinact) of chloroperoxidase from Caldariomyces fumago evaluated from the analysis of complete kinetic curves of chlorination or bromination of monochlorodimedon were found to follow the rate law kinact = k[H2O2]/(K + [H2O2]) with k = 0.009±0.002 and 0.0095±0.0010 s‐1 and K = (13±4)×10‐3 and (9±2)×10‐3 M in the presence of 0.01 M chloride and bromide, respectively, at pH 2.75 and 25 °C. The data show that chloroperoxidase investigated is more than by a factor of 10 less resistant toward hydrogen peroxide compared to horseradish peroxidase. The possible reason for it and the biotechological implications are briefly discussed.
Coordination of copperpolyamine complex with imidazoles potentiates its superoxide dismutase mimicking activity and abolishes its interaction with albumin
Authors: Athar Mohammad , Sharma Som D. , Iqbal Mohammad , Sultana Sarwat , Pandeya K. B. , Tripathi I. P.
Published: July 2007

Mixed ligand complexes of copper polyamine with biomolecules such as imidazole, substituted imidazoles or pyridine have been synthesized and characterized. These molecules were used because of their low toxicity and high activity. These complexes were found to possess a distorted octahedral microenvironment with a potential SOD mimicking activity. The IC50 values for these complexes were of the order of 2‐90 μM. Pyridine and imidazole complexes were most effective as they possess the lowest IC50 values of 2.1 and 6 μM respectively which are higher than the IC50 value of polyamine copper complex. Based on the uric acid estimations, it has also been ascertained that these complexes dismute O2.‐ without inhibiting xanthine oxidase activity. The presence of increasing concentrations of albumin had no effect on the SOD mimicking activity of mixed ligand complexes. Polyamine complex, however lost approximately 80% of SOD mimicking activity in the presence of albumin (1 mg). These results suggest that coordination of polyamine copper complex with imidazoles/pyridine may abolish their binding affinity for albumin while potentiating their SOD mimicking activity.
Use of transport mutants to examine the identity and expression of GLUT isoforms in rat cardiac myoblasts
Authors: Lu Zhan , Zaniewska Ewa , Lo Theodore C. Y.
Published: July 2007

Two different spontaneous glucose transport (GLUT) mutants were used to examine the identity and properties of proteins involved in rat cardiac myoblast glucose transport processes. The parental clone, H9C2, possessed a high (HAHT) and a low (LAHT) affinity hexose transport process, and the GLUT 1, 3 and 4 isoforms. Mutant RCM was devoid of HAHT, the GLUT 3 transcript, and a 41 kDa protein recognizable by an anti‐mouse GLUT 3 Ab. Mutant EZ‐4 was impaired in the GLUT 3 and 4 isoforms, and in HAHT and LAHT. These studies demonstrated a close association of the GLUT 3 and 4 isoforms with the HAHT and LAHT processes, respectively. Both GLUT 3 and 4 isoforms were regulated in opposite ways during myogenesis, and both GLUT 3‐ mutants were impaired in myogenesis. Despite its normal GLUT 1 transcript level, mutant EZ‐4 was devoid of an efficient carrier‐mediated glucose transport process, thus suggesting that the GLUT 1 transporter was inoperative in rat cardiac myoblasts. Unlike the rat skeletal L6 GLUT 1 isoform, expression of the rat cardiac GLUT 1 isoform was not affected by glucose starvation, and was not reduced in multinucleated myotubes. These studies demonstrated the usefulness of transport mutants in determining the identity, expression and property of GLUT isoforms and their association with specific transport processes.
Utilization of alcohols by plant and mammalian phospholipase D
Authors: Ella Krishna M. , Meier Kathryn E. , Kumar Anil , Zhang Yanzhi , Meier G. Patrick
Published: July 2007

The transphosphatidylation reaction is a unique property of phospholipase D (PLD). In this study, the abilities of plant and mammalian PLDs to utilize straight chain and branched alcohols for transphosphatidylation were analyzed and compared. PLD from peanut utilizes C1 to C8 primary alcohols and gives maximal reaction with butanol. In contrast, PLD from A7r5 vascular smooth muscle cells gives maximal reaction with pentanol and does not utilize octanol. Secondary and tertiary alcohols are not substrates for either enzyme. For branched alcohols, activity increases with distance from the alcohol to the branch point. Competition studies indicated that secondary alcohols cannot access the binding pocket. Thus, PLDs have a water/alcohol binding site with defined steric and hydrophobic parameters.
Involvement of a tyrosine residue in the ADP binding site of creatine kinase. A second‐derivative UV‐spectroscopy study
Authors: Leydier Chantal , Clottes Eric , Couthon Fabienne , Marcillat Olivier , Vial Christian
Published: July 2007

Second‐derivative spectroscopy was used to determine the percentage of tyrosine residues that are exposed to solvent in rabbit MM‐creatine kinase. Six residues, among the ten present per monomer, are solvent‐exposed. The presence of creatine in the incubation medium does not modify this value. However, this number is decreased by one when the enzyme is incubated with saturating concentrations of MgADP. A dissociation constant for MgADP can be estimated and the obtained value (0.085 mM) is comparable to the Km for this substrate. Thus, a tyrosine residue is located near the MgADP binding site or is masked during protein conformational change induced by adenyl nucleotide binding.
Glycolytic enzyme binding in Otala lactea hepatopancreas: Effect of taxol, colchicine and cytochalasin B and D on the in vivo enzyme distribution
Authors: Brooks S. P. J. , Storey K. B.
Published: July 2007

The effect of cytoskeleton modulators on glycolytic enzyme binding was examined in the hepatopancreas of Otala lactea in an attempt to identify potential cellular binding sites. Binding was followed by measuring phosphofructokinase (PFK), aldolase (ALD), glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) and pyruvate kinase (PK) distribution between low speed pellets (12,000xg), high speed pellets (100,000xg) and high speed supernatants. Taxol (which stabilizes microtubules), colchicine (which destabilizes microtubules) and cytochalasin B and D (which destabilize F‐actin filaments) were added to the homogenate prior to centrifugation. Addition of taxol increased the amount of PFK associated with the high speed pellet. Cytochalasin B and D reduced the binding of PFK and PK to the low speed pellet. ALD and GAPDH binding were unaffected by any treatment. Lowering the pH of the crude homogenate increased PFK binding to the low speed pellet by 33%. This effect could be reversed by addition of cytochalasin B and D suggesting that pH influences the PFK‐F‐actin interaction in vivo. The differential binding response of PFK, PK, ALD and GAPDH to added effectors suggests that, in the cell, PFK and PK are bound to different subcellular structural elements than are ALD and GAPDH.
Characterization of a 34 kDa immunoreactive peptide with the anti protein kinase C antibody which can recognize part of the C4 region
Authors: Ayaki Masako , Hashimoto Chieko , Inui Yukiharu
Published: July 2007

Among the immunoreactive peptides from hamster tissues with the antibody (Ab‐1) which can recognize part of the C4 conserved region of protein kinase C (PKC), a peptide with 34 kDa (34 kDa species) is constitutively detected in the soluble and the nuclear but not in the particulate fractions. The partially purified 34 kDa species demonstrates no phospholipid‐dependent or independent PKC activity and no phorbol 12,13 dibutyrate (PDBu) binding, and is not phosphorylated under the assay condition of PKC activity. The species inhibits the enzymic activity of the partially purified PKC from hamster brain in an uncompetitive manner with Ki of 200±23 nM.
Two pathways of the nitric oxide‐induced cytotoxycal action
Authors: Turpaev Kyril T. , Amchenkova Aleftina M. , Narovlyansky Alexander N.
Published: July 2007

Nitric oxide is a diffusible messenger with multiple biological functions. We show here that NO‐generating compound, S‐nitrosoglutathione (GSNO) induces apoptosis in human chondrocytes and causes necrosis‐like celt death in human epithelial CaOv cell line. Pretreatment of chondrocytes with low‐dose GSNO or with γ‐interferon enhances their tolerance to the second high‐concentration GSNO exposure. On the contrary, in CaOv cells low‐dose GSNO pretreatment diminishes the resistance and increases cytolysis at the second GSNO exposure. We conclude that human chondrocytes possess specific and inducible mechanism preventing celt killing by nitric oxide.
Characterization of a soluble IL‐6 receptor α mutant C277D/H280I expressed in E. coli
Authors: Duan Jubao , Wang Jiaxi , Cai Xin , Wang Lihong , Liu Haitian , Zou Minji
Published: July 2007

The pleiotropic cytokine interleukin‐6(IL‐6) triggers the formation of a high affinity receptor complex constituted by the ligand‐binding subunit IL‐6 receptor α(IL‐6Rα) and the signal transducer gpl30. To construct the antagonist of IL‐6, a DNA segment encoding the soluble human IL‐6Rα(shlL‐6Rα) mutant with two substitutions C277D/H280I was generated by overlap extension polymerase chain reaction. The DNA segment was then subcloned into vector pET‐3b and expressed in E.coli at a high level. The refolded and purified recombinant protein, which was designated as DM650, exhibited an increased IL‐6‐binding capability by 8‐fold. DM650 antagonized IL‐6 perfectly, resulting in the growth‐inhibition of human myeloma AF‐10 cells. DNA fragmentation assay proved that the growth of AF‐10 cells was inhibited through the induction of apoptosis suppressed by IL‐6.
The structural and functional essentiality of the N‐terminal α‐helix in the phospholipase A2 of the Taiwan banded krait
Authors: Chang Long‐Sen , Chang Chun‐Chang , Wu Pei‐Fung
Published: July 2007

In order to identify the structural and functional essentiality of the N‐terminal α‐helix of Bungarus multicinctus PLA2 for its enzymatic activity, comparative studies of the biochemical properties of native and recombinant PLA2 were made. It was found that the appearance of a Met residue preceding the N‐terminus Asn‐1 of the recombinant protein appreciably affected PLA2 activity and the hydrophobic character of the ANS‐binding site. Additionally, the charged state and the hydrophobicity of the molecular surface changed as well. However, removal of the N‐terminal Met‐1 from the recombinant PLA2 resulted in the production of a fully active PLA2, whose biochemical properties were indistinguishable from those of the native enzyme. These observations, together with the findings that the helical wheel plot of the N‐terminal α‐helix showed distinct hydrophobic and hydrophilic faces and that the ANS‐binding site was the active site of PLA2 enzymes, suggest that the hydrophobic face of the N‐terminal α‐helix in native PLA2 should be in the interior of the enzyme molecule for binding with ANS and the phospholipid/substrate.
DL‐propranolol negatively regulates the transcription of proliferating cell nuclear antigen (PCNA)‐gene and thereby suppresses DNA synthesis in regenerating rat liver
Authors: Hong Jeong‐Ho , Hwang Eun Sook , Lee Chang Ho , Lee Yong Hee , Lee Seung Ki
Published: July 2007

Previous reports have suggested that dl‐propranolol (PRL) suppresses DNA synthesis by blocking cAMP‐mediated signaling in rat liver after partial hepatectomy (PH). Here, we examined if PRL negatively regulates the expression of genes involved in cell cycle progression. Immunoblotting assays showed that the protein levels of cyclins A and E, Cdk2, p21WAF1, and p27KIP1 did not significantly change in liver tissues from either vehicle‐ or PRL‐injected rats after PH. However, the levels of PCNA and PCNA‐mRNA markedly decreased in the remnant liver in response to PRL‐injection. Similarly, PCNA‐CRE binding activity of nuclear 43kDa CREB was suppressed, although the protein levels were not altered. We suggest that PRL negatively regulates the PCNA‐gene transcription by interfering with the cAMP/PKA‐mediated induction of CREB binding to the CRE‐sequences and thereby suppresses DNA synthesis in regenerating rat liver.
Mutational analysis for the role of C‐terminal region of ecotin, a dimeric inhibitor of pancreatic serine proteases
Authors: Seong Ihn Sik , Lee Cheol Soon , Park Hye Kyung , Suh Se Won , Kang Man‐Sik , Chung Chin Ha
Published: July 2007

Ecotin is a dimeric molecule that is capable of inhibiting a variety of serine proteases. To clarify the role of the C‐terminal region, mutagenesis was performed to delete the C‐terminal residues from 130 to 142. The mutant inhibitor behaved as a monomer upon cross‐linking analysis followed by gel filtration. The mutation also resulted in a significant increase of the Ki of ecotin on trypsin, chymotrypsin, and elastase (i.e., by 1 to 2 order of magnitude). The mutant ecotin was slightly more sensitive to heating at 100°C than the wild‐type ecotin, but became much more sensitive to the heat treatment upon reduction of the intra‐chain disulfide bond in its subunits. In addition, treatment with 4 M urea resulted in complete loss of the activity of the mutant ecotin but not that of the wild‐type inhibitor. Thus, the C‐terminal region of ecotin seems to be required not only for dimerization of the subunits but also for optimal interaction with target proteases and for maintenance in its structural stability, particularly under reducing or denaturing conditions.
Linear gene fusions of antibody fragments with streptavidin can be linked to biotin labelled secondary molecules to form bispecific reagents
Authors: Pearce Lesley A. , Oddie Geoffrey W. , Coia Gregory , Kortt Alexander A. , Hudson Peter J. , Lilley Glenn G.
Published: July 2007

Monomeric single chain antibody (scFv) fragments lack both the avidity of the bivalent IgG, or (Fab′)2 fragment, and the effector functions conferred by the Fc domain. For certain diagnostic or therapeutic applications it may be desirable to link these molecules to other proteins, antibodies, enzymes or peptide ligands, and chemical or recombinant methods have been developed to produce many of these crosslinked reagents. One approach has been to link an antibody fragment to streptavidin which can bind a second biotinylated molecule to create a higher affinity, bifunctional or bispecific molecule. To demonstrate the applicability of this technology, an anti‐neuraminidase NC10 scFv‐streptavidin fusion was expressed in E. coli and the product was refolded and purified to homogeneity from 6 M guanidine hydrochloride. Analysis in a BIAcoreTM biosensor showed that the NC10 scFv moiety reacted with immobilised neuraminidase and that the core streptavidin moiety was able to bind biotinylated anti‐ferritin Fab' to produce a new model bispecific reagent which bound ferritin. Conceptually, this design principle can be applied to the creation of useful diagnostic and possibly therapeutic molecules.
Involvement of caspases in apoptosis induced by ultraviolet B irradiation in A431 human epithelioid tumor cells
Authors: Iwasaki Kazumi , Izawa Masao , Mihara Motoyuki
Published: July 2007

Six hours after ultraviolet B (UVB) irradiation (11.6 mJ/cm2), the viability of A431 cells decreased, and, at the same time, fragmentation of genomic DNA into nucleosomal units was observed. Z‐Asp‐CH2‐DCB (100 μM), an inhibitor of interleukin‐1 β‐converting enzyme (caspase‐1 ) and caspase‐1‐like proteases, markedly inhibited UVB‐induced cell death and DNA fragmentation. Both YVAD‐CMK, an inhibitor of caspase‐1, and DEVD‐CHO, an inhibitor of caspase‐3, moderately inhibited the UVB‐induced cell death. A combination of YVAD‐CMK and DEVD‐CHO acted additionally in inhibiting cell death. These observations suggest strongly the cooperative involvement of caspases in the apoptosis induced in A431 cells by UVB.
Isolation and characterization of 5′‐regulatory region of mouse activin ßa subunit gene
Authors: Yoshida Eisaku , Tanimoto Keiji , Murakami Kazuo , Fukamizu Akiyoshi
Published: July 2007

We isolated genomic clones that contain the 5′‐flanking region of the mouse activin ßa subunit gene. The nucleotide sequence determination of the 5′‐flanking region of the gene and the comparison of that with the reported mouse cDNA structure identified the putative 5′ regulatory region, a novel first exon and a part of the first intron of the gene within this region. The putative 5′ regulatory region of the mouse activin ßa subunit gene directed the expression of CAT gene in transfected HT1080 cells. Successive deletions of this region demonstrated a 400‐bp region that exerts a strong positive effect on promoter activity of the mouse activin ßa subunit gene.
Involvement of a third histidine in the ferrous active site of isopenicillin N synthase of Cephalosporium acremonium repudiated by recombinant double histidine mutants
Authors: Tan D. S. H. , Ang S. G. , Sim T. S.
Published: July 2007

Site‐directed mutagenesis studies have shown that the isopenicillin N synthase of Cephalosporium acremonium (cIPNS) requires two essential histidine residues (H216, H272) for activity. The determination of iron bound to the wildtype cIPNS and its absence in the mutants lacking histidine at positions 216 and 272 clearly supports the essential role these two histidines play in iron binding. However, nuclear magnetic resonance (NMR) studies have indicated that there could be three histidine residues that possibly coordinate the essential iron at the active site. To search for a presumed third histidine ligand, mutant cIPNS genes containing mutations at two histidine codons were created by in vitro cloning of fragments from the expression vectors bearing the respective cIPNS genes each with a single histidine mutation at positions H49, H64, H116, H126 and H137. All ten possible double histidine mutant cIPNS constructs were subsequently expressed in Escherichia coli. If a third histidine had a participatory role in the iron active centre of cIPNS, then one of the constructed double histidine mutants would have lost its enzymatic activity. However, analysis of the cIPNS activities of these recombinant double histidine mutants indicated that none of them was totally inactivated. Thus, the involvement of a third histidine can be repudiated.
In vitro processing of cucumber chloroplast tRNALeu (CAA) precursor in a pea chloroplast soluble extract
Authors: Hande Shailaja , Jayabaskaran C.
Published: July 2007

To study the sequential steps in the processing pathway of the chloroplast monocistronic intronless tRNA precursors, we examined cucumber chloroplast tRNALLeu(CAA) processing in a cucumber or pea chloroplast soluble extract. The tRNALeu(CAA) precursor synthesized from SP6 RNA polymerase‐directed transcription system, was used as a substrate. Incubation of the tRNA precursor with the pea extract resulted in processing of tRNALeu(CAA) via 5′‐ and 3′‐endonucleolytic cleavages followed by final trimming of extra 3′nucleotides by 3′ exonuclease(s). No preferred order for endonucleolytic cleavages has been observed during the in vitro tRNALLeu processing and the simultaneous occurrence of the intermediates consisting of leader + tRNALeu and tRNALeu + trailer, indicate that either 5′‐ or 3′‐ endonucleolitic cleavage can occur as the first step in vitro.
Characterization of extracellular menadion‐catalyzed H2O2 production by NTH/3T3 cells
Authors: Yamashoji S.
Published: July 2007

The extracellular menadione‐catalyzed H202 production by NIH/3T3 cells was expected to depend on plasma membrane‐bound NAD(P)H:quinone oxidoreductase. This enzyme was estimated to be a flavoprotein with the molecular mass of 70 KDa. Km values of plasma membrane‐bound NAD(P)H:quinone oxidoreductase producing H20 2 were 60 μM for NADH and 150 μM for NADPH. Ca2+ ionophore A23187 controlled menadione‐catalyzed H2O2 production by the cells in time‐ and concentration‐dependent manner.
Photoinactivation studies on adenosine deaminase
Authors: Lupidi G. , Colao C. , Marmocchi F. , Cristalli G.
Published: July 2007

Reinvestigation of direct modification of histidine residues in calf intestine adenosin deaminase resulted in loss of the activity towards substrates as adenosine, 2′‐deoxyadenosine and 6‐hydroxyl‐aminopurin riboside. Photo‐oxidation of the enzyme in the presence of methylene blue led to a complete loss of enzymatic activity and the presence of inhibitors such as purin riboside, EHNA, coformycin, showed protection against methylene blue oxidation. Kinetic analysis of the inactivation by diethylpyrocarbonate, indicated that enzyme inactivation results from the modification of at most one essential histidine residue. Photoinactivation of adenosine deaminase from Aspergillus Orizae reduces the activity of the enzyme but the accessibility of histidine residues in the active site seems to be lower as compared to that shown from mammalian adenosine deaminase. The results obtained are in agreement with the important role played by the “bridge” between an enzyme histidine residue and the 5′‐OH of the ribose mojety of substrate in the transition state stabilization in the two enzymatic forms observed. Upon ultraviolet irradiation of calf intestine adenosine deaminase in the presence of 2,2,2‐thrichloroethanol, the trasformation of fluorescent tryptophan residues occurs with the reduced enzymatic catalytical activity and confirms the likely location of tryptophans near the binding site of the enzyme.
Variation of phosphoribosylpyrophosphate synthetase activity in human cells
Authors: Okada Naomi Nagatake , Ishizuka Toshiharu , Isogai Eriko , Suzuki Nobuo , Tatibana Masamiti
Published: July 2007

The behavior of phosphoribosylpyrophosphate (PRPP) synthetase during the cell cycle was studied using RSa cells derived from human fetal fibroblasts. The cells were synchronized by the double thymidine block method. PRPP synthetase activity in cell extracts increased 2‐fold in the S phase. Metabolic flux through phosphoribosylpyrophosphate into nucleotides, which represents the in situ activity of PRPP synthetase, increased 3‐fold in the S phase. The mRNA levels of PRPP synthetase subunit I and II genes (PRPS1 and PRPS2 respectively) increased 3‐fold and the increase preceded elevation of enzyme activity in the S phase. Thus variation in PRPP synthetase activity during the cell cycle is ascribed mainly to varying amounts of the enzyme. Degradation of PRPS1 and PRPS2 mRNA was significantly suppressed in the early S phase and was presumably related to periodic accumulation of the two mRNAs. In earlier work, it was found that the expression of PRPP synthetase isoform genes PRPS1 and PRPS2 is regulated in a tissue specific manner, thereby suggesting a functional difference between them. We have now obtained evidence that there is no apparent difference between the expression of PRPS1 and PRPS2 genes in the cell cycle.
Amino acid residue 250 has a functional role in the assembly of rabbit liver microsomal cytochrome P450 2B4
Authors: Schulze Johannes , Lehnerer Michael , Lewis David F. V. , Hlavica Peter
Published: July 2007

Cytochrome P450 2B4 lacking amino acids 2‐27, CYP2B4 (Δ2‐27), was mutated at position 250 and expressed in E.coli fused toglutathione S‐transferase. Expression of the E250S variant (holo‐plus apoenzyme) proceeded to an extent comparable with that of CYP2B4 (Δ2‐27), while the protein level of the E250P mutant averaged 42% that of the control pigment. Comparison of these data with the corresponding reduced CO difference spectra of the various CYP2B4 (Δ2‐27) forms revealed that, in the control and E250S preparations, about 90% and 44%, respectively, of the total amount of hemoprotein present existed in the form of holoenzyme, whereas the E250P derivative failed to produce a reduced carbonyl complex. Thus, replacement of the negatively charged E250 with an uncharged, polar serine residue substantially hampered assembly of CYP2B4 (Δ2‐27); introduction of an α‐helix‐disrupting proline completely blocked the formation of holoenzyme. These phenomena suggested that the negative charge of E250, residing in the putative G helix, underwent pairing with some positively charged group, possibly H285 located in the I helix. Deletion of the negative charge obviously perturbed the active‐site geometry such as to affect both the incorporation and/or retention of the heme ligand and the spectral binding of substrates such as hexobarbital.
Effect of Vitamin E on antioxidant enzymes and nitric oxide in ischemia‐reperfused kidney injury
Authors: Uysal Fevziye , Girgin Ferhan K. , Tüzün Sevgi , Aldemir Sevil , Sözmen Eser Yildirim
Published: July 2007

The effects of Vitamin E administration on antioxidant enzyme activities and nitrite‐nitrate levels of the reperfused rat kidney tissues were investigated by performing a 60 min ischemia followed by 24 and 72 hours of reperfusion. Vitamin E administration or the placebo (SF) was applied as 100 mg/kg BW. As expected, catalase (CAT) (p<0.05) and superoxide dismutase (SOD) (p<0.05) activities of ischemia/reperfused (I/R) kidney tissue were lower and malondialdehyde (MDA) levels were higher than control kidneys in both SF and vitamin E treated groups following 24 h reperfusion. During reperfusion of long term (72 h), vitamin E triggered a decrease in the MDA levels in the ischemic tissue, while it did not provoke a significant effect on SOD and catalase activities. Total nitrite levels of ischemic tissues in both of the groups were higher than matched control kidneys and this elevation was more clear in the vitamin E treated group. Our results showed that vitamin E has a protective effect on I/R injury, by a direct chain breaking effect on lipid peroxidation (LPO) and hence preventing the nitric oxide (NO) reservoir of ischemic tissue. Alfa‐tocopherol may be a promising agent for the prevention of tissue injury caused by free oxygen radicals.
Soluble guanylate cyclase in Saccharomyces cerevisiae
Authors: Kuo W. N. , Kanadia R. N. , Mcnabb M.
Published: July 2007

After removing nonspecific immunoreactivities from crude extract by immunoaffinity chromatography, an immunoreactive‐band at 40kDa of soluble guanylate cyclase (SGC) from Saccharomyces cerevisiae was detected by Western blot using rabbit anti‐β1 subunit of SGC. Cyclic GMP level and SGC activity was measured by ELISA. Immunoprecipitated yeast SGC was activated by sodium nitroprusside, whereas inhibited by 1H‐(1,2,4) oxadiazolo(4,3‐A)quinoxalin‐1‐one. Increased cyclic GMP level was also noted when intact yeast cells were incubated with s‐nitrosoglutathione, a NO donor. The result implies that NO can be utilized intracellularly and extracellularly. Moreover, the presence of SGC suggests the significance of NO/cyclic GMP signaling in unicellular eukaryotes.
Luminol luminescence induced by oxidants in antioxidant‐deficient yeasts Saccharomyces cerevisiae
Authors: Jakubowski Witold , Bartosz Grzegorz , Ertel Danuta , Bilinski Tomasz , Kędziora Józef
Published: July 2007

Luminal chemiluminescence induced in the presence of yeast cells and yeast cell homogenates was significantly induced by exogenous oxidants (hydrogen peroxide and menadione), tert‐Butyl hydroperoxide did not stimulate chemiluminescence by itself but augmented menadione‐induced chemiluminescence. Comparison of yeast strains deficient in catalase, superoxide dismutase or glutathione showed that only glutathione‐deficient strains showed elevated chemiluminescence in this system. These results support the idea that more reactive species than hydrogen peroxide and superoxide are critical in the induction of luminal chemiluminescence.
Hydroxyl group of insulin A19Tyr is essential for receptor binding: Studies on (A19Phe) insulin
Authors: Du Xin , Tang Jian‐Guo
Published: July 2007

One mutant proinsulin gene was constructed through PCR with the code of A19Tyr changed into that of Phe. The mutant proinsulin, (A19Phe)‐lysproinsulin (F19KPI) was expressed in E. coli and purified. After trypsin and carboxypeptidase B cleavage and ResourceTM Q separation, (A19Phe)‐human insulin (F19HI) was obtained. With native insulin as standard, activity assay and structural analysis were carried out. It was found that the conformation of F19HI is very similar to that of native insulin according to the data from native PAGE, reverse‐phase FPLC and circular dichroism spectrum, which are also in agreement with the result of radioimmune assay. F19HI retains almost full immune activity, but displays only 13.3% of receptor binding activity. These results suggest that the hydroxyl group of insulin A19Tyr is essential for receptor binding.
Purification of recombinant human pepsinogens and their application as immunoassay standards
Authors: Aoki Takashi , Tomaki Emiko , Satoh Miyoko , Takashiro Miho , Onagi Hitoshi , Itoh Masao , Teramoto Tetsuya , Morikawa Junji , Watabe Hiroyuki
Published: July 2007

Human pepsinogen (PG) A and C were cloned in Escherichia coli, but the levels of expression were low and unstable. When these were fused to maltose‐binding protein (MBP), the fusion proteins (MBP‐PGA and MBP‐PGC) were expressed as the major products. Although these fused products were almost totally recovered from the insoluble fraction, the renaturation and purification procedures were easy and simple. MBP‐PGA and the PGA segment obtained by factor Xa digestion (designated as r‐PGA) possessed proteolytic activities equivalent to native PGA purified from gastric tissue (t‐PGA). For PGCs (MBP‐PGC, r‐PGC and t‐PGC) also, the specific activities were almost the same. However, the activities of PGCs were about 3‐ to 4‐hold higher than those of PGAs. In PGA and PGC immunoassay systems, r‐PGs (r‐PGA and r‐PGC) and the EIA kit standard PGs (gastric mucosal PGs) exhibited a good correlation. From these results, r‐PGs would seem to be applicable as assay standards without compromising the sensitivity of the immunoassay systems.
The effects of N‐thiophosphoryl amino acids on the activity of green crab (Scylla Serrata) alkaline phosphatase
Authors: Chen Qing‐Xi , Zhu Chun‐Ming , Zhou Hai‐Meng , Lu Hai‐Yan , Lin Hai‐Ning
Published: July 2007

Green crab (Scylla Serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme which catalyzed the nonspecific hydrolysis of phosphate monoesters. In the present paper, the effects of several N‐thiophosphoryl amino acids on the activity of green crab alkaline phosphatase have been studied. The results show that these derivatives of amino acids can lead to reversible inactivation. The equilibrium constants for inhibitors binding with the enzyme and/or the enzyme‐substrate complexes have been determined. The obtained results show that both N‐thiophosphoryl‐Cys and N‐thiophosphoryl‐Glu were non‐competitive inhibitors, while other five N‐thiophosphoryl amino acids were un‐competitive inhibitors. For the un‐competitive inhibitors, the inhibition strength follows the order N‐thiophosphoryl‐Ile>‐Val>‐Lys>‐Ala>‐Tyr. Compared with respective free amino acids, it can be seen that N‐thiophosphorylation of the amino acids increased their inhibition strength except the N‐thiophosphoryl‐Cys.
Effect of prooxidants on mitochondrail permeability transition and cell death in Ehrlich ascites tumour cells
Authors: Teplova Vera V. , Kudrjavtsev Andrew A. , Odinokova Irina V. , Evtodienko Yuri V. , Saris Nils‐Erik L.
Published: July 2007

Ca2+ retention in mitochondria, opening of the Cysclosporin A‐ sensitive permeability transition pore and cell death were studied in Ehrlich ascites tumour cells in the presence of different prooxidants. Low concentrations (1‐20 μM) of the prooxidants (menadione, cumenehydroperoxide, t‐butylhydroperoxide) induced pore‐opening in permeabilized cells at threshold Ca2+ load. Incubation of cells with low concentrations of prooxidants was able to induce cell cycle disturbance and cell death. Under the prooxidant effect, mitochondrial membrane potential drop and Ca2+ retention decrease in mitochondria were found to precede death of Ehrlich ascites tumour cells.
Glycated protein‐iron chelate increases lipid peroxide level in cultured aortic endothelial and smooth muscle cells
Authors: Nishigaki Ikuo , Yagil Kunio , Tanimoto Akihide , Sasaguri Yasuyuki
Published: July 2007

Formation of an iron chelate of glycated protein was demonstrated by the appearance of an absorption peak at approximately 270 nm after mixing glycated bovine serum albumin with FeCl3. This peak disappeared and a new peak appeared at approximately 420 nm to form an isosbestic point at approximately 340 nm by the addition of deferoxamine mesylate, an ironchelating agent, to the mixture, thus confirming the formation of the iron chelate of the glycated protein in the mixture. The lipid peroxide level was increased markedly in endothelial cells and slightly in smooth muscle cells from bovine aorta incubated in the medium containing glycated fetal bovine serum‐iron chelate. Morphological observation by phase‐contrast microscopy and scanning electron microscopy revealed that the glycated fetal bovine serumiron chelate caused intense damage to the endothelial cells. These results indicate that glycated protein‐iron chelate provokes lipid peroxidation, which explains at least in part the mechanism of atherogenesis found in diabetic patients.
Association of pp60cc‐src with αIIbβ3 in resting platelets
Authors: Kralisz Urszula , Cierniewski Czeslaw S.
Published: July 2007

To detect whether 125I‐αIIbβ3 is associated with tyrosine kinases in platelets, antibodies specific to pp60c‐src, pp54/58lyn, and pp62Fyn were used to precipitate their homologous antigens. In contrast to Lyn and Fyn kinases, pp60c‐src appears to be complexed with αIIbβ3. Both proteins, pp60c‐src and αIIbβ3, coprecipitated when antibodies to pp60c‐src were used in the immunoprecipitation experiments. This conclusion was further supported by immunoprecipitation of αIIbβ3 from Triton X‐100 extracts of nonlabelled platelets with P2 antibodies. There was no pp60c‐src detectable in immunoprecipitates obtained with antibodies specific to α2β1 or GPIb. Since PGE1 was used to prevent platelet activation in buffers throughout all procedures and there was no phosphorylation of pp72syk we assume that the platelets were in the resting state. Therefore, we conclude that αIIbβ3 and pp60c‐src can form a complex in resting platelets suggesting that pp60c‐src is directly involved in initiating the outside‐in signaling cascades in blood platelets.
A proposed estimate of the tumor aggressiveness of human breast cancer using radiorespirometry
Authors: Mayaudon Jacques , Yan Shou‐He , Wallon Joëlle
Published: July 2007

The relationships between carcinomatous aggressiveness and the glucolytic metabolism, namely the rate of 14CO2 production from [U‐14C] glucose, are obtained from human breast tissues using radiorespirometry. The values are estimated as the initial velocity (V) expressed in η14CO2×min‐1×g‐1 of fresh tissues by [U‐14C] glucose metabolism. The aggressiveness of the breast carcinomatous is diagnosed by the SBR grade system. As two control normal tissues, (V) are 0.86 to 0.90 from non‐cancer patients. In carcinomatous tissues (Vc), there is an increase from 1.53 to 3.14, but in the corresponding surrounding non‐cancer tissues (Vn) these show a decrease from 2.20 to 0.22 for SBR I, SNR II to SBR III. The ratio between (Vc) and (Vn) are found, according to carcinomatous aggressiveness, as 1.45 to 1.54, 1.69, 2.35 to 2.86 and 4.82 to 10.38 respectively for SBR I, lobular carcinoma, SBR II and SBR III; while the ratio is 1.04 for the normal tissue which come from non‐cancer patients. The above results suggest the possibility of assessing the carcinomatous aggressiveness by radiorespirometry before a histopathological diagnosis, even in a lower aggressiveness as in SBR I cases which are difficult to diagnose and manage.
Constitutive nitric oxide synthase in Saccharomyces cerevisiae
Authors: Kanadia R. N. , Kuo W. N. , Mcnabb M. , Botchway A.
Published: July 2007

After removing nonspecific immunoreactivities from crude extract by immunoaffinity chromatography, an immunoreactive‐band at 60kDa of constitutive nitric oxide synthase (cNOS) from Saccharomyces cerevisiae was detected by Western blot using mouse monoclonal anti‐neuronal NOS (cNOS). The activity of yeast cNOS, which was prepared by either histone‐agarose chromatography or anti‐neuronal NOS immunoprecipitation, was monitored by the formation of citrulline. Yeast cNOS was activated in the presence of calmodulin and arginine, whereas it was inhibited by L‐NAME, a mammalian NOS inhibitor. Moreover, actinomycin‐D decreased the extracellular and the intracellular levels of nitrate and nitrite which had been converted from NO. The results suggest that cNOS occurs in unicellular eukaryotes and the enzyme activity can be regulated.
Glutathione metabolism and glutathione S‐conjugate export ATPase (MRP1/GS‐X pump) activity in cancer
Authors: de Bittencourt Paulo Homem , Curi Rui , Williams John F.
Published: July 2007

Mg2+‐dependent vanadate‐sensitive glutathione S‐conjugate ATPase (GS‐X pump) activity is a common feature of some ATP‐binding cassette (ABC) transporters, such as the multidrug resistance‐associated protein (MRP1) gene product, that exports biologically active electrophiles after their conjugation with intracellular glutathione (GSH) from normal and cancer cells. Antitumor electrophiles (e.g. naturally occurring cyclopentenone prostaglandins and anticancer chemicals) can be intracellularly conjugated with GSH via a glutathione S‐transferase catalyzed reaction and be eliminated through GS‐X pumps thus threatening cancer chemotherapeutics. Since different sensitivities to antitumor electrophiles are shown by different cell types, the ability of several human cancer cell lines to produce and export S‐(2,4‐dinitrophenyl)‐glutathione (DNP‐SG) conjugate through the GS‐X pump, using whole cells and inside‐out membrane vesicle preparations, is investigated. Different cancer cell lines exhibited characteristically different GS‐X pump activity. In particular, HEp‐2 larynx carcinoma cells possess an elevated DNP‐SG export rate through the GS‐X pump compared with HeLa, K562, U937 or HL‐60 cells, which exhibit the lowest activity. The differences in DNP‐SG export rates are not due to decreased glutathione S‐transferase activity or impaired de novo synthesis of GSH. The findings suggest that the GS‐X pump may be involved in the modulation of the biological activity of both naturally occurring electrophiles and anticancer drugs. The differential expression of GS‐X pumps may lead to an improved understanding of multidrug resistance and may be exploited in the development of new therapeutic strategies for the treatment of cancer patients.
Various forms of rabbit plasma α‐1‐antiproteinase
Authors: Saito Akio , Sinohara Hyogo
Published: July 2007

Amino acid sequencing of the ficin‐derived C‐terminal fragments of α‐1‐antiproteinase (also called α‐1‐antitrypsin or α‐1‐proteinase inhibitor) prepared from rabbit plasma revealed the presence of the E isoform, which had been confirmed in the cDNA library in addition to the F and S‐1 forms. The S‐2 form was identified in inflamed rabbit plasma. The ficin digest of human plasma α‐1‐antitrypsin resulted in a major fragment of the M type. Multiple forms of α‐1‐antiproteinase in the rabbit plasma implicate the unknown functions other than the inhibition of neutrophil elastase.
Peroxide production and apoptosis in cultured cells carrying mtDNA mutation causing encephalomyopathy
Authors: Zhang Jin , Yoneda Makoto , Naruse Keiji , Borgeld Harm‐Jan W. , Gong Jian‐Sheng , Obata Shu‐ichi , Tanaka Masashi , Yagi Kunio
Published: July 2007

When cybrids with a point mutation, which locates in the tRNALeu(UUR) gene of mtDNA and causes a mitochondrial encephalomyopathy (MELAS syndrome), were exposed to a high concentration of oxygen (95%), the peroxide production markedly increased by 6 h of oxygen exposure, whereas the peroxide production was similar among the cybrids under a normal concentration of oxygen. The peroxide production by oxygen exposure was enhanced particularly in cybrids with high proportions of the mutant mtDNA and low respiratory capacities. The appearance of apoptotic cells by oxygen exposure was high in cybrids with the impaired respiratory function due to the mutation. An antioxidant NAC successfully suppressed both the peroxide production and apoptosis. These results imply that the peroxide production plays an important role in inducing apoptosis in cells carrying the mtDNA mutation causing encephalomyopathy.