bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of a beta‐lactamase from Mycobacterium smegmatis SN2
Authors: Basu Dhiman , Narayankumar D. V. , Van Beeumen Josef , Basu Joyoti
Published: July 2007

Beta‐lactamases have been reported to be largely responsible for beta‐lactam resistance in Mycobacteria. We report the characterization of a cell‐associated beta‐lactamase from Mycobacterium smegmatis. The enzyme hydrolyzed the “beta‐lactamase‐stable” oximinocephalosporins. Nitrocefin was the best substrate. 6‐Beta‐iodopenicillanate, clavulanate and sulbactam were effective inhibitors, whereas the Ki value for aztreonam was high. From its substrate and inhibitor profile, the enzyme appeared to be a cephalosporinase of group 2e.
Regulation of CREB phosphorylation by cAMP and Ca2 in parotid acinar cells
Authors: Takuma Taishin , Tajima Yoshifumi , Ichida Tokuro
Published: July 2007

Since various secretory stimuli regulate not only secretion but also protein, RNA, and DNA syntheses in salivary glands, we evaluated the effect of secretory stimuli on the phosphorylation state of CREB (cAMP response element‐binding protein). Isoproterenol, forskolin, and CPS‐cAMP markedly stimulated the phosphorylation of CREB in parotid acinar cells, and PKA inhibitors H‐8 and H‐89 dose‐dependently inhibited it. In contrast, carbachol (CCH) and A23187 decreased CREB phosphorylation, but CCH did not decrease it in the absence of extracellular Ca2+. Although protein phosphatase inhibitor calyculin A alone markedly increased the phosphorylation, it could not prevent CCH‐induced dephosphorylation of CREB. CaM kinase IV, a putative protein kinase for CREB in response to Ca2+ elevation, was undetectable in parotid acinar cells.
Differential inhibition of aflatoxin B1 oxidation by gestodene action on human liver microsomes
Authors: Kim Bok Ryang , Oh Hyun Sook , Kim Dong‐Hyun
Published: July 2007

Human cytochrome P450 (P450) 3A is known to be involved in the formation of both aflatoxin B1‐exo‐8,9‐epoxide (exo‐epoxidation) and aflatoxin Q1 (3α‐hydroxylation). Gestodene, a known inactivator of P450 3A4, inhibited the formation of AFB1 metabolites in a variety of ways depending on the incubation condition. Preincubation of gestodene with human liver microsomes prior to the addition of AFB1 inhibited both exo‐epoxidation and 3α‐hydroxylation whereas simultaneous incubation of gestodene with AFB1 only inhibited 3α‐hydroxylation. These results suggest that two independent substrate binding sites exist in P450 3A4, and AFB1 binds to both of the binding sites. Gestodene selectively binds to one of the binding sites leading to the formation of AFQ1, whereas it does not affect the formation of exo‐epoxide via the other binding site.
Effect of polyglutamate on the thermal stability of ferricytochrome c
Authors: Bágel'ová Jaroslava , Antalík Marián , Tomori Zoltán
Published: July 2007

The effect of saturated solutions of polyglutamate on the thermal stabilities of the Met‐80‐heme iron bond and of the ferricytochrome c as a whole were studied by absorption spectroscopy and differential scanning calorimetry at pH 7.0. According to spectral data the midtransition temperature of the cleavage of the sulfur‐iron bond was 57.4±0.5 °C and 66.8±0.5 °C for cytochrome c and cytochrome c‐polyglutamate complex, respectively. Addition of polyglutamate to cytochrome c at pH 7.0 alters the denaturation properties of the protein. As follows from DSC scans, the denaturation temperature for cytochrome c is decreased from 85.4±0.2 °C to 68.7±0.2 °C in the presence of the saturated amount of polyglutamate. The protein stability in terms of Gibbs energy change at protein unfolding amount to ΔG(25°C)=22.7±2.7 and 32.0±2.2 kJ/mol, for cytochrome c and cytochrome c‐polyglutamate complex, respectively, at pH 7.0. It is evident that polyglutamate increases the thermal stability of the sulfur‐iron bond and decreases the denaturation temperature of the cytochrome c molecule as a whole. The complex thermal stability in terms of Gibbs energy is not lower than that of cytochrome c in the range of physiological temperatures.
Inhibition of the proliferative effect of transforming growth factor‐α by c‐myc antisense DNA in human ovarian cancer cells
Authors: Park Hae‐Young
Published: July 2007

In search of critical genes associated with the mechanism of transforming growth factor‐α (TGFα) action in human ovarian cancer, it was found that TGFα stimulates c‐rnyc gene expression in human ovarian NIH:OVCAR‐3. The role of c‐myc in TGFα‐stimulated growth of NIH:OVCAR‐3 cells was examined by the use of the synthetic antisense‐myc phosphorothioate oligonucleotide (OPT). Prior exposure of NIH:OVCAR‐3 cells to an antisense‐myc OPT inhibited TGFα‐stimulated cell growth and DNA syntbesis in a dose‐dependent and sequence‐spocific manner over 4 days. c‐Myc protein expression was down‐regulated in the antisense‐myc treated cells. These results demonstrate both the specific and durable effects of the antisense‐myc OPT. Furthermore, the results suggest a role for c‐myc in TGFα‐stimulated cell proliferation.
Fatty acid composition of brown adipose tissue in dietary obese rats
Authors: Lladó Isabel , Pons Antoni , Palou Andreu
Published: July 2007

The effects of both dietary obesity and a food deprivation period of 24 hours on fatty acid composition of brown adipose tissue have been investigated. Long time exposure to a hypercaloric high‐fat diet such as the cafeteria diet induced an important tissue fatty acid accumulation, mainly for the major saturated and monounsaturated fatty acids. Notable metabolic differences have been observed in the behaviour of control and obese rats facing a food deprivation period: a preferential utilization of the most abundant saturated fatty acids in control rats and a minor response in obese rats, with a greater fat accumulation in the interscapular brown adipose tissue.
Evidence for the transfer in culture of [14C]‐labelled fatty acids from macrophages to lymphocytes
Authors: Peres C. M. , de Bittencourt P.I. Homem , Costa M. , Curi R. , Williams J. F.
Published: July 2007

[14C]‐labelled palmitic acid (PA), oleic acid (OA), linoleic (LA) and arachidonic (AA) acids were transferred from macrophages (Mφ) to lymphocytes (LY) when equal numbers of the two cell types were co‐cultured. The relative degree and amounts of the fatty acids transferred from Mφ to LY are as follow: AA (368.57±21.62)=OA (274.52±15.41)>LA (42.11±8.31)=PA (36.53±2.45). The transfer units are nmol/1010 Mφ/1010 LY and the values are mean±SEM for 7 experiments. The [144C]‐radioactivity transferred was mainly directed to the phospholipid fraction of the lymphocytes (85% by PA, 86% by LA, 83% by OA and 79% by AA). In the same order as above, phosphatidylcholine was the phospholipid moiety most heavily labelled (82% by PA, 71% by LA, 66% by OA and 47% by AA). The amount of [14C]‐radioactivity transferred to stimulated lymphocytes of thioglycollate treated animals remained unchanged for LA, PA and AA but reduced for OA (71%). The significance of these observations for the immune functions of the cells and resolution of the question of whether some of the [14C]‐isotope transfer involves a component of exchange or is unequivocally net fatty acid mass transfer are still being investigated.
Quantification of prostaglandin D synthetase in cerebrospinal fluid: A potential marker for brain tumor
Authors: Saso Luciano , Leone Maria Grazia , Sorrentino Claudio , Giacomelli Sabrina , Silvestrini Bruno , Grima Josephine , Li Jonathan C. H. , Samy Eileen , Mruk Dolores , Cheng C. Yan
Published: July 2007

Prostaglandin D synthetase (PGD‐S; prostaglandin‐H2 D‐isomerase, EC 5,3,99,2), a 30 kDa glycoprotein also known as β‐trace protein that catalyzes the formation of prostaglandin D2 (PGD2) from PGH2, was purified to apparent homogeneity from human cerebrospinal fluid (CSF) using a two‐step procedure involving HPLC on a Vydac C8 reversed‐phase column and high performance electrophoresis chromatography (HPEC) using a 10% T SDS‐polyacrylamide gel. The purity of PGD‐S isolated from CSF was confirmed by silver stained SDS‐polyacrylamide gel and direct protein microsequencing (NH2‐APEAQVSVQPNFQ). A highly specific polyclonal antibody was prepared against this protein for immunoassay development. Using an ELISA, it was found that the concentration of PGD‐S in CSF did not alter significantly in different pathological conditions of the central nervous system (CNS). These include dementia (n=9), hydrocephalus (n=4), neuropathy (n=11), optic neuritis (n=4), multiple sclerosis (n=11), and demyelinating syndrome (n=11), when compared to normal individuals (n=12); however, the level of PGD‐S in the CSF obtained from patients with brain tumor (n=11), was reduced by as much as 2‐fold when compared to control samples (n=12) illustrating PGD‐S is a potentially useful marker for brain tumor.
Relationship between rate and extent of catechin absorption and plasma antioxidant status
Authors: Pietta Piergiorgio , Simonetti Paolo , Gardana Claudio , Brusamolino Antonella , Morazzoni Paolo , Bombardelli Ezio
Published: July 2007

Flavonoids are described to exert a large array of biological activities, which are mostly ascribed to their radical‐scavenging, metal chelating and enzyme modulation ability. Most of these evidences have been obtained by in vitro studies on individual compounds and at doses largely exceeding those dietary. Little is known about a possible relationship between rate and extent of the absorption and modifications of plasma antioxidants. To elucidate this aspect, human volunteers were supplemented with single doses of green tea catechins in free (Greense‐lect™) or phosphotipid complex form (Greenselect™ Phytosomer®) equivalent to 400 mg epigallocatechingallate (EGCg). EGCg was chosen as biomarker for green tea catechin absorption, and its time course plasma concentration was correlated to the subsequent percent variations of plasma ascorbate, total glutathione, α‐tocopherol, β‐carotene and Total Radical Antioxidant Parameter (TRAP). Green tea catechins were absorbed more extensively when administered as phospholipid complex rather than as free catechins. Single dose intake of both forms of catechins produced a transient decrease (10‐20%) of plasma ascorbate and total glutathione and an increase of plasma TRAP (16‐19%). These variations were consistent with the plasmatic levels of EGCg, ascorbate and total glutathione.
The carboxyl‐terminal fragment of osteopontin suppresses arginine‐glycine‐asparatic acid‐dependent cell adhesion
Authors: Takahashi Kazuhisa , Takahashi Fumiyuki , Tanabe Kenneth K. , Takahashi Hideki , Fukuchi Yoshinosuke
Published: July 2007

Osteopontin (OPN) is a secreted glycoprotein implicated in cell adhesion. It contains the arginine‐glycine‐asparatic acid (RGD) cell adhesive domain and the thrombin cleavage sequence. Although thrombin cleavage of OPN has been shown to be of physiological importance, the function of C‐terminal OPN fragment cleaved by thrombin remains unknown. To determine its role, we performed cell adhesion assays using glutathione S‐transferase‐OPN fusion protein fragments and full‐length OPN fusion protein. The N‐terminal fragment containing RGD motif promoted enhanced adhesion of mouse and human fibroblasts by 2.9 and 2.8 folds in comparison with full‐length OPN, respectively. The enhanced adhesion of both cells mediated by N‐terminal fragment was significantly suppressed by addition of C‐terminal fragment lacking RGD motif that has less cell adhesive property than full‐length OPN. These results suggest that the C‐terminal domain may play a pivotal role in regulating OPN functions by suppressing the RGD‐dependent cell adhesion.
Human milk lactoferrin binds ATP and dissociates into monomers
Authors: Semenov Dmitry V. , Kanyshkova Tatyana G. , Buneva Valentina N. , Nevinsky Georgy A.
Published: July 2007

The physiological role of lactoferrin (LF) is still unclear, but it has been suggested to be responsible for primary defence against microbial infections. Many different unique functions have been attributed to LF, including DNA and RNA binding, and transport into the nucleus, where LF binds to specific DNA sequences and activates transcription. Here we present evidence that in addition to the above (and below) mentioned functions LF binds ATP with a stoichiometry of 1 mole of nucleotide per mole of the protein and a Kd=0.3 mM. The ATP‐binding site is localized in the C‐terminal domain of LF, in contrast to the antibacterial and polyanion‐binding sites, which are located in the N‐terminal domain. Binding of ATP by LF leads to dissociation of its oligomeric forms and to a change of the protein's interaction with polysaccharides, DNA and proteins.
Transcriptional regulation of the CDK inhibitor p16INK4a gene by a novel pRb‐associated repressor, RBAR1
Authors: Kaneko Satoshi , Nishioka Junji , Tanaka Minoru , Nakashima Kunio , Nobori Tsutomu
Published: July 2007

p16, also known as INK4a, CDKN2, or MTS1, plays an important role in the control of the cell cycle progression, and retinoblastoma protein (pRb) is suggested to be involved in the transcriptional regulation of p16. However, it is not fully understood how pRb regulates transcription of the p16 gene. Nuclear proteins prepared only from the pRb‐nonfunctional human tumor cells were found to bind to the 5′‐flanking sequence of the p16 gene in the presence of Zn2+ by electrophoretic mobility shift assay (EMSA). EMSA using mutagenized 5′‐flanking sequences as competitors suggested that the sequence at position ‐97 to ‐87 relative to first ATG of the p16 gene was critical for protein binding. Transient reporter assay indicated that the sequence identified by EMSA acted as a silencer element in the pRb‐nonfunctional tumor cells, showing the presence of a transcriptional repressor associated with functional pRb (RBAR1).
Effects of sulfhydryl compounds on the inhibition of erythrocyte membrane Na ‐K ATPase by ozone
Authors: Bilgin Ramazan , Gül Sermin , Tükel S. Seyhan
Published: July 2007

Exposure of human erythrocyte membranes to ozone (5 μmol/10 min) resulted in the inhibition of erythrocyte membrane Na+‐K+ ATPase (EC.3.6.1.39). It was determined that, the degree of enzyme inhibition in the directly ozone exposed membranes was greater than that of membranes obtained from ozone exposed intact erythrocytes. In the presence of varying concentrations (0‐1.0 mM) of dithiotrethiol or mercaptoethanol Na+‐K+ ATPase activities of both types of ozone exposed membranes were increased almost proportionally with the concentration of dithiotrethiol or mercaptoethanol however, the activities were still lower than the normal Na+‐K+ ATPase value.

The results indicate that, dithiotrethiol or mercaptoethanol prevent the enzyme inhibition by ozone in vitro. This suggests that the membrane thiol groups are primary targets for ozone and thereby preventing the oxidation of essential functional groups of enzyme, protein.
Cloning and expression of bovine herpesvirus‐1 glycoprotein C
Authors: Gupta Praveen K. , Saini Mohini , Gupta L. K. , Bandyopadhyay S. K. , Garg S. K.
Published: July 2007

Glycoprotein C (gC) of Bovine Herpesvirus‐1 (BHV‐1) is expressed at high levels on surface of infected cells and on virus envelope. It is relatively immunodominant in antibody response to BHV‐1 infection and protective in immunized bovines against BHV‐1 challenge. In an attempt to express gC in mammalian cells, the 2.4 kb BamHI‐EcoRI fragment, containing complete coding sequence of the gC gene was excised from a recombinant plasmid and cloned under the control of RSV‐LTR. The resultant plasmid pRSV‐gC was transfected into MDBK cells and expression of gC was detected by indirect immunofluorescence. The non‐permeabilized cells revealed surface expression of gC.
20S proteasome, hsp90, p97 fusion protein, PA28 activator copurifying oligomers and ATPase activities
Authors: Montel Valérie , Gardrat Florence , Azanza Jean‐Louis , Raymond Jacques
Published: July 2007

Whether hsp90 acts in an ATP‐depeadent or independent way is of crucial importance for understanding the molecular mechanism of this chaperone and, to day, the involvement of ATP hydrolysis in hsp90 function is still a contreversial subject. ATPase activities may be detected in partially purified hsp90′s preparations from rabbit muscle. We demonstrate that the major contaminant associated with hsp90 is the p97 fusion protein and that these oligomeric structures are copurifying together with the 20S proteasome and its PA28 activator. Improving the purification procedure permits to separate hsp90 and p97 to homogeneity. Then, our attempts failed to detect any significant ATPase activity in the hsp90 fraction. Thus, p97 would be principally responsible for the ATPase activity detected in partially purified hsp90 preparations from rabbit muscle.
Evaluation of kidney and liver subacute toxicity induced by bezalip ‐ pravastatin. Lopid antihyperlipidaemic compounds in rats
Authors: Pispirigos K. , Simopoulos K. , Kouskoukis K. , Kounis N. , Avramopoulos A.
Published: July 2007

Renal and hepatic subacute toxicity induced by the antihyperlipidaemic drugs: Bezalip ‐ Pravastatin and Lopid was investigated in rats using serum biochemical parameters. Toxicological evaluation was performed in serum samples following the administration of the therapeutic dose regimens of the compounds that were previously shown to be effective in inhibition of 3‐hydroxy‐methylgiutaryl coenzyme A (HMG CoA) reductase, the enzyme controlling the rate‐limiting step in the synthesis of cholesterol, and acyl‐ CoA cholesterol acyl transferase (ACAT) which converts intracellnlar free cholesterol to cholesterol ester. Renal and hepatic subacute toxicity was evaluated by measuring enzyme activity or concentrations of: alanine aminotransferace, alkaline phosphatase, asparate aminotransferase, γ‐glutamyltransferase, glucose, potassium, sodium, blood urea nitrogen, uric acid and creatinine. The use of the above serum biochemical parameters indicated that the overall toxicity impact of antihyperlipidaemic drugs was Bezalip = Pravastatin < Lopid.

We have found that the Pravastatin ‐in contrast to the above antihyperlipidaemic drags‐ only transiently affects the biochemical parameters associated with toxicity, but, it affects some of the biochemical parameters associated with hepatic and renal toxicity, up to a significantly lower extent than the antihyperlipidaemic drags.
Isolation and characterization of an arginine ester hydrolase from Bothrops jararacussu venom which induces contractions of the isolated rat uterus
Authors: Andrião‐Escarso S. H. , Soares A. M. , Rodrigues V. M. , Mancin A. C. , Reis M. L. , Ballejo G. , Giglio J. R.
Published: July 2007

The isolation and partial characterization of a serine protease with arginine ester hydrolase activity from Bothrops jararacussu snake venom are described. The purification procedure consisted of a gel filtration of the crude venom on Sephadex G‐75 followed by an ion‐exchange chromatography of the active fraction on DEAE‐cellulose and a rechromatography on Bio‐Rex 70 resin. The esterase fraction (DI‐III), Mr=25,000 by SDS‐PAGE, showed proteolytic activity on fibrinogen and casein. After 2hr incubation, the Aα and Bβ chains of fibrinogen were intensely hydrolysed, while the γ, chain kept apparentely intact, even after 20hr of incubation. In spite of that, DI‐III did not clot fibrinogen. DI‐III induced edema in the rat paw. Although unable to release bradykinin, it induced contractions of the isolated rat uterus. DI‐III did not catalyse the hydrolysis of bradykinin. Its arginine ester hydrolase activity was completely inhibited by diisopropyl fluorophosphate after 1 hr incubation, but not by phenylmethylsulfonyl fluoride under the same conditions.
A Geopolitics of Identity: Drawing the Line Between Russia and Estonia
Authors: Merritt Martha
Published: July 2007

Although narratives of national identity feature territory, myths, and historical memories presumably shared by members,1national identity formation is an ongoing process with changeable membership and boundaries.2One of the more complex challenges to a national “imagined community” has been the significant presence in Estonia of cultural Russians3after the collapse of the Soviet Union in 1991, at that time one-third and now some 28% of the small country's population. The often grudging accommodation of cultural Russians by the titular nationality continues to draw attention from international organizations, scholars, and policymakers alike. Undoubtedly the commentary least welcome to Estonian governments in the last decade, however, has been the thunder of denunciation from Russia. In a great spillover from domestic concerns about Slavic identity to international relations, Estonia has been ranked as Russia's greatest enemy, and political figures across the spectrum have condemned Estonian citizenship and language policies.4
Conflict and Catharsis: A Report on Developments in Dagestan Following the Incursions of August and September 1999
Authors: Kisriev Enver , Ware Robert Bruce
Published: July 2007

There was a fateful inevitability to the military actions in Dagestan that began on 2 August 1999 and concluded on 16 September. During the 2 years preceding, tensions within Dagestan's Islamic community had been building between fundamentalist Wahhabis and traditionalists. These tensions were exacerbated by Dagestan's sharp economic decline. Unemployment, which was running at 80% by August, contributed to growing dissatisfaction, especially in Dagestan's rural regions. These tensions reached critical proportions in the Botliksky rayon, particularly among young men belonging to the Andi ethno-linguistic sub-group of the Avars. Many of the latter were attracted to military training camps operated in Chechnya by Emir al Khattab, leader of the Wahhabite Islamic djamaat (village or connected group of villages) at Karamakhi, Chabanmakhi and Kadar, and by Shamyl Basayev, leader of the Islamic Congress of the Peoples of Ichkeria and Dagestan. In these camps rural Dagestani alienation met Chechen militancy and international Islamic fundamentalist support. Meanwhile Wahhabism grew increasingly influential in Chechnya as rival political leaders appealed to puritanical Islam in order to bolster their claims to authority and legitimize their political agendas.
S‐Nitrosylation of Viral Proteins: Molecular Bases for Antiviral Effect of Nitric Oxide
Authors: Colasanti Marco , Persichini Tiziana , Venturini Giorgio , Ascenzi Paolo
Published: July 2007

Nitric oxide (NO) is considered an important signaling molecule implied in various different physiological processes, including nervous transmission, vascular regulation, and immune defence, as well as the pathogenesis of several diseases. NO reportedly also has an antiviral effect on several DNA and RNA virus families. The NO‐mediated S‐nitrosylation of viral and host (macro)molecules appears to be an intriguing general mechanism for the control of the virus life cycle. In this respect, NO is able to nitrosylate cysteine‐containing enzymes (e.g., proteases, reverse transcriptase, and ribonucleotide reductase). Moreover, zinc‐fingers and related domains present in enzymes (e.g., HIV‐1 encoded integrase or herpes simplex virus type‐1 heterotrimeric helicase primase complex) or nucleocapsid proteins may beconsidered as NO targets. Also, NO may regulate both host (e.g., nuclear factor‐kappaB) and viral‐encoded (e.g., HIV‐1 tat protein or Epstein‐Barr virus Zta) transcriptional factors that are involved in virus replication. Finally, NO‐mediated S‐nitrosylation of cysteine‐containing glycoproteins and hemagglutinin may also occur. Here, NO targets are summarised, and the molecular bases for the antiviral effect of NO are discussed.
Effects of Genetic and Diet‐Induced Obesity on Lipid Metabolism
Authors: Libinaki Roksan , Heffernan Mark , Jiang Woei‐Jia , Ogru Esra , Ignjatovic Vera , Gianello Robert , Trickey Lisa , Taylor Michelle , Ng Frank
Published: July 2007

C57BL/6J obese (ob/ob) and lean mice fed ad libitum on a normal mouse chow diet (Normal), were compared with lean mice of the same age and strain fed ad libitum on a high‐fat diet, consisting of the Normal diet with the addition of beef lard (Lard), from age 3 months for 34 days. The lard‐fed mice were seen to have significantly higher (P < 0.05) body weight in this 34‐day period than that of the other two groups fed on the Normal diet. Epididymal fat depot and adipocyte cell size were significantly larger (P < 0.05) in the Lard‐fed lean mice and in the obese (ob/ob) mice than were those of the Normal‐fed lean mice. Dietary Lard intake did not significantly affect concentrations of plasma triglyceride although those of plasma cholesterol were significantly increased (P < 0.05). The development of obesity in these Lard‐fed mice appeared to be accelerated and significant.
New Chemiluminescence Assay for Linamarin
Authors: Ruengprapavut Sutarnthip , Chulavatnatol Montri
Published: July 2007

A new chemiluminescence assay was developed for the quantitative determination of linamarin, a cyanogenic glucoside present in cassava. The assay involved hydrolysis of linamarin by a specific enzyme, linamarase, to release glucose, which was then quantitated by a chemiluminescence system consisting of glucose oxidaseperoxidase‐luminol.The new assay was more sensitive than the conventional spectrophotometric method for quantitating linamarin in cassava extracts. However, the following agents were found to interfere with the new assay:Vanadate,Mg2+,and Cu2+,were inhibitory to the luminescence of the H2O2‐peroxidase‐luminol system used in the coupling reaction, whereas EDTA and EGTA activated the system. In addition, Hg2+, which inhibits glucose oxidase, and Tris ion, which inhibits linamarase, both interfered with the new assay.
Evolutionary Conservation of the Membrane Fusion Machine
Authors: Poumbourios Pantelis , Center Rob J. , Wilson Kirilee A. , Kemp Bruce E. , Kobe Bostjan
Published: July 2007

Recent structural studies of proteins mediating membrane fusion reveal intriguing similarities between diverse viral and mammalian systems. Particularly striking is the close similarity between the transmembrane envelope glycoproteins from the retrovirus HTLV‐1 and the filovirus Ebola. These similarities suggest similar mechanisms of membrane fusion. The model that fits most currently available data suggests fusion activation in viral systems is driven by a symmetrical conformational change triggered by an activation event such as receptor binding or a pH change. The mammalian vesicle fusion mediated by the SNARE protein complex most likely occurs by a similar mechanism but without symmetry constraints.
Biochemical Properties of the Protein Tyrosine Kinase of the Bacterium Acinetobacter johnsonii
Authors: Grangeasse C. , Vincent C. , Doublet P. , Cozzone A. J. , Duclos B.
Published: July 2007

The biochemical properties of the autophosphorylating protein tyrosine kinase of Acinetobacter johnsonii were analyzed in vitro. The study shows that the optimal pH value for the phosphorylation reaction is 7. The enzyme activity is stimulated by magnesium and, to a lesser extent, by manganese ions, whereas calcium ions have no effect. The phosphorylation process is rapid reaching a maximum in <2 min, and the enzyme is modified at multiple sites. Interestingly, the bacterial enzyme is insensitive to a series of molecules known to affect the activity of eukaryotic protein tyrosine kinases: genistein, quercetin, tosyllysine chloromethyl ketone, and vanadate. We concluded that, even though the overall phosphorylation reaction catalyzed by the A. johnsonii enzyme is identical to that occurring in eukaryotes, this bacterial kinase exhibits a number of specific properties and therefore probably belongs to a separate group in the general family of protein tyrosine kinases.
Real‐Time Oligonucleotide Hybridization Kinetics Monitored by Resonant Mirror Technique
Authors: Koval Vladimir V. , Gnedenko Oksana V. , Ivanov Yuri D. , Fedorova Olga S. , Archakov Alexander I. , Knorre Dimitri G.
Published: July 2007

The kinetics of hybridization of 11‐meric and 14‐meric oligonucleotides, dTGGGAAGAGGG (ODN‐11) and dTGGGAAGAGGGTCA (ODN‐14), with 14‐meric oligonucleotide dpTGACCCTCTTCCCA (p14) attached to the surface of a cuvette was studied by the resonant mirror method. The treatment of the experimental curves with exponential equations leads to the following values for association (kas) and dissociation (kdis) rate constants at 25 °C: kas = 219 +/‐ 39 and 183 +/‐ 162 M‐1s‐1, kdis = (2.0 +/‐ 0.4) 10‐3 and (4 +/‐ 1) 10‐4s‐1 for the duplexes (p14) (ODN‐11) and p14 (ODN14), respectively. The oligonucleotide dTGCCTTGAATGGGAAGAGGGTCA (ODN‐23), which forms a hairpin structure, does not associate with p14. The data were compared with the results of melting curve detection and temperature‐jump experiments. The association rate constants for ODN‐11 and ODN‐14 are much slower than those values in homogeneous aqueous solution. The dissociation rate constants have the same magnitude values as estimated by using association constants measured from melting curves but differ from the values estimated in temperature‐jump experiments.
Cloning and Characterization of Manganese Superoxide Dismutase Gene from Vibrio parahaemolyticus and Application to Preliminary Identification of Vibrio Strains
Authors: Shyu Yu‐Chiau , Lin Fu‐Pang
Published: July 2007

The sodA gene coding for manganese superoxide dismutase (Mn‐SOD) from the marine microorganism Vibrio parahaemolyticus was cloned, sequenced, and overexpressed in Escherichia coli by use of the pET20b (+) expression vector. The full‐length gene consisted of a 588‐bp open reading frame and encoded a polypeptide of 196 amino acid residues, with a calculated molecular mass of 21 713 Da. The recombinant enzyme was efficiently purified from crude E. coli cell lysate by metal ion affinity chromatography. The recombinant VPMn‐SOD resisted thermo‐denaturation up to 60 °C and was insensitive to such inhibitors as EDTA, NaN3 and diethyldithiocarbamic acid. The specificity of V. parahaemolyticus Mn‐SOD gene probe was analyzed by cross‐species polymerase chain reaction to provide information for Vibrio strain identification.
Substitution of Surface‐Exposed Framework Residues Alters Secretion of Recombinant Fusion Protein Fv/Tumor Necrosis Factor in Escherichia coli
Authors: Yang Junbao , Jia Zongchao , Xiang Jim
Published: July 2007

Substitution of hydrophobic residues with hydrophilic ones at surface‐exposed positions may influence the yield of antibody fragment expression in Escherichia coli by reducing its aggregation potential. We introduced such substitutions at 8 surface‐exposed framework region residues of a fusion protein Fv/Tumor Necrosis Factor, which resulted in the expression of 10 mutant fusion proteins (Mut 1‐10) in E. coli. Our results showed that expression levels of Mut 1‐3, with mutations of A9S, T18K, and G41D, respectively, decreased by 4‐ to 8‐fold, whereas expression levels of Mut 4, 9, and 10, with mutations of S60D/S70D, T28D, and G65D, respectively, were not affected. In contrast, mutation of F83A at light‐chain residue 83, which is usually buried at the variable/constant domain interface of antibody molecules but becomes surface‐exposed in recombinant Fv molecules, increased the expression level of Mut 5 by 4‐fold. Our results suggest that this important substitution of a hydrophobic residue with a hydrophilic one may also be applied to increase the secretion of other recombinant Fv molecules in E. coli periplasm.
A Mitochondrial Model for Premature Ageing of Somatically Cloned Mammals
Authors: Allen John F. , Allen Carol A.
Published: July 2007

Cloned sheep have recently been discovered to have an unexpectedly advanced biological age. We propose that the explanation is a simple consequence of inheritance of acquired, free radical‐induced cellular damage with somatic mitochondria that contribute to the mitochondrial population of cloned cells but not to zygotes produced by fertilization in normal sexual reproduction. Each increment of ageing in cloning experiments is therefore predicted to be maternally inherited. The hypothesis suggests practical ways of decreasing the effect. The hypothesis is itself a prediction of the recent proposal that mitochondria of the female germ line function primarily as genetic templates.
Activity of pp60c‐src and Association of pp60c‐src, pp54/58lyn, pp60fyn, and pp72syk with the Cytosḱeleton in Platelets Activated by Collagen
Authors: Kralisz Urszula , Cierniewski Czeslaw S.
Published: July 2007

Collagen stimulation of platelets induced an increase in the specific activity of pp60c‐src immunoprecipitated from the Triton‐soluble fraction. The earliest time after collagen stimulation that an increase in pp60c‐src activity was observed was 30 s. However, the maximum activity of pp60c‐src in the Triton‐soluble fraction was observed 60 s after collagen stimulation. At this time an approximately twofold increase of pp60c‐src activity towards phosphorylation of KVEKIGEGTYGVVKK specific peptide and enolase and a 4.5‐fold increase towards phosphorylation of pp60c‐src itself was measured. Furthermore, the majority of pp60c‐src as well as pp54/58lyn, pp60fyn, and pp72syk were found in the Triton‐soluble fraction in resting platelets. Collagen induced, to different extents and velocities, translocation of all of these proteins from the Triton‐soluble fraction to the Triton‐insoluble, cytoskeleton‐rich, platelets fraction. These results provide direct evidence that collagen stimulation of platelets increases the tyrosine kinase activity of pp60c‐src and suggest that the platelet cytoskeleton plays an important role in collagen‐induced signal transduction by localizing signaling molecules.
Cloning and Characterization of a Novel cDNA Sequence Encoding the Precursor of a Novel Venom Peptide (BmKbpp) Related to a Bradykinin‐Potentiating Peptide from Chinese Scorpion Buthus martensii Karsch
Authors: Zeng Xian‐Chun , Li Wen‐Xin , Peng Fang , Zhu Zhi‐Hui
Published: July 2007

Based on the amino acid sequence of a bradykinin‐potentiating peptide (Bpp) (peptide K‐12) from scorpion Buthus occitanus, a full‐length cDNA sequence encoding the precursor of a novel venom peptide (named BmKbpp) related to this Bpp, has been isolated and analyzed. The cDNA encodes a precursor of 72 amino acid residues, including a signal peptide of 22 residues and an extra Arg‐ArgArg tail at the C‐terminal end of the precursor, which have to be removed in the processing step. The C‐terminal region (21 residues) of the precursor is homologous (57% identical) with the sequence of peptide K‐12. Thus, according to the primary structure of the BmKbpp precursor, there may be a propeptide between the signal peptide and the putative mature BmKbpp at the C‐terminal region of the precursor.
The p66shc Protein: A Mediator of the Programmed Death of an Organism?
Authors: Skulachev Vladimir P.
Published: July 2007

Recently knockout of the gene encoding an adaptor protein (p66shc) was shown both to prolong the life span of an animal and to prevent apoptosis of cells in response to added H2O2 (Migliaccio et al. [1999] Nature 402, 309?313). A hypothesis is put forward in which p66shc is assumed to be involved in phenoptosis, i.e., programmed death of an organism, mediated by the reactive oxygen species?dependent massive apoptosis in an organ of vital importance. The reactive oxygen species are suggested to oxidize phosphatidyl serine in the inner leaflet of the cell plasma membrane, resulting in appearance of this phospholipid in the outer membrane leaflet, an effect recognized by a special receptor and causing the p66shc phosphorylation at a serine residue. Serine‐phosphorylated p66shc there is proposed to block mitosis and initiate apoptosis. The large‐scale apoptosis leads to phenoptosis and, hence, shortens the life span of the organism.
Reverse Transcription of a Naturally Occurring Nonretroviral RNA Produces a Precise Deletion in the Majority of Its cDNA Products
Authors: Johnson Karyn N. , Gordon Karl H. J. , Hanzlik Terry N.
Published: July 2007

A precise, reproducible deletion made during in vitro reverse transcription of RNA2 from the icosahedral positive‐stranded Helicoverpa armigera stunt virus (Tetraviridae) is described. The deletion, located between two hexamer repeats, is a 50‐base sequence that includes one copy of the hexamer repeat. Only the Moloney murine leukemia virus reverse transcriptase and its derivative Superscript I, carrying a deletion of the carboxy‐terminal RNase H region, showed this response, indicating a template‐switching mechanism different from one proposed that involves a RNase H? dependent strand transfer. Superscript II, however, which carries point mutations to reduce RNase H activity, does not cause a deletion. A possible mechanism involves the enzyme pausing at the 3′ side of a stem‐loop structure and the 3′ end of the nascent DNA strand separating from the template and reannealing to the upstream hexamer repeat.
An RNA Polymerase II Complex Containing Capping Enzymes and Viral Oncoproteins
Authors: Cabrejos Maria Eugenia , Maldonado Edio
Published: July 2007

The aim of this work was to identify proteins from Adenovirus 2‐infected HeLa cell extracts that interact with the carboxyl‐terminal domain of the largest subunit of RNA polymerase II. First, a mammalian RNA polymerase II complex was isolated from Adenovirus 2‐infected HeLa cell extracts by affinity chromatography against the carboxyl‐terminal domain of the largest subunit of RNA polymerase II, followed by chromatography on a Mono S fast protein liquid chromatographic column. Second, the isolated complex was further characterized by Western blot analysis, the formation of a GMP‐protein complex, and transcriptional activity. The isolated complex contains general transcription factors, chromatin‐remodeling factors, histone acetyltransferases, Srbs, capping enzymes, and E1A viral oncoproteins. The RNA polymerase II complex is active in transcription when supplemented with recombinant transcription factor IIB.
Interaction of Bacterial Flagellum Filaments with Skeletal Muscle Myosin
Authors: Novikova Nadezhda A. , Levitsky Dmitrii I. , Metlina Antonina L. , Poglazov Boris F.
Published: July 2007

Interaction of isolated bacterial flagellum filaments (BFF) and intact flagella from E. coli MS 1350 and B. brevis G.‐B.p+ with rabbit skeletal myosin was studied. BFF were shown to coprecipitate with myosin (but not with isolated myosin rod) at low ionic strength, that is, under conditions of myosin aggregation. The data of electron microscopy indicate that filaments of intact bacterial flagella interact with isolated myosin heads (myosin subfragment 1, S1), and this interaction is fully prevented by addition of Mg2+‐ATP. Addition of BFF inhibited both K+‐EDTA‐and Ca2+‐ATPase activity of skeletal muscle myosin, but had no effect on its Mg2+‐ATPase activity. Monomeric flagellin did not coprecipitate with myosin and had no effect on its ATPase activities. BFF were shown to compete with F‐actin in myosin binding. It is concluded that BFF interact with myosin heads and affect their ATPase activity. Thus, BFF composed of a single protein flagellin are in many respects similar to actin filaments. Common origin of actin and flagellin may be a reason for this similarity.
Changes in working with gender equality in management in Sweden
Authors: Wahl Anna , Höök Pia
Published: July 2007

Purpose

The article is based on a study of the changes in the methods used in working with gender equality issues in management in Sweden from the early 1990s until the early 2000s.

Design/methodology/approach

The material consists of interviews with 20 people who work either as employees in organisations or as business owners/consultants in the field of increasing the numbers of women at executive levels. In the article, they are referred to as “equality professionals”.

Findings

The changes that have taken place may be described in two ways – partly changes in how the work is pursued within organisations, and partly through the work having moved to other arenas. When summarising the material, four overriding trends can be identified that characterise the changes in gender equality work: (1) a more radical understanding of gender equality among groups positive to change; (2) a professionalisation of gender equality work; (3) a shift from organisational projects to working with individuals; (4) a shift from organisational projects to influencing organisations from other public arenas.Research limitations/implications – Its focus on Sweden as well as its focus on a specific, quite small (20) group of people.

Practical implications

Useful and interesting for those working with gender equality, especially as Sweden is sometimes put forward as “the world's most gender equal country”.

Originality/value

The article has value because it shows the Swedish context as well as the longitudinal approach, i.e. that the study focuses on a 10‐year period (1993‐2002) and includes comparative material.
Identification of a QTL on BTA20 affecting susceptibility toMycobacterium aviumssp.paratuberculosisinfection in US Holsteins
Authors: Gonda M. G. , Kirkpatrick B. W. , Shook G. E. , Collins M. T.
Published: July 2007

The objective of this study was to identify QTL affecting susceptibility toMycobacterium paratuberculosisinfection in US Holsteins. Twelve paternal half‐sib families were selected for the study based on large numbers of daughters in production and limited relationships among sires. Serum and faecal samples from 4350 daughters of these 12 sires were obtained for disease testing. Case definition for an infected cow was an ELISA sample‐to‐positive ratio ≥0.25, a positive faecal culture or both. Three families were selected for genotyping based on a high apparent prevalence (6.8–10.4% infected cows), high faecal culture prevalence (46.2–52.9% positive faecal cultures) and large numbers of daughters tested for disease (264–585). DNA pooling was used to genotype cows, with an average of 159 microsatellites within each sire family. Infected cows (the positive pool) were matched with two of their non‐infected herdmates in the same lactation (the negative pool) to control for herd and age effects. Eight chromosomal regions putatively linked with susceptibility toM. paratuberculosisinfection were identified using aZ‐test (P < 0.01). Significant results were more rigorously tested by individually genotyping cows with three to five informative microsatellites within 15 cM of the significant markers identified with the DNA pools. Probability of infection based on both diagnostic tests was estimated for each individual and used as the dependent variable for interval mapping. Based on this analysis, evidence for the presence of a QTL segregating within families on BTA20 was found (chromosome‐wideP‐value = 0.0319).
Molecular simulations to estimate thermodynamics for adsorption of polar organic solutes to montmorillonite
Authors: Aggarwal V. , Chien Y.‐Y. , Teppen B. J.
Published: July 2007

Ideally, one could use molecular mechanics or quantum mechanics to predict the magnitude of organic solute adsorption from water to soil minerals. Reproduction and/or prediction of mineral and interfacial structures remains challenging, but calculation of meaningful energy relations through computational chemistry techniques is even more difficult than structural calculations. This paper attempts to define the necessary and relevant components for an overall scheme that allows translation of computed interaction enthalpies to experimental adsorption enthalpies and vice versa. While the scheme could be applied to quantum calculations, we test it for the possibility of using empirical molecular mechanics to estimate relative energies for the adsorption of non‐ionic organic solutes in clay mineral‐water‐solute systems. We used molecular dynamics’ simulations to estimate relative clay–organic interaction enthalpies for a series of nitro‐aromatic solutes and hydrated, K‐saturated montmorillonite, for comparison with experimental adsorption isotherm data for the same clay‐nitroaromatic systems. The trend of computed interaction enthalpies (e.g. −234 ± 17 kJ mol−1 for trinitrobenzene and −154 ± 16 kJ mol−1 for p‐nitrobenzene) agreed modestly well with the trend of adsorption maxima from the experiments. Furthermore, we developed several variants on a thermodynamic cycle framework for comparing computed interaction energies with experimentally determined adsorption enthalpies. The algorithms, which include estimates for enthalpy changes both in bulk solution and in the clay interlayer, show promise: for p‐dinitrobenzene and for 1,3,5‐trinitrobenzene, the overall predicted adsorption enthalpies (e.g. −13 ± 22 and −67 ± 23 kJ mol−1, respectively) were in modest agreement with experiments (−18 ± 1 and −28 ± 4 kJ mol−1, respectively). We discuss shortcomings of the methods, in hopes of encouraging better estimates for the various energy terms, improvement of the algorithms, and more valid comparisons between quantum mechanical or molecular mechanical interaction energies and experimental enthalpies.
Patterns of histological change in liver disease: my approach to ‘medical’ liver biopsy reporting
Authors: Bateman A C
Published: July 2007

The aim of this review was to emphasize the benefit of a methodical approach to ‘medical’ liver biopsy reporting and to illustrate that recognition of patterns of disease can greatly aid correct interpretation of these often‐complex specimens. This applies both to assessment of the liver architecture and to evaluation of the likely disease aetiology. Therefore, a great deal of information can be obtained by examining biopsy material at low‐ and medium‐power magnification, prior to confirming detailed features with high‐power examination. A range of recommended special histochemical stains that personal experience has shown to be valuable is included. Emphasis is also placed on the absolutely key requirement for appropriate knowledge of the clinical situation within which the biopsy specimen was taken.
OsRAD21‐3, an orthologue of yeast RAD21, is required for pollen development in Oryza sativa
Authors: Tao Jiayi , Zhang Liangran , Chong Kang , Wang Tai
Published: July 2007

In contrast to animals, in which products of meiosis differentiate directly into sperm, flowering plants employ a specific mechanism to give rise to functional sperm cells, the specifics of which remain largely unknown. A previous study revealed that, compared to yeast and vertebrates, which have two proteins (Rad21 and its meiosis‐specific variant Rec8) that play a vital role in sister chromatid cohesion and segregation for mitosis and meiosis, respectively, the rice genome encodes four Rad21/Rec8 proteins (OsRad21s). In this paper, phylogenetic and immunostaining analyses reveal that OsRad21‐3 is an orthologue of yeast Rad21. OsRAD21‐3 transcript and protein accumulated preferentially in flowers, with low levels in vegetative tissues. In flowers, they persisted from the stamen and carpel primordia stages until the mature pollen stage. OsRAD21‐3‐deficient RNAi lines showed arrested pollen mitosis, aberrant pollen chromosome segregation and aborted pollen grains, which led to disrupted pollen viability. However, male meiosis in these RNAi lines did not appear to be severely disrupted, which suggests that the main involvement of OsRAD21‐3 is in post‐meiotic pollen development by affecting pollen mitosis. Furthermore, of the four OsRAD21 genes in the rice genome, only OsRAD21‐3 was expressed in pollen grains. Given that the mechanism involving generation of sperm cells differs between flowering plants and metozoans, this study shows, in part, why flowering plants of rice and Arabidopsis have four Rad21/Rec8 proteins, as compared with two in yeast and metozoans, and gives some clues to the functional differentiation of Rad21/Rec8 proteins during evolution.