bluuebunny/crossref_metadata_2025 huggingface dataset:
WESTMEAD CENTRE DRUG AND ALCOHOL UNIT: THE FIRST 12 MONTHS
Authors: Batey R.
Published: July 2007

The establishment and development of the Drug & Alcohol Unit at the Westmead Centre is described in this review. A summary of the experiences of the first 12 months is presented.
An Approach to Evaluating the Drug and Alcohol Problem in Prisoners
Authors: Ellard John
Published: July 2007

Abstract: In this paper a perspective on the drug and alcohol problems experienced in a specific group, namely, prisoners, is offered. The roles of the medical profession as assessors of drug and alcohol problems, and as educators, is briefly discussed.
Price as a determinant of alcohol consumption
Authors: Maynard Alan
Published: July 2007

Abstract: The formulation and execution of alcohol policy is considered, particularly in relation to the impact of economic variables, such as price, on consumption of alcohol. Trends in consumption levels and their effects are outlined. Available econometric evidence suggests that price has a significant impact on consumption, with other influential variables being income and access, and to a lesser extent, advertising.
Stored Energy and its Role in Recrystallization Process
Authors: Wierzbanowski K. , Tarasiuk J. , Bacroix B. , Sztwiertnia K.
Published: July 2007

The stored energy, present in a material after deformation in the form of dislocation density, plays a crucial role in the recrystallizaton process. It can be evaluated directly using electron diffraction or indirectly using X-ray and neutron diffraction (peak profile study). The stored energy can be also calculated, using deformation models. Knowledge of the stored energy distribution is necessary in quantitative studies of recrystallization. The distribution of stored energy can explain the highly oriented nucleation (case of most of b.c.c. metals) as well as the highly oriented growth (case of most of f.c.c metals).
Residual Strain Tensor Determination within Highly Plastically Deformed Torsion Samples Using High Energy Synchrotron Radiation
Authors: Martins R.V. , Lienert U. , Margulies L. , Pyzalla A.
Published: July 2007

The residual strain tensor is measured within strongly plastically deformed torsion samples using high energy synchrotron radiation. The diameter of the solid AlMg3 samples is 5mm. A low speed deformation with free ends to γ = 150% is carried out at room temperature. The strain mapping technique utilizes a micro-focussed incident X-ray beam, a conical slit cell, and a two dimensional detector. Extremely fast data acquisition is achieved by the simultaneous observation of six diffraction rings emanating from the same local gauge volume within the sample. The radial dependence of the complete residual strain tensor is evaluated from the deformation of the A1 422 diffraction rings.
Vascular Dysfunction in Obesity and Insulin Resistance
Authors: Frisbee Jefferson C.
Published: July 2007

ABSTRACT

With the growing prevalence of obesity and impaired glycemic control, and the correlation between these conditions and an elevated predisposition for the development of vascular disease, research emphases are increasingly being targeted to the mechanistic bases and functional outcomes of these relationships. Given this, the current issue of Microcirculation, presents a series of reviews that summarize knowledge on an array of topics relevant to obesity, insulin resistance, and vascular dysfunction. The first chapters discuss altered patterns of blood flow regulation, vascular reactivity, microvascular density, and vascular wall mechanics. The second grouping details alterations to coronary, renal, and hepatic circulations and the implications of these effects for organ function. Additionally, one article presents knowledge and outlines future research directions for the study of endothelial permeability and barrier function within insulin resistance. The last group of articles discusses the effects of inflammation with obesity and insulin resistance on vascular function, and also details the role of perivascular adipose tissue in contributing to vascular dysfunction. The final review extends this general topic to the effects of the metabolic syndrome on microvascular dysfunction, wherein obesity and impaired glycemic control are contributing elements to a larger constellation of systemic pathologies. The authors hope that this Special Topics Issue will be informative for its readers and will provide a basis for future investigation into microvasculopathy in obesity and insulin‐resistance.
The Enemy Next Door: Italian Collaboration in Deporting Jews during the German Occupation of Rome
Authors: Wildvang Frauke
Published: July 2007

About 2,000 Jews were deported from Rome during the nine months of German occupation, half of them after the infamous Germanrazziaof 16 October 1943. Who took part in their identification and arrest? Italian historiography has most commonly focused on a few ardent Fascist collaborators, while the majority of Italians were proclaimed to be engaged in rescue operations for persecuted Jews. Due to the long-standing hegemony of the notion of ‘italiani brava gente’ and the taboo against discussing Italian collaboration, almost no studies of the Jewish persecution during the German occupation in Italy have been undertaken. The analysis of over 50 trials against Fascist collaborators offers insight into thecaccia all'ebreoon the micro-level of occupied Rome. Elaborating on different forms of denunciations characterizing the persecution as well as the diverse motives of the Italian perpetrators, this article presents a comprehensive picture of the collaboration between German occupation forces and the population of Rome.
Division and Conflict in the Partisan Resistance
Authors: Dondi Mirco
Published: July 2007

The article sheds light on the power struggles at the heart of the Italian Resistance movement. From June 1944, as the movement grew rapidly, the leadership positions, both at national and local level, became ever more important and contested. The most significant roles in the Resistance, such as the national and regional leadership, but also the provincial commands, depended on the military strength of the various formations and on the power of the anti-Fascist parties. The re-formed political parties attempted to occupy important positions in the Resistance movement, hoping that these roles would help them out in any future settlement. In fact the rules of the game turned out to be far more complex and the political role played by any particular party did not determine its future success. The Anglo-Americans' influence over the power balance within the Resistance movement was to be decisive. The Allies managed to orchestrate the appointment of Raffaele Cadorna, who was not looked on favourably by the parties of the left and the Action Party, as military commander. In this way the Allies fostered the growth of moderate military formations frequently linked to Christian Democracy. In order to understand the Resistance in all its complexity, it is therefore necessary to return to the concept of internal conflict. The power struggles were better managed at national rather than local level, where they frequently led to violence.
Inhibitory effect of 4‐(2,6‐dichlorophenyl)‐1,2,5‐thiadiazol‐3‐yl‐N‐methyl, N‐ethylcarbamate on replication of human immunodeficiency virus type 1 and the mechanism of action
Authors: Ijichi Katsushi , Fujiwara Masatoshi , Hanasaki Yasuaki , Katsuura Kimio , Shigeta Shiro , Konno Kenji , Yokota Tomoyuki , Baba Masanori
Published: July 2007

In the search for effective antiviral agents, we have found 4‐(2, 6‐dichlorophenyl)‐1, 2, 5‐thiadiazol‐3‐yl‐N‐methyl, N‐ethylcarbamate (RD4‐2025) to be a highly potent and selective inhibitor of human immunodeficiency virus type 1 (HIV‐1) in vitro. The 50% effective concentration of RD4‐2025 for HIV‐1‐induced cytopathic effect in MT‐4 cells was 37 nM, yet no antiviral activity was observed against HIV‐2. In HIV‐1 reverse transcriptase (RT) assays, RD4‐2025 inhibited both RNA‐dependent and DNA‐dependent DNA polymerase activities of a recombinant HIV‐1 RT with 50% inhibitory concentrations of 0.11 and 3.5 µM, respectively. However, the compound did not affect the activity of human DNA polymerase α. Kinetic studies revealed that the inhibition was noncompetitive with respect to dGTP as the substrate and poly(C)/(dG)12‐18 as the template/primer. These results were in accordance with those of nonnucleoside RT inhibitors (NNRTIs), such as R89439 (an α‐anilinophenylacetamide derivative) and nevirapine, indicating that RD4‐2025 also belongs to the family of NNRTIs.
Enhancement of barrier function of human aortic endothelial cells by activators of protein kinase C
Authors: Yamada Yoshiji , Yokota Mitsuhiro
Published: July 2007

We investigated the role of protein kinase C (PKC) in regulating the permeability of human and bovine aortic endothelial cells (AECs) cultured on micropore filters. AECs cultured in the upper chamber were incubated with medium containing a PKC agonist, phorbol 12‐myristate 13‐acetate (PMA) or 1‐oleoyl‐2‐acetyl‐glycerol (OAG) for 15 min to 8 h, after which the cells were incubated with albumin for 1 h. The amount of albumin that had transferred through AECs was determined. Activation of PKC by exposure to PMA or OAG inhibited albumin permeability in human AECs, but increased it in bovine AECs. While the PKC inhibitor, staurosporine, did not itself influence endothelial permeability, it reduced the decrease or increase in permeability induced by exposure to PMA or OAG in human and bovine AECs, respectively. Activation of PKC thus enhanced the barrier function of human AECs, but reduced it in bovine AECs.
A novel guanyl‐preferable ribonuclease of Bacillus polymyxa: Isolation and characterization of the enzyme
Authors: Dementiev A. A. , Mirgorodskaya O. A. , Moiseyev G. P. , Yakovlev G. I. , Shlyapnikov S. V. , Kirpichnikov M. P.
Published: July 2007

Three forms of the extracellular alkaline ribonuclease of B. polymyxa (RNases Bpo I, Bpo II and Bpo III) were obtained in the homogeneous states. Relative molecular weights, N‐ and C‐terminal amino acid sequences of the proteins were determined. The two higher‐molecular weight forms of the enzyme (RNases Bpo I and Bpo III) were shown to be precursor molecules which differed from the mature B. polymyxa RNase (RNase Bpo II) by the presence of a extra twelve and six amino acid residues at the amino terminus, respectively. The study of catalytic properties of the enzyme elucidated that the extent of specificity of B. polymyxa RNase in respect to high‐polymeric substrates is different from that of other natural Bacillus RNases. Amino acid composition of B. polymyxa RNase was established and structural similarity within family of Bacillus RNases is discussed. B. polymyxa RNase was shown to be inhibited by intracellular protein inhibitor of B. amyloliquefaciens RNase with the dissociation constant of 2.7×10‐12 M.
Properties of 26S proteasome purified from rat skeletal muscles: Comparison with those of 26S proteasome from the rat brain
Authors: Akaishi Takahiro , Sawada Hitoshi , Yokosawa Hideyoshi
Published: July 2007

A ubiquitin/ATP‐dependent proteolytic complex (26S proteasome) was highly purified from rat skeletal muscles and its enzymatic properties were compared with those of the brain 26S proteasome. The purified 26S proteasome comprises 22‐110 kDa subunits characteristic of the typical 26S proteasome on the basis of SDS‐PAGE. The two‐dimensional PAGE (NEPHGE and SDS‐PAGE) pattern revealed that the pI values and molecular masses of the muscle 26S proteasome subunits were similar but not identical to those of the subunits of 26S proteasome purified from the rat brain. The enzymatic properties of the muscle 26S proteasome were very similar to those of the brain enzyme in substrate specificity and inhibitor susceptibility. The specific activities of the muscle 26S proteasome toward three fluorogenic peptide substrates were indistinguishable from those of the brain enzyme.
Phosphorylation of different human milk proteins by human catalytic secretory immunoglobulin A
Authors: Kit Yury Ya. , Semenov Dmitry V. , Nevinsky Georgy A.
Published: July 2007

The ability of secretory immunoglobulin A (sIgA) from milk of healthy mothers to phosphorylate various milk proteins in the presence of γ‐[32P]‐ATP was shown to be a property of the antibodies. The polyclonal sIgA was purified by sequential chromatography on Protein‐A Sepharose and DEAE‐cellulose, and then separated by chromatography on the ATP‐Sepharose. The preparations containing all milk proteins except for protein kinases [integrated milk proteins (IMP)] were obtained by extraction of the kinase activities from the milk using their affinity to the insoluble crosslinked staphylococcus. Addition of sIgA fractions (having a different affinity to ATP) to the IMP led to phosphorylation of casein and several other milk proteins with different efficiencies.
Bisindolylmaleimide inhibits the PMA‐induced down‐regulation of collagen synthesis in fibroblasts
Authors: Park Rang‐Woon , Kim In‐San , Jo Joon‐Seung
Published: July 2007

We assessed the role of protein kinase C (PKC) in the regulation of collagen synthesis. Two PKC activators, Phorbol 12‐myristate 13‐acetate (PMA) and 1‐oleyl 2‐acetyl‐sn‐glycerol (OAG), decreased the relative rate of collagen synthesis in a dose‐ and time‐dependent manner in fibroblasts, whereas an inactive phorbol ester, 4α‐phorbol didecanoate failed to affect the collagen synthesis. In PKC‐depleted cells both PMA and OAG were unable to inhibit collagen synthesis. Bisindolylmaleimide, a specific inhibitor of PKC, completely abrogated PMA‐induced inhibition of collagen synthesis in a dose‐dependent fashion while two other PKC inhibitors with low specificity, H7 and staurosporin failed to block PMA effect on collagen synthesis. The results provide evidence that collagen synthesis is regulated through the signal pathway involving PKC activation.
Ginsenoside‐Rg1 positively regulates cyclin E‐dependent kinase activity in human hepatoma SK‐HEP‐1 cells
Authors: Lee Kwang Youl , Lee Seung Ki
Published: July 2007

In the present study, we describe that ginsenoside‐Rg1 (G‐Rg1) stimulates the proliferation of cultured human hepatoma SK‐HEP‐1 cells. The incorporation of [3H] thymidine into DNA was increased in the cells in response to G‐Rg1. The stimulatory effect of G‐Rg1 on DNA synthesis in SK‐HEP‐1 cells require newly synthesized proteins, since cycloheximide blocked the DNA synthetic activity stimulated with G‐Rg1. Thus, we examined whether G‐Rg1 induces the intracellular protein levels of regulatory proteins for cell cycle progression using immunoblottings. The results from immunoblottings showed that G‐Rg1 induced the levels of cyclin E and cdk2 proteins in the cells. Furthermore, the results from immuno‐complex kinase assays for cyclin E‐dependent kinase showed that G‐Rg1 up‐regulates the kinase activity in a dose‐dependent manner. These results suggest that G‐Rg1 stimulates cell‐growth of SK‐HEP‐1 cells by inducing the intracellular levels of cyclin E/cdk2 complex, which in turn up‐regulates cyclin E‐dependent kinase activity.
Comparison of the heparanase enzymes from mouse melanoma cells, mouse macrophages, and human platelets
Authors: Graham Lloyd D. , Underwood P. Anne
Published: July 2007

The intracellular heparanases from mouse macrophage and melanoma cells are very similar in terms of their size (60‐80 kDa), pI (5.3‐4.1), pH optimum (<‐5.5), and interactions with heparin. These proteins are therefore likely to be identical, suggesting that tumour and blood cells utilise the same heparanase enzyme. The human platelet enzyme is similar to the mouse enzymes in terms of pH optimum (č‐5.5) and pI value (5.3‐4.8), but appears to be smaller in size (40‐60 kDa). It also seems to differ from the mouse enzymes in aspects of its surface charge, and in its interactions with heparin. There was no indication that proteolysis was of significance for the enzymes, nor that they contained any sialic acid residues.
Isolation of a developmentally‐regulated expressed sequence tag from bladder tissue using the mRNA differential display
Authors: Chaqour Brahim , Howard Pamela S. , Macarak Edward J.
Published: July 2007

In order to gain insight into the molecular and cellular events that govern the structural and the functional properties in developing organs, we have conducted a study to identify genes that have a temporally‐restricted expression in the bladder wall during fetal development. We utilized the mRNA differential display technique and compared the pattern of gene expression during the first, the second and the third trimester of gestation. We cloned and sequenced a cDNA fragment (bld‐10) which was expressed during the second and third trimester but consistently absent during the first trimester. The bld‐10 sequence is not related to any known gene in the GenBank database but has significant homology (89%) with human expressed sequence tag (EST) that has been cloned from human fetal heart and brain libraries. When used in Northern‐blot hybridization as a probe, the fragment bld‐10 generates two hybridization signals of 3.1 and 4.0 kb, that are minimally expressed during the first trimester of gestation and up‐regulated in the second and third trimester. Differential expression of this gene may be responsible for some of the profound changes which occur during organ development.
Identification and characterization of a mitochondrial endonuclease from yeast, Schizosaccharomyces pombe
Authors: Ikeda Shogo , Maeda Noriaki , Ohshima Tomoaki , Takata Norifumi
Published: July 2007

Schizosaccharomyces pombe mitochondria were isolated from the cells treated with Novozyme 234, and purified in a Percoll gradient. A zymographic assay in a SDS‐polyacrylamide gel containing single‐stranded DNA revealed that an endonuclease of 32 kDa is associated with the mitochondria. The endonuclease was extracted from the mitochondria with 0.5 M KCI and was partially purified. The 32‐kDa enzyme degraded both DNA and RNA at a weak alkaline pH, but preferred single‐stranded DNA. The enzyme required Mg2+ or Mn2+, but not Ca2+ or Zn2+ for activity, and was inhibited by 50% with a 150 mM salt solution. Nicks generated by the enzyme could be resealed with T4 DNA ligase, indicating that the enzyme produces 5′‐P and 3′‐OH ends.
Modification of Tyrosine‐3 (63) and Lysine‐6 of Taiwan cobra phospholipase A2 affects its ability to enhance 8‐Anilinonaphthalene‐1‐sulfonate fluorescence
Authors: Chang Long‐sen , Lin Shinne‐ren
Published: July 2007

The Tyr‐3 (63) and Lys‐6 of Naja naja atra phospholipase A2 (PLA2) were modified with p‐nitrobenzenesulfonyl fluoride and 4‐chloro‐3,5‐dinitrobenzoate, respectively. Although the ability of the Lys‐modified derivative to enhance the ANS fluoresence was lower than that observed with native PLA2, the ANS‐binding affinity of the Lys‐modified derivative was similar to that of the native enzyme. Modifications on Tyr‐3 or/and Tyr‐63 of PLA2 resulted in the complete loss of its ability to enhance the ANS fluorescence. Nevertheless, the extent of O‐sulfonylation of Tyr residues was not effectively reduced by the addition of ANS. This suggests that Tyr‐3 and Tyr‐63 interact with the bound ANS in an aromatic ring‐stacking manner. Alternatively, the incorporation of a carboxydinitrophenylated group on Lys‐6 may notably perturb the nonpolarity of the ANS‐binding site.
Structure of bacteriochlorophyll aggregates in chlorosomes of green bacteria: A spectral hole burning study
Authors: Novoderezhkin Vladimir I. , Fetisova Zoya G.
Published: July 2007

Exciton level structure, homogeneous absorption and hole‐burning spectra were calculated for different models of bacteriochlorophyll aggregation in chlorosomal antennae of green bacteria. It was demonstrated that none of the earlier proposed models of noninteracting linear bacteriochlorophyll aggregates and linear bacteriochlorophyll chains assembling in tubular aggregates with high density of packing, exhibits the in vivo exciton level structure revealed by hole‐burning experiments on intact cells of green bacteria. The models of linear exciton‐coupled bacteriochlorophyll chains with a low packing density, approximating that in vivo, were proposed as alternative, to obtain the main spectral features found in natural antennae.
Protein kinase C inactivation by Fenton's‐reaction at discrete CU binding sites
Authors: Traverso Nicola , Ricciarelli Roberta , Domenicotti Cinzia , Menini Stefano , Pronzato M. Adelaide , Cottalasso Damiano , Marinari Umberto M.
Published: July 2007

The consequence of direct exposure to HO radical (chemically generated by Fenton's reaction) of partially purified rat liver PKC has been evaluated in this work. PKC inhibited Fenton‐dependent HO generation, probably due to the binding of copper ions to the enzyme. PKC activity was inhibited by H2O2. Copper ions were able to increase the H2O2‐mediated damage to the enzyme, but only beyond a concentration threshold.

The possible interactions between PKC and Fenton's reagents, in particular copper ions, is discussed.
Activation of cathepsin D by polyanionic compounds
Authors: Watabe Shoji , Ikeda Tadao , Yago Nagasumi
Published: July 2007

We examined the nature of the activation of cathepsin D by polyanionic compounds. Tripolyphosphate, a model compound for polyanions, decreased the Km value of porcine cathepsin D for bovine serum albumin without affecting Vmax. Half‐maximal activation was achieved at 0.2 mM free tripolyphosphate. Electrophoretic mobility of cathepsin D decreased as the tripolyphosphate concentration was increased, and the enzyme had no net charge at 50 mM triP. The concentration for the half‐maximal mobility change of cathepsin D (0.18 mM) was similar to that for half‐maximal activation. These results suggest that tripolyphosphate increased affinity of the enzyme for its substrate by cancelling positive charges on cathepsin D and thus decreasing the electrostatic repulsion.
Expression of catalytically active human multifunctional glycogen‐debranching enzyme and lysosomal acid alpha‐glucosidase in insect cells
Authors: Wu Jer‐Yuarn , Van Hove Johan L. K. , Huang Yuan‐Sheng , Chen Yuan‐Tsong
Published: July 2007

Glycogen debranching enzyme and acid α‐glucosdase are responsible for glycogen degradation in human. The formal enzyme is a mulifunctional enzyme with two independenent catalytic activities occuring on a single polypeptide, while the latter is a lysosomal enzyme which matures through extensive glycosylation and phosphorylation and proteolytic processing. Deficiency of glycogen debranching enzyme and acid α‐glucosidase cause glycogen storage disease type III and II, respectively. Baculovirus/insect expression system was used to produce both GDE and GAA. Both enzymes were found to be catalytically and antigenically active. The majority of recombinant GDE is present in the medium (70%). Uptake experiment indicated that GAA produced in the insect cells could not be absorbed into the GSD type II patient fiboblasts through mannose‐6‐phosphate receptor mediated endocytosis. Uptake experiment combined with immunoblot analysis indicated there are differences in the posttranslational modification and processing between insect cells and mammalian cells.
Identification of a Zn2 ‐sensitive component of Ehrlich cell plasma membrane redox system by CHAPS‐agarose‐polyacrylamide electrophoresis and in situ staining of activity
Authors: Rodríguez‐Caso Luis , Rodríguez‐Agudo Daniel , Castillo‐Olivares Antonio del , Márquez Javier , de Castro Ignacio Núñez , Medina Miguel Ángel
Published: July 2007

A procedure based on CHAPS‐agarose‐polyacrylamide electrophoresis and in situ staining of activity was used to detect a Zn2+‐sensitive component of Ehrlich cell plasma membrane redox system. The procedure is so powerful that it allows to use crude plasma membrane fractions and can be easily adapted for use in an electrophoretic approach to the purification of this protein.
Essential fatty acid metabolism in cardiomyocytes grown in media enriched with different N‐6/N‐3 fatty acid combinations
Authors: Hrelia Silvana , Biagi Pier L. , Lorenzini Antonello , Jimenez Josè A. Lopez , Horrobin David F. , Bordoni Alessandra
Published: July 2007

We have evaluated the effects of three different 18:3n‐6, 20:5n‐3 and 22:6n‐3 fatty acid combinations on essential fatty acid (EFA) metabolism in rat cultured cardiomyocytes. The desaturating/elongating activities for linoleic (LA) and α‐linolenic acid (ALA) were evaluated by radlolabehng the cells with 1‐[14C]LA or 1‐[14C]ALA and the fatty acid pattern of cardiomyocytes was assessed by gas chromatography. LA and ALA conversion to more unsaturated metabolites was reduced by increasing respectively n‐3 and n‐6 fatty acid concentration in the media. The all three combinations used reduced the saturated and increased the polyunsaturated fatty acid content of cardiomyocytes. The n‐6/n‐3 fatty acid ratio did not change compared to control cells in cardiomyocytes receiving the highest amount of 18:3n‐6 and the lowest amounts of n‐3 fatty acids This combination may be suitable for modifying EFA desaturating/elongating activities without altering the physicochemical parameters which are related to the correct balance between n‐6 and n‐3 fatty acid content.
Cytokine profile in autosomal dominant polycystic kidney disease
Authors: Merta M. , Tesař V. , Zima T. , Jirsa M. , Ryšavá R. , Žabka J.
Published: July 2007

To elucidate the pathogenetic mechanism of renal parenchymal injury in autosomal dominant polycystic kidney disease (ADPKD) patients, typically characterized by renal cystic changes paralleled by interstitial inflammation and gradual fibrotic changes, the role of selected inflammatory mediators was evaluated in a group of ADPKD patients with normal glomerular filtration rate. The plasma concentrations of IL‐6, IL‐8, ICAM‐1 and VCAM‐1 (which may reflect systemic response to inflammation/infection) were increased in the ADPKD patient group. Coupled with decreased urinary excretion of the IL‐1 receptor antagonist (which exerts an anti‐inflammatory role), these results suggest that even in overt infection free status, the proinflammatory system is more activated and anti‐inflammatory defence system weakened in ADPKD subjects. Our data support the current view that cytokines are candidate contributors to pathogenesis of ADPKD.
Adsorption of Bacillus subtilis endo‐β‐1,4‐glucanase to cellulosic materials
Authors: Kim Hoon , Ahn Dong Ho , Jung Kyung Hwa , Pack Moo Young
Published: July 2007

The intact and the truncated endo‐β‐1,4‐glucanase expressed in Bacillus megaterium by a B. subtilis gene were purified and its adsorption characteristics to cellulosic materials were investigated. The intact enzyme was purified by affinity towards insoluble cellulose and Mono‐Q chromatography. The truncated enzyme was purified by gel filtration and chromatofocusing. The molecular activities of these enzymes towards the soluble substrates were identical. Optimum pH and temperature of these two types of enzyme were same, 5.5 and 60°C, respectively. However, the insoluble substrate, Avicel, was hydrolyzed slowly by the intact endoglucanase while the truncated endoglucanase did not hydrolyze the Avicel. Isoelectric points of the intact and the truncated enzyme were 6.2 and 5.6, respectively. In a Sephadex column the large form of intact endoglucanase was eluted later than the small form of truncated enzyme. Only the intact enzyme was strongly adsorbed to Avicel. The practical maximum adsorption was about 20 mg/g of Avicel at pH 6.0 and 4°C.
Inositol 1,4,5‐Trisphosphate distribution in Lycopersicon esculentum mill. seedlings cultivated “in vitro” under different conditions
Authors: Piacentini Maria Piera , Ricci Donata , Fraternale Daniele , Piatti Elena , Manunta Antonio , Accorsi Augusto
Published: July 2007

Measurements of inositol 1,4,5‐trisphosphate (Ins 1,4,5‐P3 in cotyledons, epicotyls and roots of tomato seedlings grown “in vitro” either in the light or in the dark indicated that higher concentrations of this signal‐transducing molecule are contained in hypogeous vs. epigeous tissues. The same was observed in induced cotyledon explants grown in the light in the presence of growth regulators. Data concerning phosphatidylinositol metabolism in seedling roots are also reported. Taken together, our results may be helpful in understanding the role of the polyphosphoinositide signal system in plants.
Isolation and characterization of isoforms of retinol binding protein by isoelectrofocusing
Authors: Minic Zoran , Hranisavljevic Jovan , Vucelic Dusan
Published: July 2007

Retinol binding protein prepared from human urine was fractionated by chromatofocusing into four isoforms: two retinol‐containing (holo‐) and two retinol‐free (apo‐) species. The pI values of the isoforms ascertained by isoelectrofocusing with an immobiline pH gradient were: holo(I) 4.79‐4.77; apo(II) 4.61‐4.56; holo(III) 4.63 and apo(IV) 4.46‐4.41. In vitro aging experiments with apo(II) under conditions favoring deamidation (37°C, pH 7‐10, 3‐28 days) resulted in formation of the more acidic apo(IV)‐isoform. The aging rate was consistent with pH increase. It appears that the urinary RBP mixture is composed of two apo‐holo pairs: a native form with genuine protein structure and an acidic form generated upon aging.
D‐glucose metabolism in dimethyl suberimidate‐treated tumoral pancreatic islet cells
Authors: Vanhoutte C. , Malaisse W. J.
Published: July 2007

After protein cross‐linking by dimethyl suberimidate, tumoral insulin‐producing cells of the RINm5F line were either exposed to digitonin for measurement of hexokinase activity in the resulting cell pellet and supernatant, or incubated in the presence of D‐[5‐3H]glucose, D‐[U‐14C]glucose or L‐[U‐14C]glutamine to assess the metabolism of these nutrients. After digitonin treatment, the activity of hexokinase recovered in the cell pellet was about 40 % higher in cross‐linked than control RINm5F cells. Although failing to affect the metabolism of L‐[U‐14C]glutamine, and severely decreasing the oxidation of D‐[U‐14C]glucose, the cross‐linking of proteins accentuated the increase in D‐[5‐3H]glucose utilization and D‐[U‐14C]glucose conversion to acidic metabolites resulting from a rise in hexose concentration from 2.8 to 16.7 mM. The latter change represents a mirror image of that previously found in cross‐linked pancreatic islets. Taking into account the vastly different participation of glucokinase to hexose phosphorylation in RINm5F cells and normal islet cells, the present findings further support, therefore, the regulatory role of protein‐to‐protein interaction in the control of glucokinase catalytic activity in these fuel‐sensing cells.
Functioning of oligomeric‐type light‐harvesting antenna
Authors: Timpmann K. E. , Taisova A. S. , Novoderezhkin V. I. , Fetisova Z. G.
Published: July 2007

In our previous work, we developed, for the first time, a theory of excitation energy transfer within an oligomeric‐type light‐harvesting antenna and, in particular, within the chlorosome of green bacteria (Biophys. J., 1996, vol.71, pp.995‐1010). The theory was recently developed for a new original exciton model of aggregation of chlorosomal pigments, bacteriochlorophylls (BChl) c/d/e (Biochem. Mol.Biol.Int., 1996, vol.40, No.2, pp. 243‐252). In this paper, it was demonstrated with picosecond fluorescence spectroscopy that this theory explains the antenna‐size‐dependent kinetics of fluorescence decay in chlorosomal antenna, measured for intact cells of different cultures of the green bacterium Chlorobium limicola with different chlorosomal antenna size determined by electron microscopic examination of the ultrathin sections of the cells. According to our model, the energy transfer dynamics within the chlorosome imply the formation of a cylindrical exciton, delocalized over a tubular aggregate of BChl c chains, and inductive‐resonance‐type transfer of such a cylindrical exciton between the nearest tubular BChl c aggregates and to BChl a of the chlorosome.
ATP/ADP antiporter‐ and aspartate/glutamate antiporter‐mediated fatty acid‐induced uncoupling of liver mitochondria in incubation media differing in ion composition
Authors: Samartsev Victor N. , Mokhova Elena N.
Published: July 2007

Substitution of potassium cloride for sucrose in the incubation medium of liver mitochondria or addition of magnesium cloride to the sucrose medium produces a small (if any) effect on the palmitate‐induced uncoupling and its sensitivity to carboxyatractylate and aspartate, but it exerts a pronounced increase in the recoupling effect of glutamate on this uncoupling. 20‐40 μM cetyltrimethyl ammonium bromide increases the recoupling effect of glutamate and aspartate but decreases that of carboxyatractylate. The data are in line with suggestion that both ATP/ADP antiporter and aspartate/glutamate antiporters are involved in the fatty acid‐induced uncoupling.
Evaluation of antisense inhibition in a B cell model
Authors: McCall M. N. , Kuchel M. G. , Basten A.
Published: July 2007

An in vitro model has been developed in an attempt to optimise antisense inhibition in B cells as a prelude to transgenic studies. The hypotheses tested were that i) the 3′‐untranslated region would be an appropriate target for antisense inhibition; 2) the immunoglobulin heavy chain intronic enhancer could be used to enhance antisense inhibition via increased production of antisense transcripts; and 3) the mouse metallothionein‐1 promoter would allow induction of antisense inhibition in B cells. Secreted IgM protein and mRNA were monitored following the stable transfection of a B cell line, HO‐2.2, with a series of plasmid constructs containing antisense or sense target sequence DNA under the control of either the mouse metallothionein‐1 promoter or homologous (ie same promoter as target sequence) immunoglobulin heavy chain promoter. The 3′‐untranslated region proved to be an appropriate target resulting in 70% inhibition of IgM secretion. Compared with untransfected and sense controls, significant decreases in IgM secretion (and RNA levels) were detected in clones transfected with antisense constructs utilising the mouse metaltothionein‐1 promoter and the immunoglobulin heavy chain intronic enhancer elements. These clones exhibited a further significant reduction in secreted IgM production upon zinc induction. Hybridisation studies demonstrated that decreased protein production was most likely attributable to reduction in RNA levels. In contrast, transfection with antisense constructs had no effect on membrane IgM protein levels which not only confirmed the specificity of antisense action but meant that the B cell remained sensitive to receptor ligation. We conclude that reasonable antisense inhibition of gene product expression can be achieved in B cells by targeting the 3′‐untranslated region and using both an inducible promoter (mouse metallothionein‐1) and the IgH enhancer to aid antisense RNA production.
Binding of the bovine caseinoglycopeptide to the platelet membrane glycoprotein GPIbα
Authors: Chabance Béatrice , Qian Zu‐Yuan , Migliore‐Samour Danièle , Jollès Pierre , Fiat Anne‐Marie
Published: July 2007

The bovine caseinoglycopeptide (residues 106‐169), the C‐terminal part of κ‐casein, inhibited the von Willebrand factor‐dependent platelet aggregation in a dose‐dependent manner. An affinity matrix made of the caseinoglycopeptide selectively bound the platelet membrane glycoprotein GPIbα which contains the von Willebrand factor binding site. The amino acid residues of GPIbα participating in the caseinoglycopeptide binding were located after residue Glu90.
Simple and reliable procedure for PCR amplification of genomic DNA from yeast cells using short sequencing primers
Authors: Haaning Jesper , Oxvig Claus , Overgaard Michael Toft , Sottrup‐Jensen Lars
Published: July 2007

Yeast is widely used in molecular biology. Heterologous expression of recombinant proteins in yeast involves screening of a large number of recombinants. We present an easy and reliable procedure for amplifying genomic DNA from freshly grown cells of the methylotrophic yeast Pichia pastoris by means of PCR without any prior DNA purification steps. This method involves a simple boiling step of whole yeast cells in the presence of detergent, and subsequent amplification of genomic DNA using short sequencing primers in a polymerase chain reaction assay with a decreasing annealing temperature.
Selective excitation of tryptophans in OmpF: A fluorescence emission study
Authors: Pattnaik Bikash Ranjan , Ghosh Subhendu , Rajeswari Moganty R.
Published: July 2007

The fluorescence studies of E. coli porin OmpF at pH 7.5 were carried out using excitation at 280 and 305 nm. Similar studies were performed in presence of a denaturant (urea) and a quencher (KI). Results show that both the tryptophans present in OmpF (residue numbers 61 and 214) contribute to fluorescence at 280 nm excitation, whereas only one residue shows fluorescence emission when excited at 305 nm. Based on these findings and the available crystal structure, it is speculated that tryptophan 61 of OmpF is selectively excited at 305 nm. The present studies point out some interesting features of the tryptophan microenvironments in OmpF.
Role of H‐ras gene in chronic liver damage in mice. By using transgenic mice carrying a human c‐H‐ras proto‐oncogene without mutations
Authors: Tsunematsu Satoshi , Saito Hidetsugu , Sato Reiko , Morizane Toshio , Ishii Hiromasa
Published: July 2007

Hepatic tumors including hepatocellular carcinoma were generated by carbon tetrachloride in transgenic mice carrying a human c‐H‐ras gene (rasH2 mice). RasH2 mice express 2 to 3 times more ras protein (ras p21) in the liver than do non‐Tg mice. When carbon tetrachloride was administered, the rasH2 mice produced about 5 times as many hepatic tumors than did the non‐transgenic mice. However, neither the 10‐100 times higher ras p21 expression required for murine fibroblast transformation by itself nor the mutational activation of the H‐ras gene was observed in carbon tetrachloride‐induced hepatic tumors. These results show that H‐ras proto‐oncogene expression in the murine liver, even if it is not high enough to transform cells, also causes liver tumors when CCl4 are repeatedly given.
Determination of sugar specificity of jackfruit lectin by a simple sugar‐lectin binding assay using microtiter plate
Authors: Wetprasit Nuanchawee , Chulavatnatol Montri
Published: July 2007

Sugar‐lectin binding assay was developed as a simple method which employed direct coating of microtiter plate with galactose‐binding lectins. Biotin‐galactose conjugate was used to bind to the immobilized lectins. The bound conjugate was then detected using streptavidin‐horseradish peroxidase. Using the assay in conjunction with various competing carbohydrates, jackfruit lectin from Artocarpus heterophyllus was found to be specific for α‐anomer of galactoside with an aromatic residue.
Cloning, expression and purification of the coat protein of encephalitis virus (DIEV) infecting Dicentrarchus labrax
Authors: Sideris Diamantis C.
Published: July 2007

The coat protein gene from encephalitis virus infecting Dicentrarhuslabrax (DIEV) has been cloned by gene amplification, sequenced and expressed in Escherichia coli. DNA sequencing has revealed an open reading frame of 1017 bases encoding a polypeptide of 338 amino acids. The sequence similarities between the DIEV coat protein gene and the same gene in five encephalitis viruses infected other fish species were over 71.5% at the nucleotide level and over 79.5% at the amino acid level. These results indicate that the nodaviruses that cause encephalopathy and retinopathy in fishes are very closed related. E. coli cells harbouring the plasmid containing the DIEV gene can produce the viral coat protein. An efficient purification scheme using a Sepharore‐Ni+2 column is presented. This, gives approx. 10 mg of more than 95% pure protein per gr of E. coli culture.
Recombinant CTFR detection in CF tracheal epithelial cells following in vitro liposomeme‐mediated gene transfer
Authors: Colosimo Alessia , Scarpino Stefania , Sangiuolo Federica , Sario Sabrina Di , Mossa Giuseppe , Novelli Giuseppe , Dallapiccola Bruno
Published: July 2007

The efficacy of CFTR gene transfer mediated by cationic liposomes Dc‐Chol/DOPE into cystic fibrosis (CF) tracheal epithelial cells carrying defective processing mutations (S549N/N1303K), was assessed by studying mRNA and protein expression of the recombinant product. Appreciable levels of mRNA transcripts were detected 48 h after transfection, while complete translocation of the recombinant CFTR to the apical membrane of epithelial cells was observed after 72 h following transfection. Our results suggest that in vitro restoration of a normal CFTR processing and migration to the cell plasmalemma requires 72 h at least as demonstrated by immunocyto‐fluorescence using the monoclonal antibody MATG 1016. These findings are relevant onto gene transfer phase I clinical studies.
Efficient production of active TNF‐α by albumin signal peptide
Authors: Maeda Yuu , Soda Mariko , Ito Keizo , Sato Kenzo
Published: July 2007

TNF‐α is initially synthesized as a membrane‐anchored precursor protein and processed proteolytically by a matrix metalloproteinase (MMP)‐like enzyme. In order to establish an efficient expression system of TNF‐α in mammalian cells without involvement of the extracellular enzyme, an expression plasmid (pCN‐alb‐TNF) was constructed with a signal sequence of the rat albumin gene as a module for secretion. The highest level of production of TNF‐α was observed in the clone CT‐3 by SDS‐PAGE and Western blot analysis. Biological activity of the secretion was revealed by repression of catalase gene expression in hepatoma cells and cytotoxity to L929 cells. Attachment of the signal peptide to mature form resulted in the enhancement of production of the cytokine.
Isoform‐specific monoclonal antibodies against HSP90
Authors: Nemoto Takayuki , Roi Rina , Matsusaka Tomonori , Iwanari Hiroko , Yamashita Hisahiko , Kyakumoto Seiko , Sato Nobuko
Published: July 2007

The purpose of this study was to develop monoclonal antibodies (mAbs) that distinguish between the two isoforms of human 90‐kDa heat shock protein (HSP90), i.e HSP90α and β. Human HSP90α and β isoforms expressed in Escherichia coil were separately used as antigens for developing the mAbs. Twenty‐three and ten mAbs were obtained by immunization of mice with HSP90α and β, respectively. Among them, ten and three mAbs specifically recognized HSP90α and β isoforms, respectively, on the criteria of both enzyme‐linked immunosorbent and immunoblotting analyses. Immunochemical analysis by use of the mAbs revealed that both of the HSP90 isoforms were present in human cells even under unstressed conditions and that the expression of HSP90α was more strongly induced when the cells were exposed to arsenate. This is the first report of the development of the mAbs discriminating between the two isoforms of HSP90. The mAbs specific for HSP90 isoforms should be useful for the regulational and functional analyses of HSP90 isoforms.
A recombinant human fibronectin fragment facilitates retroviral mediated gene transfer into human hematopoietic progenitor cells
Authors: Dutt Parmesh , Hanenberg Helmut , Vik Terry , Williams David A. , Yoder Mervin C.
Published: July 2007

Gene transfer into hematopoietic progenitor cells is increased when these cells are adherent to the carboxy‐terminal chymotryptic fragment of human plasma fibronectin (FN30/35) containing the heparin binding domain (HBD) and the alternatively spliced type three connecting segment (IIICS) region. We report herein the production of a recombinant human fibronectin fragment comprised of the HBD and IIICS regions expressed by Sf9 insect cells following recombinant baculovirus infection. The resulting isolated peptide, HuBacFN, facilitated gene transfer into human hematopoietic cell lines and primary human hematopoietic progenitors to a level achieved with the purified human plasma FN30/35 peptide.
Nonenzymatic reduction of brucine N‐oxide by the heme group of cytochrome P450
Authors: Takekawa Koji , Sugihara Kazumi , Kitamura Shigeyuki , Tatsumi Kiyoshi
Published: July 2007

Evidence showing that cytochrome P450‐mediated reduction of brucine N‐oxide to brucine by rat liver microsomes proceeds nonenzymatically in the presence of both a reduced pyridine nucleotide and FAD is presented. The microsomal N‐oxide reduction appears to proceed in two steps: The first step is reduction of FAD by NADPH or NADH either enzymatically or nonenzymatically. The second step is nonenzymatic reduction of the tertiary amine N‐oxide by the reduced flavin and is nonenzymatically catalyzed by the heme group of cytochrome P450.
Effects of vitamin E and selenium on antioxidant defense in rat heart
Authors: Scholz R. W. , Minicucci L. A. , Reddy C. C.
Published: July 2007

Heart mitochondria, isolated from rats fed diets deficient or supplemented with vitamin E (E) and/or selenium (Se), were subjected to time‐course assays of lipid peroxidation stimulated by ascorbate/ADP/Fe3+. Mitochondria depleted of α‐tocopherol (α‐TH) peroxided rapidly as assessed by formation of thiobarbituric acid reactive substances (TBARS). Formation of TBARS was strongly inhibited in mitochondria from rats fed diets supplemented with E. Selenium deficiency, reduced glutathione (GSH), glutathione disulfide (GSSG) or GSH + GSSG did not affect the course of lipid peroxidation in mitochondria from rats supplemented or deficient in E. Combined E and Se deficiency resulted in significantly lower total (oxidized + reduced) mitochondrial coenzyme Q‐9 (CoQ‐9) concentration compared with control rats supplemented with dietary E and Se. Time‐course changes in mitochondrial α‐TH and total CoQ‐9 during oxidizing conditions were minor in +E rats. Total CoQ‐9 was reduced substantially, however, during the course of lipid peroxidation in mitochondria depleted of α‐TH. Selenium‐dependent glutathione peroxidase (Se‐GSHPx) activity was reduced by approximately 96% in heart cytosol, and to a somewhat lesser extent in mitochondria, by dietary Se deficiency. Non‐Se GSHPx activity was not detected in heart cytosol but was detected in very small amounts in heart mitochondria. Glutathione S‐transferase activity of heart cytosol was decreased in E and/or Se deficiency. The results of these experiments indicate that membrane α‐TH was most effective in inhibiting lipid peroxidation in heart mitochondria.
Purification and biochemical characterisation of human placental acid α‐glucosidase
Authors: Chadalavada Durga M. , Sivakami S.
Published: July 2007

An acid α‐glucosidase (EC 3.2.1.3) has been purified to electrophoretic homogeneity from the soluble fraction of the human term placenta. In the presence of SDS, two doublets of 79 and 67 kDa were seen in addition to other bands of extremely low intensity. Each of these bands was seen to cross‐react with polyclonal antiserum raised to the purified enzyme, thus confirming the homogeneity of the preparation. The purified enzyme exhibited a broad substrate specificity. The kinetic data revealed the possible presence of multiple substrate binding sites. Chemical modification using group specific reagents indicated the presence of a carboxyl group and tryptophan at the active site. Based on these results a possible structure for the active site of the human term placental acid α‐glucosidase has been proposed.