bluuebunny/crossref_metadata_2025 huggingface dataset:
S and G2 phase histone biosynthesis of HEp‐2 cells after the influence of the bisalkylating agent, chlorambucil
Authors: Sourlingas Thomae G. , Sekeri‐Pataryas Kalliope E.
Published: July 2007

Chlorambucil was previously found to be a specific inhibitor of total histone synthesis without affecting total cellular protein synthesis. In this study, we used S and G2 phase HEp‐2 cancer cells to further analyze and specifically localize this effect. One hour (S phase), 4 hours (S phase) and 9 hours (G2 phase) after release from an aphidicolin double block synchronization procedure, cells were preincubated for 60 min with 30 μM chlorambucil and then radiolabeled for another 60 min in the continued presence of the agent. At the end of each of these time intervals, cells in almost mid‐S phase, late S phase and toward the end of the G2 phase were obtained for analysis. It was found that chlorambucil partially inhibits total histone synthesis nonspecifically as to the variants being synthesized (S phase and basal variants) but only during the first half of the S phase. DNA synthesis is also inhibited partially, but during the second half of the S phase. The position during the S phase where chlorambucil exerts its effect on total histone synthesis, the degree of this effect and its uncoupling with DNA synthesis inhibition, indicate that it is temporally linked with the onset of S phase histone
Antioxidants and cataract: (Cataract induction in space environment and application to terrestrial aging cataract)
Authors: Bantseev Vladimir , Bhardwaj Ratan , Rathbun W. , Nagasawa H. , Trevithick John R.
Published: July 2007

The effect of several antioxidants and cysteine‐elevating precursor drugs (prodrugs) was tested on lens damage occurring after in vitro exposure to low levels of 60Co‐y‐irradiation, to simulate in vitro the exposure to radiation in vivo of (1) astronauts (2) jet crews (3) military radiation accident personnel. Tocopherol (100 μM), ascorbic acid (1mM), R‐α‐lipoic acid (1mM), and taurine (0.5 mM) protected against radiation‐ associated protein leakage. MTCA and ribocysteine protected lenses against opacification, LDH and protein leakage, indicating that antioxidants and prodrugs of cysteine appear to offer protection against lens damage caused by low level radiation.
Expression of progesterone receptor mRNA in the first trimester human placenta
Authors: Shanker Y. Gopi , Sharma S. C. , Rao A. Jagannadha
Published: July 2007

The human placenta produces large quantities of progesterone, the function and target tissues of action of which during pregnancy are not completely understood. Although it has been suggested that placenta itself could be a potential target tissue for progesterone action, there is no conclusive evidence for the presence of progesterone receptors in the human placenta. We provide evidence for the expression of progesterone receptor mRNA in the human placenta by reverse transcription coupled to polymerase chain reaction (RT‐PCR). This was further confirmed by Southern hybridization, restriction analysis and sequencing of the PCR amplified fragment.
A comparison of the hydroxyl radical scavenging properties of the shark bile steroid 5β‐scymnol and plant pycnogenols
Authors: Macrides Theodore A. , Shihata Amal , Kalafatis Nicolette , Wright Paul F. A.
Published: July 2007

The hydroxyl radical (OH·) quenching abilities of the following compounds were compared in the deoxyribose degradation system (initiated by the ferrous‐ascorbate Fenton reaction): (a) 5β‐scymnol, the hepatoprotective shark bile sterol, and its mono‐ and di‐sulfate esters; (b) three marketed pycnogenol preparations (syn: proanthocyanidin ‐ natural plant‐derived polyphenolic bioflavonoids) extracted from pine tree (Pinus maritima) bark and grape (Vitis vinifera) seeds; and (c) two known hydroxyl radical scavengers, dimethyl sulfoxide and mannitol, and the peroxyl radical scavenger TroloxTM (the α‐tocopherol analogue). 5β‐scymnol was a more potent OH· quencher than dimethyl sulfoxide, mannitol and Trolox, and markedly more potent than the pycnogenol preparations. Increased sulfation of 5β‐scymnol progressively reduced its free radical scavenging activity, thus clearly attributing the potent OH· quenching properties to its novel tri‐alcohol‐substituted aliphatic side chain. The favourable interaction of these bile steroids with reactive oxygen species in an aqueous environment, makes them attractive candidates for evaluation as protective agents against disorders in which oxidative stress is implicated.
Developmental study of hepatic glutamine synthetase in a mouse model of congenital hyperammonemia
Authors: Skarpetas Andrew , Mawal Yogesh , Qureshi Ijaz A.
Published: July 2007

The development of hepatic glutamine synthetase (GS; EC 6.3.1.2) activity and expression was studied in 1 to 112 day old sparse‐fur (spf) mutant mice, with X‐linked ornithine transcarbamylase (OTC, EC 2.1.3.3.) deficiency. The spf/Y mutant mice were found to have a smaller body weight (p<0.01) yet possessed a larger liver (p<0.01‐0.05) in comparison to normal male mice (+/Y). The neonatal hepatic GS activity was retarded in the spf/Y mice (p<0.01) but reached normal values by the 28th day of age, after which it increased as compared to the control CD‐I mice (p<0.01). The spf GS activity remained constant from 28 to 56 days, whereas the CD‐I GS activity decreased. A further significant increase in the spf GS activity was observed from 56 day to 112 day indicating its adaptation. The decrease of GS mRNA in the spflY mice from 28 to 112 days of age (3.72±0.25 vs 1.68±0.32, p<0.01) suggests translational and post‐translational modifications in the regulation of GS activity. The changes in the activity and expression patterns of GS could be due to an effect of the OTC mutation on the hepatic ammonia metabolism. This may be indicative of the adaptational processes in the spf mutant mice, which may play a specific role in this animal model to help it to survive with its hyperammonemia.
cAMP‐dependent phosphorylation activates phosphofructokinase from mantle tissue of the mollusc Mytilus galloprovincialis. Identification of the phosphorylated site
Authors: Fernández Montserrat , Cao Jesús , Vega Félix V. , Hellman Ulf , Wernstedt Christer , Villamarín J. Antonio
Published: July 2007

Phosphofructokinase from mantle tissue of the sea mussel Mytilus galloprovincialis was phosphorylated in vitro by a protein kinase isolated from the same tissue, homologous to mammalian cAMP‐dependent protein kinase; the maximal level of phosphorylation achieved was around 1 mol of Pi/mol of phosphofructokinase subunit. The covalent incorporation of phosphate leads to a notable increase in the enzyme activity assayed at near‐physiological concentrations of substrates and allosteric modulators and neutral pH. Tryptic digestion of labeled phosphofructokinase released a phosphopeptide whose sequence was Lys‐Asp‐Ser(P)‐Ile‐Trp‐Ile‐Gln‐Thr‐Gly‐Arg. This sequence showed high homology with the phosphopeptides from other invertebrates whose phosphofructokinase is also activated by cAMP‐dependent phosphorylation.
The nitric oxide donor S‐nitroso‐N‐acetyl‐D,L‐penicillamine degrades heparan sulfate and heparin
Authors: Ghael Dineshchandra , Mileva Mayia , Dweck Harry S. , Rosenfeld Louis
Published: July 2007

Nitric oxide (NO) is a powerful vascular and neural regulator. One of the breakdown products of nitric oxide is nitrite which converts to nitrous acid, a reagent routinely used for the degradation of heparin and heparan sulfate. We have recently shown that nitric oxide gas degrades heparin and heparan sulfate through a nitrous acid mechanism (Vilar et al, 1997, Biochemical Journal, 324, 473‐479). The purpose of the present study is to confirm these findings using the nitric oxide donor S‐nitroso‐N‐acetyl‐D,L‐penicillamine (SNAP) under conditions that are close to those found in vivo. The results show that 2 mM SNAP releases a steady‐state level of nitrite of over 200 μM. This level substantially degrades heparin and heparan sulfate at a pH of up to 5.0. This reaction may be important in breakdown of the glycosaminoglycan components of the extracellular matrix during normal and pathological conditions.
Iron as a possible mediator of the oxic‐to‐anoxic transition in Paracoccus denitrificans
Authors: Kucera Igor , Mat'chová Ivana
Published: July 2007

Expression of the lacZ gene from the Fnr‐regulated FF‐melR promoter on a plasmid in iron‐deprived Paracoccus denitrificans cells required not only a decreased oxygen tension but also supplementation with iron. The levels of β‐galactosidase and 5‐aminolevulinate synthase showed comparable responses to changes in iron availability. The presence of soluble and particulate enzymes catalyzing the reduction of Fe(III) by NADH suggests a hypothesis in which the redox state of the cytoplasmic NAD‐couple determines the concentration of free Fe(II) and thereby modulates the activity of a common regulator of the Fnr type.
cDNA cloning and genomic organization of enhancer of split groucho gene from nematode Caenorhabditis elegans
Authors: Sharief Farida S. , Tsoi Stephen C. ‐M. , Li Steven S. ‐L.
Published: July 2007

This first genomic Enhancer of split groucho (ESG) gene and its full length complementary DNA (cDNA) from nematode C. elegans were cloned and sequenced via homology with the corresponding Drosophila groucho cDNA. The cDNA of 2.1‐Kb encodes a protein of 612 amino acids, and the nematode ESG protein is the smallest and most different in structure compared to all ESG related proteins. The gene isolated is 4,246‐bp in size, including 1,219‐bp promoter region. A putative TATA‐box at position ‐1166, two consensus sequence of ACTGG, characteristic of leader binding protein‐1 (LBP‐1) binding motifs at position ‐563 and ‐211 and nine CAAT boxes were found in the promoter region of ESG gene. The protein‐coding sequence is interrupted by five introns. The length of introns 1 to 5 is 52, 252, 87, 53 and 518 bp, respectively. The overall structural relationships of the ESG‐related proteins among human, mouse, rat, Xenopus, Drosophila and nematode were also analyzed.
Binding characteristics of sle anti‐DNA autoantibodies to modified DNA analogue
Authors: Arjumand Sadia , Ali Asif
Published: July 2007

Native calf thymus DNA was modified with furoconmarins and reactive oxygen species (ROS). The modifications were probed by UV & Fluorescence spectroscopy, thermal denaturation and hydroxyapatite chromatography. In CD spectroscopy changes in the ellipticity of the DNA molecule were observed as a result of modification. The binding specificity of naturally occurring anti‐DNA antibodies with modified DNA molecules was assessed by ELISA and quantitative precipitin titration. The affinity ofanti‐DNA antibodies for modified conformers was found to be quite high.
Expression of chloroplastic genes during autumnal senescence in a deciduous tree Populus deltiodes
Authors: Reddy M.S. Srinivasa , Trivedi Prabodh K. , Tuli Rakesh , Sane Prafullachandra V.
Published: July 2007

In Populus deltoides, a deciduous tree, the development on new leaves starts in the month of March, the leaves reach maturity by October and fall by December. Changes in the composition and function of the photosynthetic apparatus were analysed during autumnal senescence. With the progress of senescence, there was an initial increase followed by a decrease in the steady state levels of psbA, psbD/C and psaA/B gene transcripts. Decrease in the steady state level of D1 protein was faster than that of Cytochrome f. The decline in LHCP level was seen only during late senescence. Although the leaves continue to look green and healthy till late November, the electron transport driven by individual photosystems started declining by October end suggesting the onset of senescence.
Cloning and functional analysis of rat cyclin D2 promoter: multiple prolactin‐responsive elements
Authors: Yang Miao , Hosokawa Yoshitaka , Hu Ying , Kaneko Satoshi , Kaneko Hiroshi , Tanaka Minoru , Nakashima Kunio
Published: July 2007

Structure and function of the promoter region of rat cyclin D2 gene were investigated by cloning, sequence analysis and DNA mobility shift assay using nuclear extracts of prolactin‐stimulated Nb2 cells. The results show that the expression of rat cyclin D2 gene is not dependent on the TATA‐box but is stimulated by multiple transcription factors induced by prolactin at an immediate‐early stage of the cell cycle.
Stimulation of cytosolic phospholipase A2‐catalyzed arachidonic acid liberation by low dose tert‐butyl hydroperoxide without an influence on the enzyme activity in rabbit platelets
Authors: Akiba Satoshi , Nagatomo Ryo , Hayama Misako , Sato Takashi
Published: July 2007

The effect of lipid peroxide on the hydrolytic action of cytosolic phospholipase A2 (cPLA2) in rabbit platelets was investigated. Ionomycin‐stimulated arachidonic acid liberation and lysophosphatidylcholine formation were significantly potentiated when platelets were pretreated with tert‐butyl hydroperoxide (BHP) and FeSO4, and then washed. Under the conditions, oxidizing reagents did not enhance the increase in cPLA2 activity by ionomycin or the basal activity in unslimulated cells. Furthermore, the treatment of a platelet lysate with BHP and FeSO4 did not affect Ca2+‐induced translocation of cPLA2 to the membranes. However, with a membrane fraction, arachidonic acid liberation catalyzed by the partially purified cPLA2 was synergistically enhanced by BHP and FeSO4. These results suggest that oxidative stress may potentiate the hydrolytic action of cPLA2 on membrane phospholipids without an influence on the processes leading to the enzyme activation.
Cloning and characterization of a rhamnogalacturonan hydrolase gene from Botrytis cinerea
Authors: Chen Huey‐Jen , Smith David L. , Starrett David A. , Zhou Dingbo , Tucker Mark L. , Solomos Theophanes , Gross Kenneth C.
Published: July 2007

Rhamnogalacturonan hydrolase (Rgase A) cleaves α1→2 linkages between rhamnosyl and galacturonosyl residues in pectin. A 1.9 kb RGase A cDNA clone (BCRHGA) was isolated from a B. cinerea cDNA library using a PCR‐amplified Aspergillus aculeatus RGase A probe. It's 1.7 kb open reading frame had 62% identity at the amino acid level with A. aculeatus RGase A. Northern blots of B. cinerea total RNA probed with BCRHGA revealed a 2 kb band, suggesting the cDNA clone is full or nearly‐full length. To determine mRNA expression of the gene, B. cinerea was grown in media containing 0.5% apple pectin, 0.5% rhamnogalacturonan‐I and 1% glucose carbon sources. Northern analysis revealed the BCRHGA gene was expressed on all carbon sources, but with different patterns of expression. B. cinerea RGase A appeared to be coded for by a single or low copy number gene based on Southern analysis.
Nitrogen starvation in a Saccharomyces cerevisiae strain deleted in the trehalose‐6‐phosphate synthase complex
Authors: Fernandes Patricia M. B. , Trugo Luiz C. , Panek Anita D.
Published: July 2007

Little is known about the mechanisms by which nutrient limitation leads to G1 arrest in Saccharomyces cerevisiae. We have shown that mutant cells deleted in the trehalose‐6‐phosphate synthase gene able to grow on glucose, when starved for nitrogen, did not arrest in the G1 phase of the cell cycle. We attribute this effect to an increase in the activity of cAMP‐dependent protein kinase. When grown on maltose without nitrogen they arrested properly in G1. Tests with a mutant cell deleted in the specific trehalose phosphatase proved that the presence of the trehalose precursor, trehalose‐6‐phosphate, was sufficient to trigger the negative sign during nitrogen starvation, leading the cells to arrest in the G1 phase of the cell cycle.
Effect of detergent treatment on the cross‐linking of purified chromaffin granule membranes with F‐actin
Authors: Morita Kyoji , Pollard Harvey B.
Published: July 2007

Chromaffin granule‐microfilament interaction has previously been proposed to play a potential role in the regulation of dopamine uptake into the granules as well as catecholamine secretion in the adrenal medullary cell. However, the microfilament‐binding site on the granule surface has not yet been identified. To establish the conditions for solubilization of the binding site, the effects of different‐type detergents on the cross‐linking of chromaffin granule membranes with F‐actin were examined. The cross‐linking activity was abolished by treatment of the granule membranes with deoxycholate (DOC), but not Triton X‐100 (TX100) and CHAPS. The addition of DOC‐extract decreased the viscosity of F‐actin solution, suggesting that solubilized proteins bind to F‐actin, but not cause the cross‐linking of actin filaments. These results indicate that the actin‐binding site can be solubilized from chromaffin granule membranes by DOC treatment.
Regional heterogeneity of plasma membrane proteins in rat brain
Authors: Babu Phanithi Prakasa
Published: July 2007

Plasma membranes separated and purified from rat cerebral cortex and cerebellum showed varying protein profiles on SDS‐PAGE scanning densitometry. Using highly sensitive two‐dimensional gel electrophoresis these proteins were further resolved and an apparent regional heterogeneity of plasma membrane associated proteins was demonstrated. These proteins might be involved in region specific structural and functional aspects in the brain.
Effect of n‐3 fatty acids on VLDL production by hepatocytes is mediated through prostaglandins
Authors: Anil K. , Jayadeep A. , Sudhakaran P. R.
Published: July 2007

The mechanism of the hypolipidemic effect of n‐3 fatty acids was studied using isolated rat hepatocytes maintained in culture. EPA and DHA caused a significant reduction in the incorporation of 3[H]‐leucine into apoB associated with the VLDL produced by hepatocytes in culture when compared to that in presence of palmitic acid. Presence of indomethacin, an inhibitor of cyclo‐oxygenase reversed the effect of EPA on VLDL synthesis while diethyl carbamazine an inhibitor of lipoxygenase did not show any effect suggesting that the effect of EPA may be mediated through prostaglandins. This was further tested by invivo experiments where animals were fed fish oil containing diet with and without aspirin, which inhibits formation of prostaglandins. The incorporation of 3[H] ‐leucine into apo B and 14[C]‐ acetate into cholesterol of VLDL produced by heypatocytes from aspirin treated animals were significantly high. The reversal of the effect of n‐3 fatty acids by agents which inhibit the formation of prostaglandin suggests that the n‐3 fatty acids may exert their effect on VLDL production by liver cells through prostaglandins.
The effects of a selection of alkaloids on the invertase activity of some higher plants
Authors: Rojo Hebe P. , Quiroga Emma N. , Vattuone Marta A. , Sampietro Antonio R.
Published: July 2007

The effects of several alkaloids on the invertase activities of Ricinus communis, Solanum tuberosum, Oryza sativa and Carica papaya were studied. The following alkaloids: caffeine, berberine, strychnine, morphine, ethyl‐narceine and nomicotine, have different inhibitory effects on invertase activity. Kinetic studies of the alkaloid inhibition revealed that different alkaloids exhibited diverse types of inhibition of invertase. It was found that alkaloids form reversible complexes with invertases. However cocaine and potato glycoalkaloids were activators of some invertase. The sugar moiety of potato glycoalkaloids were also activators. It is concluded that alkaloids may be effectors of higher plant invertase activity.
Aziridinyl steroid‐induced lesions in DNA and apoptosis in promyelocytic leukemia cells
Authors: Quadri Shafat A. , Qadri Ariful N. , Ahmad Masood , Islam Shabana
Published: July 2007

Variety of synthetic steroids are reported to be mutagenic as well as carcinogenic. The mutagenic and carcinogenic nature of these compounds have been related to their potential of being reactive to genetic material and production of reactive oxygen species. Here we have analyzed the action of aziridinyl steroid on calf thymus DNA and human promyelocytic leukemia cell line HL‐60 under in vitro conditions. Calf thymus DNA when treated with various doses of aziridinyl steroid induced a high degree of strand separation and sensitivity/susceptibility to S1 nuclease hydrolysis. The treatment also induced an increasing number of strand breaks per molecule of DNA as determined by alkaline unwinding assay. Relatively higher doses of steroid, however, displayed a reduced susceptibility to S1 nuclease hydrolysis and did not increase the number of strand breaks in DNA. Moreover, the high dose treatments result increased melting temperature and an enhanced rate of reanealing after thermal denaturation, indicating that interstrand crosslinks are induced at higher doses of steroid treatment. Moreover, steroid treatment caused cell death in human promyelocytic leukemia cell line HL‐60 and induced DNA degradation, characteristic of apoptosis. The test steroid has the ability to produce reactive oxygen intermediates (ROI) as determined by chemical methods. Incorporation of oxygen radical scavengers into the system blocked the damaging effect of steroid in calf thymus DNA and HL‐60 cells. These observations strongly suggest that aziridinyl steroid, a pharmaceutical, damages mammalian DNA and induces apoptosis by the production of ROI in the test system.
Epitope analysis of monoclonal antibodies to human Gc globulin (vitamin D‐binding protein)
Authors: Osawa M. , Kimura A. , Yukawa N. , Seto Y. , Saito T. , Tsuji T. , Takeichi S.
Published: July 2007

Gc globulin, also called vitamin D‐binding protein, is human 56 kD plasma protein. Antigenic epitopes of the protein recognized by 12 different monoclonal antibodies (MoAbs), including the previously prepared MoAb E12 of which binding to Gc is inhibited by actin, were analyzed in relation to the functional domains of the protein. To map the epitopes recognized by the respective antibody, the reactivities of the MoAbs were tested by immunoblotting against the recombinant Gc fragments expressed in E. coli cells, and then a competitive ELISA was performed. The results showed that the antibodies were classified into at least six groups. Furthermore, in addition to E12, actin inhibited the reactivity of MoAb 21, of which the epitope was located within residues Asp320 to Glu379, in an ELISA in the presence of the ligand. This result must indicate that this antibody‐binding site is near the site that interacts with actin. In contrast, no inhibitory effect by 25‐hydroxyvitamin D to any antibodies was observed. Furthermore, all antibodies, including E12 and 21, reacted with membrane‐bound Gc of lymphocytes by an immunofluorescence study, suggesting that Gc is unlikely to bind to the plasma membrane in its interaction with actin.
Isolation of thermostable phosphatase from the hyperthermophilic archaeon Thermococcus pacificus by immobilized metal affinity chromatography
Authors: Bannikova Galina E. , Varlamov Valery P. , Miroshnichenko Margarita L. , Bonch‐Osmolovskaya Elizaveta A.
Published: July 2007

Phosphatase was isolated from cells of the hyperthermophilic marine archaeon Thermococcus pacificus by a procedure including chromatography on Butyl‐Fractogel TSK‐650 and Ni2+‐iminodiacetic‐agarose. Enzyme activity is maximal at 90°C, and the enzyme half‐life time at this temperature is 1 h. The pH optimum of phosphatase activity is 6.0. Electrophoresis under denaturating conditions yielded a subunit molecular weight of 45 kDa. On gel‐filtration on Sephacryl S‐300 HR three peak corresponding to 295, 85 and 45 kDa were observed, suggesting that the enzyme is a homohexamer.
Efects of deleting A19 tyrosine form insulin
Authors: Du Xin , Tang Jian‐Guo
Published: July 2007

A mutant proinsulin gene was constructed through PCR mediated mutagenesis. The code of A19Tyr was deleted. The mutant proinsulin, (ΔY)19‐lys‐proinsulin [(ΔY)19KPI], was expressed in E. coli and purified. After treatment with trypsin and carboxypeptidase B, and ResourceTM Q separation, (ΔY)19‐human insulin [(ΔY)19HI] was obtained. It retains 63.6% of receptor binding activity but only 2.2% of immune activity, and shows a longer retaining time on reverse‐phase FPLC and a slower mobility by native PAGE analysis. These results suggest that the deletion of A19Tyr causes some conformational changes on insulin, which plays a minor role on the affinity of insulin to its receptor, and a major role on immunegenicity of the hormone.
Molecular cloning of a novel chemokine receptor‐like gene from early stage chick embryos
Authors: Gupta Shalley K. , Pillarisetti Kodandaram , Gray Samantha L. , Stadel Jeffrey M.
Published: July 2007

Proliferation, differentiation and regulated trafficking of cells are the hallmarks of development and embryogenesis. This led us to speculate a role for chemokines and their receptors in this process. Here, we report the molecular cloning of AvCRL1, a novel member of the G‐protein coupled receptor family from early stage 3 days old chick embryos. While the function and ligand for this receptor remain unknown, its sequence and gene structure indicates that it is most related to the family of chemokine receptors, with highest homology to the virally induced human BLR‐1 and the CXCR3 or γIP‐10/Mig‐1 receptors.
PP7, a plant phosphatase representing a novel evolutionary branch of eukaryotic protein Ser/Thr phosphatases
Authors: Andreeva Alexandra V. , Evans David E. , Hawes Chris R. , Bennett Nelly , Kutuzov Mikhail A.
Published: July 2007

We describe a novel protein Ser/Thr phosphatase from Arabidopsis thaliana, PP7, which is only 27‐32% identical in amino acid sequence to the known phosphatases and is the most divergent member of the PPP (PP1/2A/2B) family for today. Some structural features suggest more close relationship of PP7 to the PP5/rdgC subfamily. PP7 contains all of the residues essential for the phosphatase activity and possesses three major insertions in its presumable C‐terminal subdomain, which suggest its unique regulation and/or optimisation of its structure for interaction with specific substrates or regulators. A phosphatase structurally related to PP7 is expressed in rice. PP7 conservation between mono‐ and dicotyledonous plants may point to its essential role in the plant cell.
Effect of lipids on the conformation of an antigenic determinant for experimental autoimmune neuritis
Authors: Shin Hang‐Cheol
Published: July 2007

The conformation of SP‐26, the synthetic peptide (residues 53‐78) of myelin P2 protein that causes experimental autoimmune neuritis (EAN) in the peripheral nervous system, has been studied in lipid environments and in water/trifluoroethanol (TFE) mixture using circular dichroism (CD) spectroscopy and Fourier transform infra‐red (FTIR) spectroscopy. SP‐26 becomes more structured on binding with micelles formed from anionic lipids and in the presence of TFE. On the other hand, zwitterionic lipids have a denaturing effect or no effect on the conformation of SP‐26. The present results suggest that the disease‐modulating effects of various lipids are closely related to their ability to induce conformational change in the antigenic region of P2 protein.
Purification and characterization of CD38/ADP‐ribosyl cyclase from rat lung
Authors: Khoo Keng Meng , Chang Chan Fong
Published: July 2007

Purification and characterization of CD38/ADP‐ribosyl cyctase in the rat lung tissue was performed with microsomes solubilized in Triton X‐100 and the ADP‐ribosyl cyclase was then purified using sequential column chromatography. Partially purified rat lung ADP‐ribosyl cyclase was analyzed by immunoblotting using an antibody raised against a recombinant rat CD38 and showed the presence of monomer (42 kDa) and dimer (85 kDa) under non‐reducing conditions but under reducing conditions, only the monomer was detected. Both the monomer and dimer could be eluted out in a stable manner from SDS‐PAGE and the enzymatic activity was retained by the two different forms of CD38/ADP‐ribosyl cyclase. Immunohistochemical staining showed the presence of CD38 on the bronchial epithelium and the alveoli.
Involvement of available SH groups in the heterogeneity of hemoglobin from the tortoise Geochelone carbonaria
Authors: Torsoni M. A. , Souza‐Torsoni A. , Ogo S. H.
Published: July 2007

Geochelone carbonaria hemoglobin (Hb) was analyzed by polyacrylamide gel electrophoresis (PAGE) and purified by ion exchange chromatography on CM‐cellulose. Seven fractions were obtained using fresh Hb preparations. CM‐cellulose chromatography of Hb reacted with iodoacetamide, showed one minor (HbI) and one major band (HbII). Analysis of the molecular masses of recently collected Hb and of aged solutions determined by gel filtration showed that polymerization increased with the duration of storage. The reaction with oxidized glutathione changed the electrophoretic pattern of Hb, and highlighted the bands corresponding to glutathionyl‐Hb. The presence of these bands in fresh Hb solutions and in alkylated preparations suggests that they may occur in vivo. PAGE under dissociating conditions showed that the hemolysate contained 3 different polypeptide chains (G1, G2 and G3). Both Hb components shared the G1 globin chain with HbI containing G1 and G2 and HbII, G1 and G3 chains.
Purification of swine serum angiotensin converting enzyme with high recovery of activity using lisinopril coupled to epoxy‐activated sepharose 6B
Authors: Quassinti Luana , Miano Antonino , Bramucci Massimo , Maccari Ennio , Amici Domenico
Published: July 2007

The Authors describe the purification of swine serum ACE to molecular homogeneity with high recovery of activity (40 %) in a few stepsl. The purification procedure consists of affinity chromatography, using commercial activated resin (epoxy‐activated sepharose 6B) and two steps of anion exchange chromatography (Resource Q) performed at different pH (pH 9.0 and pH 6.0). Furthermore, a specific and sensitive method for the accurate quantitation of ACE activity in biological fluids was developed, based on the hydrolysis of synthetic FAPGG (N‐[3‐(2‐furyl) acryloyl] L‐phenylalanyl glycyl glycine), as substrate and following the separation of prodcts by reversed‐phase HPLC. Some kinetic parameters were determined. The Km and Kcat values for FAPGG were 0.793±0.052 mM and 5830 s‐1, respectively, and the I50 values for captopril and lisinopril, two specific ACE inhibitors, are 5.7±0.67 nM and 1.0±0.29 nM, respectively.
DNA gyrase from Streptomyces noursei and comparison with gyrases from other sources: Gyr B subunit may affect the pH‐dependence of the enzymic activity
Authors: Simon Hannelore , Zimmer Christoph
Published: July 2007

Purified subunits of DNA gyrase from Streptomyces noursei have been obtained with relatively high yield. By comparing the activity of the reconstituted enzyme (supercoiling and cleavage reaction) with other gyrase species and hybrid enzymes it is shown that the reactivity of gyrases in vitro may differ significantly with pH, an effect not previously reported. The pH‐effects on gyrase and hybrid enzyme activity seems to be largely determined by the Gyr B protein.
Steady state levels of D1 protein and psbA transcript during UV‐B inactivation of photosystem II in wheat
Authors: Chaturvedi R. , Shyam R. , Sane P. V.
Published: July 2007

UV‐B induced alterations in the pigment composition and photosystem II (PS II) electron transport activity was studied in laboratory grown seedlings of wheat cv. RR21. Subsequent to UV‐B exposure, the chlorophyll, carotenoids, anthocyanin and flavonoids, showed an increase in their levels. A time dependent decrease in the photosystem II electron transport activity was evident in the UV‐B treated plants. Reduction in the steady state levels of transcript of the psbA gene and its translation product the D1 protein was concurrent with the loss of PS II activity in the wheat plants exposed to UV‐B.
Surface hydrophobicity and thermal aggregation of adenylate kinase
Authors: Yan‐Ling Zhang , Xian‐Ming Pan , Jun‐Mei Zhou
Published: July 2007

The fluorescence probe, 8‐anilinonaphthalene‐1 sulfonate (ANS), is used to study the thermal denaturation of adenylate kinase (AK). Native AK has one ANS binding site. With increasing temperature, the ANS binding fluorescence intensity increases, showing that the enzyme undergoes a thermally induced conformational change that causes the exposure of the hydrophobic surfaces. With the exposure of the hydrophobic surfaces, the partially denatured enzyme proceeds to aggregate. This result suggests that the formation of the aggregates may be correlated with increases in the surface hydrophobicity of the intermediates. The thermally unfolded AK become resistant to aggregation in the presence of ammonium sulfate or ethylene glycol. These observations are discussed in terms of a model where the ethylene glycol enhances the hydrophobicity of the solvent and increases the solubility of the intermediates.
Inhibition of epidermal growth factor‐induced stat1 activation by lysophosphatidic acid is mediated by pertussis toxin‐sensitive g proteins and tyrosine phosphatase in human epidermoid carcinoma A431 cells
Authors: Suzuki Yasuyuki , Ozawa Yasuhiro , Murakami Kazuo , Miyazaki Hitoshi
Published: July 2007

We previously reported that pretreatment of A431 cells with G protein‐coupled receptor agonists which activate phospholipase C, such as lysophosphatidic acid (LPA), inhibit epidermal growth factor (EGF)‐induced formation of Stat1‐DNA complex (SIF‐C). In this study, we examined the mechanism for this phenomenon. LPA inhibited EGF‐induced but not interferon‐γ‐induced SIF‐C formation. Ligand‐dependent tyrosine phosphorylation of the EGF receptor was markedly reduced by LPA, suggesting that the reduction of the receptor phosphorylation is probably responsible for LPA‐induced inhibition of SIF‐C formation. The LPA action was prevented by pertussis toxin and the tyrosine phosphatase inhibitor vanadate, but not by the phospholipase C inhibitor U‐73122. These results demonstrate that the inhibitory effect of LPA on EGF‐induced SIF‐C formation is mediated by Gi and/or Go proteins and tyrosine phosphatase, but not by Gq proteins coupling with a phospholipase C pathway.
Involvement of p21 in mitomycin C‐induced G2 arrest in LP1‐1 cells
Authors: Kang Kyung Hwa , Oh Won Jong , Kim Won Ho , Choi Kyung Hee
Published: July 2007

p21, a protein kinase inhibitor, was known to play a major role in the induction of G1 arrest in response to DNA damaging agent, but its role in G2/M transition was poorly understood. In this study, we investigated the involvement of p21 in mitomycin C‐induced G2 arrest in LP1‐1 cells. Treatment of mitomycin C at early G2 phase of LP1‐1 cells was found to induce G2 arrest and inhibit cdc2 kinase activity. However, expression of cyclin B and cdc2 protein were not affected by mitomycin C and an alteration of tyrosine dephosphorylation of cdc2 kinase could not be observed in the cells. On the other hand, p21 mRNA and protein were induced by mitomycin C. The binding of p21 protein to cyelin B‐cdc2 complex was increased and it was accompanied with the inactivation of cdc2 kinase after treatment of mitomycin C. These results suggest that mitomycin C‐induced G2 arrest may be mediated via increment of p21 level, elevation of binding ability with cyclin B‐cdc2 complex, and a subsequent reduction of cdc2 kinase activity.
Differential expression of mRNAS for M31 and M32, murine homologues of Drosophila heterochromatin protein 1 (HP1), during murine embryogenesis
Authors: Miyado Kenji , Sato Masahiro , Kimura Minoru
Published: July 2007

Mice have two structural homologues of the Drosophila HP1 gene, termed M31 and M32, which have two structurally conserved domains, the chromo (C) and chromo shadow (CS) domains. In Drosophila, HP1 has been isolated as one of the components of conserved chromatin structure (heterochromatin) and is thought to bind DNA indirectly by mediating formation of a complex of nuclear proteins. However, little is known about the function of M31 and M32 in mammals. In order to assess the function of the M31 and M32 proteins, Northern blot analysis was performed in mice. M32 mRNA (with approximately 1,800 nucleotides (ntds)) was expressed strongly throughout embryonic stages examined and in TT2 embryonic stem (ES) cells, while expression of M31 mRNA (with approximately 2,300 and 1,100 ntds) was strong in embryos at embryonic day 8.5 (E8.5) and in TT2 cells, but was decreased at E12.5 and thereafter. In adults, expression of M32 mRNA was strongest in brain and brain stem among tissues examined, and was relatively weak in organs including kidney, stomach, intestine and testis. The two M31 transcripts were weakly but uniformly expressed throughout the organs examined in adults. These findings suggest that M31 and M32 may play different roles in murine embryogenesis, although they have the C and CS domains and are members of the same gene family.
Vaccinia virus‐induced changes in cytokine‐regulated acute phase plasma protein synthesis by hepatoma cells
Authors: Rokita H. , Branicki W. , Wronska D. , Borysiewicz L. K. , Koj A.
Published: July 2007

Human hepatoma cell line, HepG2, has been infected with vaccinia virus and synthesis of plasma proteins was determined by etectroimmunoassay and corresponding mRNA's measured by Northem blotting. The inhibitory effect of the virus was dose‐ and time‐ dependent. Electrophoretic mobility shift assay revealed a decrease in C/EBP binding activities in nuclear extracts isolated from the infected hepatoma cells. Supershift analysis of the C/EBP isoforms showed α and β subunit involvement in DNA binding. The treatment of the cells with interleukin‐1, interleukin‐6, and dexamethasone at the initital stage of infection appears to delay the virally induced inhibition of host cell protein synthesis. Thus, possible “protective” role of the acute phase cytokines in viral infection is proposed.
Resistance of the insulinotropic action of α‐D‐glucose and β‐L‐glucose pentaacetates to cholera and pertussis toxins
Authors: Malaisse Willy J. , Ladriere Laurence
Published: July 2007

The pentaacetate esters of α‐D‐glucose and β‐L‐glucose were recently reported to stimulate insulin release. The possible participation of G‐protein‐coupled receptors to the insulinotropic action of these esters was investigated in rat pancreatic islets either preincubated with cholera toxin or obtained from animals injected with pertussis toxin. Neither procedure affected adversely the secretory response to the esters in islets incubated in the presence of L‐leucine. Thus, in both situations, α‐D‐glucose pentaacetate and, to a lesser extent, β‐L‐glucose pentaacetate augmented insulin release evoked by the branched‐chain amino acid, whilst β‐L‐galactose pentaacetate failed to do so. These findings suggest that G‐proteins sensitive to either cholera or pertussis toxins are not involved in one of the two modalities by which these esters are thought to stimulate insulin secretion, namely that independent of the catabolic fate of their hexose moieties.
Signal transduction by the peptide which mimics the activity of thrombopoietin
Authors: Kimura Tatsuya , Kaburaki Hiroshi , Tsujino Tomomi , Watanabe Yoshinari , Kato Hideo
Published: July 2007

Thrombopoietin (TPO) plays a central role in megakaryopoiesis and platelet production. It is a ligand for c‐mpl, which is a member of the hematopoietic receptor superfamily. We have recently identified several human c‐mpl binding peptides which are distinct from TPO, from phage random peptide libraries. PK1M is one of these peptides and is an agonist of c‐mpl which is TPO receptor. We show here that PK1M induces the tyrosine phosphorylation of the Janus kinase 2 (JAK2) and the activation of the signal transducer and activation of transcription 5 (STAT5) in TPO‐dependent cells like TPO.
Branched‐chain α‐keto acid dehydrogenase kinase content in rat skeletal muscle is decreased by endurance training
Authors: Fujii Hisao , Shimomura Yoshiharu , Murakami Taro , Nakai Naoya , Sato Tasuku , Suzuki Masashige , Harris Robert A.
Published: July 2007

The activity state of branched‐chain α‐keto acid dehydrogenase complex in skeletal muscle was elevated by running exercise in trained and untrained rats, but level of this elevation was significantly greater in the former than in the latter. To elucidate the mechanism of the training effect on the exercise‐induced activation of the complex, a protein amount of branched‐chain α‐keto acid dehydrogenase kinase, which is responsible for inactivation of the complex by phosphorylation, in the muscle was measured by the Western blot analysis. Endurance training decreased the content of the kinase protein in the muscle by ∼30%, suggesting that this decrease is involved in the mechanisms for greater activation of the complex by exercise in trained rats.
A method for uni‐directional reconstitution of human erythrocyte glucose transporter
Authors: Xiao L. , Xie W. S. , Yang F. Y.
Published: July 2007

The glucose transporter of human erythrocytes (Glutl) was reconstituted into soybean phospholipid liposomes by a method of direct incorporation using the nonionic detergent n‐octyl β‐D‐glucopyranoside. The reconstituted proteoliposomes were proved to be intact and low ionic permeability. Freeze‐fracture electron microscopy study showed that the diameter of the proteoliposomes was about 150±50 nm and the protein was randomly distributed. The kinetic parameters of the reconstituted transporter were: Km=16.23 mmol/L, Vmax=34.48 nmol/sec.mg protein. Furthermore, about 90% of the glucose transporter in the reconstituted proteoliposomes were orientated inside‐out. Until now it is a more efficient method for uni‐directional reconstitution of Glut 1 with good reproducibility and higher transport activity.
Attenuated antigenicity of ribonucleoproteins modified by reactive oxygen species
Authors: Waris Ghulam , Alam Khurshid
Published: July 2007

Ribonucleoprotein (RNP) particles isolated from fresh goat fiver nuclei were exposed to hydroxyl radical which induced modification in the gross structure of RNP particles. To evaluate the effect of hydroxyl modification on the antigenic properties of RNP and possible role of ROS‐RNP in the initiation and development of SLE, enzyme immunoassays were carried out. SLE sera having high titre anti‐DNA antibodies showed enhanced binding to hydroxyl modified RNP particles in comparison to unmodified RNP particles. In competition assay none of the SLE sera or isolated IgG showed preference for ROS‐modified RNP particles over native RNP particles. These studies suggest that anti‐RNP autoantibodies observed in subpopulation of SLE patients are generated by some other intra‐or extracellular mechanisms and hydroxyl radical has probably no direct role in the initiation of antibodies.
Hormone‐induced changes in cardiolipin from leydog cells: Possible involvement in intramitochondrial cholesterol translocation
Authors: Gasnier Françoise , Rey Catherine , Graverand Marie‐Pierre Hellio Le , Louisot Pierre , Benahmed Mohamed
Published: July 2007

The rate‐limiting and hormonally regulated step in steroid hormone biosynthesis is the delivery of cholesterol from the outer to the inner mitochondrial membrane where cytochrome P450scc resides. Although the exact mechanism of intramitochondrial cholesterol translocation remains unknown, the formation of contact sites between outer and inner mitochondrial membranes appears as a necessary component for cholesterol transfer. Several pieces of evidence suggest that local formation of intermembrane contact is a consequence of a non‐bilayer arrangement of polymorphic lipids which are enriched in the junctions. As a step toward clarifying mitochondrial contact sites formation and thus cholesterol translocation in steroidogenic cells, we have undertaken studies to identify the factors which might result in non‐bilayer structure to be adopted by mitochondrial phospholipids on stimulation of MA‐10 Leydig cells. Our results demonstrate that an increase in the unsaturation of the cardiolipin acyl groups on hormonal stimulation might favor the formation of non‐bilayer adhesion points.
Methionine modification impairs the C5‐cleavage function of cobra venom factor‐dependent C3/C5 convertase
Authors: Fu Qinglan , Gowda D. Channe , McPhie Peter
Published: July 2007

The complement‐mediated lysis of guinea pig erythrocytes by cobra venom factor (CVF) decreased by 50‐60% within 2 min of treatment with 5 mM sodium periodate at 0 °C. This loss of activity paralleled modification of 3‐4 Met; other amino acids and sugar residues of the oligosaccharide chains were not affected. Treatment with N‐chlorosuccinimide or chloramine‐T under conditions that specifically modified 3‐4 readily‐oxidizable Met also caused 50‐60% loss of CVF activity. The secondary structure of CVF was not altered by these modifications. Methionine‐modified CVF (MetCVF) supported the cleavage of factor B by factor D with equal efficiency as that of untreated CVF to form C3/C5 convertase (MetCVF,Bb) of the alternative pathway. MetCVF,Bb and CVF,Bb were indistinguishable with respect to C3 cleavage. However, the C5‐cleavage ability of MetCVF,Bb was significantly lower than that of CVF,Bb. These results suggest the involvement of Met in CVF binding of C5.
The prevention of carbon tetrachloride‐induced hepatotoxicity in mice by α‐hederin: Inhibiton of cytochrome P450 2E1 expression
Authors: Jeong Hye Gwang , Park Hyeon Yong
Published: July 2007

The protective effects of α‐Hederin on carbon tetrachloride‐induced hepatotoxicities were investigated in mice. Pretreatment with α‐Hederin prior to the administration of carbon tetrachloride significantly prevented the increase in serum alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) activity and lipid peroxidation in a dose dependent manner. Hepatic glutathione levels and glutathione‐S‐transferase activities were not affected by pretreatment with α‐Hederin alone but pretreatment with α‐Hederin protects carbon tetrachloride‐induced depletion of hepatic glutathione levels. The effects of α‐Hederin on the cytochrome P450 (P450) 2E1, the major isozyme involved in carbon tetrachloride bioactivation were investigated, α‐Hederin markedly decreased the P450 2E1‐specific activities of p‐nitrophenol and aniline hydroxylation in a dose‐dependent manner. Consistent with these observations, the P450 2E1 expressions were also decreased, as determined by immunoblot analysis. These results demonstrate that treatment of mice with α‐Hederin decreases the expression and activities of P450 2E1 enzyme, and reduces biotransformation of carbon tetrachloride, and diminished carbon tetrachloride‐induced liver injury.
Cross‐species reactivity of a monoclonal antibody against glutathione S‐transferase fusion protein of human β2‐adrenergic receptor
Authors: Shin Chan Young , Kang Suk‐jo , Song Mi‐ryoung , Park Kyu Hwan , Seo Dong Ook , Cheong Jae Hoon , Ko Kwang Ho
Published: July 2007

The purpose of the present study was to produce and characterize a monoclonal antibody against human β2‐adrenergic receptor. Male BALB/c mice were immunized with glutathione S‐transferase (GST) fusion protein of the C‐terminal portion of the human β2‐adrenergic receptor which was expressed in E.Coli. The immunized splenocytes were fused with myeloma SP2/0‐Ag14 cells and the resulting monoclonal antibody was named as mAbβC02. The monoclonal antibody βC02 was determined as IgM subtype and then purified by anti‐mouse IgM‐agarose affinity chromatography. The results of ELISA, Western blot, and immunocytochemistry showed that mAbβC02 recognized human β2‐adrenergic receptor in the β2‐adrenergic receptor‐GST fusion protein and human epidermoid carcinoma cell line A431 with highly specific immunoreactivity. In addition, mAbβC02 showed cross‐species reactivity against β‐adrenergic receptor of hamster lung and rat brain as revealed by Western blot and immunohistochemistry. The monoclonal antibody βC02 may provide useful tools for the study of the β‐adrenergic receptor of human and other species including rats.