bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of an enhancer sequence in 5′‐flanking region of 1A exon of mouse liver/bone/kidney‐type alkaline phosphatase gene
Authors: Kobayashi Tatsuya , Sugimoto Toshitsugu , Kanzawa Michiko , Chihara Kazuo
Published: July 2007

The 5′‐flanking region of the 1A exon of the mouse liver/bone/kidney‐type alkaline phosphatase (L/B/K (ALP) gene was analyzed for the promoter activity. Luciferase assays revealed that a segment containing a 9 bp inverted repeat (nt‐1212 to ‐1179, designated as IRS for inverted repeat segment) had an enhancer activity. Though an E‐box at nt ‐234 did not have a prominent promoter activity, the effect of IRS required its presence. Both transcriptional activity and IRS binding to the nuclear proteins were similarly observed even in cells that did not express ALP. Bone morphogenetic protein‐2 (BMP‐2) induced the ALP expression in pluripotent C3H10T1/2 cells, but it did not increase the transcriptional activity of the promoter region. Though they do not determine cell specific or BMP‐2 inducible ALP expression, IRS and the E‐box at position ‐241 seem to be important for the basic transcriptional activity of ALP gene.
Subunit structure of lysine sensitive aspartate kinase from spinach leaves
Authors: Kochhar S. , Kochhar V. K. , Sane P. V.
Published: July 2007

The lysine‐sensitive isoenzyme of aspartate kinase was purified to homogeneity from spinach leaves and its subunit composition was studied. The purified preparation had an apparent molecular mass of 280,000 and separated into two subunits‐ a large subunit with molecular mass of 53000 and smaller subunit with molecular mass of 17,000 by urea treatment and SDS PAGE. The enzyme molecule has subunit composition of 4 large and 4 small subunits. The activity of the large subunit was stimulated more than two fold by the addition of small subunit and the stimulated activity was inhibited by EGTA. This inhibition could be reversed by Ca++. Further characteristics of the smaller subunit such as heat stability, behavior on ion exchange chromatography, elctrophoretic mobility on polyacrylamide gels, amino acid composition and pattern, presence of trimethyl lysine, its ability to activate other calmodulin stimulated enzymes and its calmodulin‐like nature in RIA tests suggested that this subunit is identical to calmodulin.
p116 transcript predicting an alternative form of sulfated glycoprotein‐2 (clusterin) is transcribed by alternative splicing mechanism
Authors: Izawa Masao
Published: July 2007

To further determine the role of androgens in the seminal vesicle physiology, we previously undertook cDNA cloning of androgen‐responsive mRNAs from rat seminal vesicles, and isolated several cDNA clones. During the process of characterization, we have isolated a distinct cDNA clone designated p116, predicting the expression of a transcript encoding an alternative form of sulfated glycoprotein‐2 (SGP‐2, clusterin). In the present study, we addressed the question of whether SGP‐2 and p116 transcripts are transcribed from an identical gene by alternative splicing mechanism or not. Results from genomic PCR and genomic Southern analyses demonstrate that p116 and SGP‐2 mRNAs are transcribed from their respective exons of an identical gene. Since the expression of SGP‐2 has been proposed as a molecular marker of apoptosis, it is important to further understand the roles of expressions of SGP‐2 and its related p 116 proteins in apoptotic cells.
Quantitative measurement of protein S gene expression by reverse transcription‐competitive PCR method; determination of mRNA level in fractionated human peripheral blood cells and various cultured cell lines
Authors: Okamoto Yoshihiro , Hara Yusuke , Ishikawa Satomi , Nishida Mikio
Published: July 2007

We developed the measurement system for human protein S mRNA (cDNA) by a reverse transcription‐competitive polymerase chain reaction (RT‐competitive PCR) method with a compact digital camera. The sensitivity of the method was 100,000 fold more sensitive than the traditional northern blot analysis. We confirmed the accuracy and usefulness of the system by measuring the absolute values of protein S mRNA in fractionated peripheral blood cells, namely platelets, mononuclear cells, granulocytes and red blood cell from healthy human and six different cultured disease cell lines tested, namely, lung carcinoma, stomach carcinoma, hepatoma, highly differentiated hepatoma, promyelocytic leukemia and monocytic leukemia. The amount of protein S mRNA in the platelet rich fraction (6.89×10‐19 mol/μg total RNA) was the largest of all fractions tested and that in the mononuclear cells was the second largest. In the fractions of granulocytes and red blood cells, no protein S mRNA was detected. The detected values in five cell lines ranged from approximately 5.6×10‐19 to 97.2×10‐19 mol/μg total RNA. The content was reduced in the order, highly differentiated hepatoma>hepatoma>lung carcinoma>stomach carcinoma>monocytic leukemia. Promyelocytic leukemia cell line had no expressed protein S mRNA. Thus, the assay system described here was sensitive, reproducible, simple and useful for the quantitative measurement of an infinitesimal level of protein S mRNA to characterize normal and/or patho‐physiological states of human blood and somatic cells.
The synthesis of HSPs in sugar beet suspension culture cells under hyperthermia exhibits differential sensitivity to calcium
Authors: Kuznetsov Vladimir V. , Andreev Igor M. , Trofimova Marina S.
Published: July 2007

Experiments aimed at testing the effect of Ca2+ on heat shock‐induced changes in protein synthesis of cultured sugar beet cells were performed. Heat shock inhibits the synthesis of non‐heat shock proteins (non‐HSPs) and promotes the synthesis of a set of HSPs. Extracellular Ca2+ appeared to be strictly required for the synthesis of non‐HSPs. Calcium was found to differentially exert its effect on the HSP synthesis: calcium induced (96 and 76 kDa), stimulated (94, 67, 58, 52, 32, 30, 26 and 22 kDa) or did not influence (82, 17 kDa) the de novo production of various HSPs. Cell injury increased if the cells were exposed to high temperature in a Ca2+‐deficient medium. Calcium supplement to Ca2+‐depleted cells resulted in the recovery of HSP synthesis and reduced cell injury by heat shock.
Pigment organization and exciton dynamics in the B808‐866 antenna of the green bacterium Chloroflexus aurantiacus
Authors: Novoderezhkin Vladimir , Taisova Alexandra , Fetisova Zoya
Published: July 2007

The model for the B808‐866 antenna of the green photosynthetic bacterium Chloroflexus aurantiacus based on femtosecond pump‐probe studies is proposed. The three‐dimensional structure of the B808‐866 antenna is assumed to be similar to the structure of the B800‐850 antenna of purple bacteria, i.e. it has the form of two concentric rings of N strongly coupled BChl866 pigments and of N/2 weakly coupled BChl808 monomers. The Qy transition dipoles of BChl808 and BChl866 molecules form the angles 43°±3° and 8°±4°, respectively, with the plane of the corresponding rings. The lowest limit of the BChl866 aggregate size is N=18. The anomalously high bleaching value of the BChl866 band with respect to the monomeric BChlS08 band provides evidence for a high degree of exciton delocalization. To account for this phenomenon, the exciton model describing exciton dynamics in the spectrally disordered circular BChl866 aggregate is developed. According to this model, the effective exciton size in this antenna (Neff) is 6‐8.
Analysis of 66 kDa toxin from Bacillus thuringiensis subsp, kurstaki reveals differential amino terminal processing of protoxin by endogenous protease(s)
Authors: Kumar N. Suresh , Venkateswerlu G.
Published: July 2007

The endogenous protease(s) activated crystal toxin from Bacillus thuringiensis subsp, kurstaki was purified and examined. The purified toxin was homogenous, as demonstrated by two‐dimensional polyacrylamide gel electrophoresis and contained 1.38 μmoles neutral sugar and 9 nmoles sialic acid per mg protein, amino terminal amino acid sequence data revealed that the toxin is a cleavage product of 132 kDa protoxin with glutamic acid‐30 of the deduced amino acid sequence of the crystal protein (Schnepf, H.E., Wong,HC and Whiteley,H.R. (1985) J. Biol. Chem. 260: 6264‐6272) at the amino terminus.
Influence of some quanternised 1,4‐Dihydropyridine derivatives on liposomes and erythrocyte membranes
Authors: Tirzite D. , Koronova J. , Plotniece A.
Published: July 2007

1,4‐Dihydropyridine derivatives possess various physiological activities but their mechanism of membranotropic action is not completely investigated yet. We have examined the membranotropic effects of 4‐β‐pyridyl‐1,4‐dihydropyridine derivatives containing different length alkyl chain substituent at N‐quaternised 4‐β‐pyridyl moiety. The results show the relation between incorporation of 1,4‐dihydropyridine derivatives in the liposomal membranes and influence on bilayer fluidity. The compound with hexadecyl (C16H33) substituent in the 4‐β‐pyridyl moiety possess the most pronounced incorporation ability and fluidizing effect. This compound causes the remarkable release of fluorescent probe calcein from liposomes and induces the hemolysis of human erythrocytes as well. The obtained results suggest that the length of alkyl chain at quaternized 4‐β‐pyridyt moiety is significant for the expression of membranotropic effects of tested compounds.
Lower archidonic acid content and preferental β‐oxidation of arachidonic acid over palmitic acid in tumour cell lines as compared to normal lymphoid cells
Authors: Barbieri B. , Alvelius G. , Papadogiannakis N.
Published: July 2007

In several studies certain polyunsaturated fatty acids (PUFA) have been shown to be selectively tumouricidal or suppressive of tumour cell proliferation. The mechanism behind this phenomenon likely involves peroxidation of the PUFA and generation of free radicals to which tumour cells seem to be more sensitive than normal cells.

In this report we have measured the total lipid content in separated lymphoid cells and several tumour cell lines, among which, T‐cell leukaemia, monocytic leukaemia, melanoma, fibrosarcoma, lung carcinoma and colon adenocarcinoma are included. Generally these tumour cell lines contain only one half to one third of the relative amount of arachidonic acid (AA) as compared to fleshly prepared lymphocytes and monocytes or lymphocytes kept in culture.

Furthermore, when we measured the β‐oxidation in long term incubation of [1‐14C] AA and compared it with that of [1‐14C] palmitic acid we found that several of the tumour cell lines showed a preference for AA over palmitic acid in the tumour cell lines whereas the opposite was observed for normal lymphoid cells.
Modulation of map kinase signaling and growth characteristics by the overexpression of protein kinase C in NIH3T3 cells
Authors: Chang Yoon‐Young , Kim Soo‐Jung , Park Tae‐Kyu , Kang Shin‐Sung , Ha Mahn Joon , Mushinski J. Fredric , Chun Jang‐Soo
Published: July 2007

This study was performed to examine effects of the overexpression of protein kinase C (PKC) isoforms (i.e., βI, βII, γ, δ,η, and ζ) mitogen‐activated protein (MAP) kinase (Erk‐1 and ‐2) signaling and growth characteristics of NIH3T3 cells. Phorbol ester (PMA) activated endogenous and ectopically expressed PKCα, βI, βII, γ, δ, ε, and η. Overexpression of the examined PKC isoforms enhanced PMA‐induced MAP kinase activation. Potentiation of MAP kinase activation was also observed upon stimulation of cells with platelet‐derived growth factor (PDGF) although there was no indication for the activation PKC isoforms by PDGF. Inhibition of PKC blocked PMA‐ but not PDGF‐induced MAP kinase activation. Thus, potentiation of PDGF‐induced MAP kinase activation appears to be independent to PKC activity, while PMA‐induced MAP kinase activation requires PKC activity. The ability of PKC isoforms to potentiate MAP kinase activation is not related to the growth characteristics of cells because individual PKC isoforms differentially regulated maximum density and proliferation of cells.
Glutathione metabolism and glutathione S‐conjugate export ATPase (MRP1/GS‐X pump) activity in cancer
Authors: de Bittencourt Paulo Homem , Senna Sueli M. , Vidor Ana C. , Miyasaka Célio K. , Curi Rui , Williams John F.
Published: July 2007

A severe complication in late‐stage cancer patients is host immunosuppression. It is suggested that overproduction of the highly cytostatic and cytotoxic antiproliferative cyclopentenone prostaglandins (CP‐PGs) within the plasma of cancer‐bearing subjects may contribute to immunosuppression. Lymphoid tissues of Walker 256 tumor‐bearing rats accumulate large amounts of CP‐PGs while the tumor tissue itself does not. Moreover, tumor cells may present differential sensitivity to CP‐PGs due to the expression of the multidrug resistance‐associated protein (MRP1) gene product which shows a Mg2+‐dependent vanadate‐sensitive glutathione S‐conjugate export ATPase (GS‐X pump) activity that extrudes CP‐PGs from cells as glutathione S‐conjugates. In this study, the possibility that deficient GS‐X pump activity in immune cells that may be involved in the accumulation of CP‐PGs is investigated. Rat lymph node lymphocytes do not exhibit any notable activity even when mitogen‐stimulated. Conversely, although rat peritoneal resident (quiescent) or thioglycollate‐stimulated (inflammatory) macrophages exhibit low GS‐X pump activity, Bacillus Calmette‐Guérin (BCG)‐activated macrophages show a notable rise in the activity of the ATPase, suggesting that the cellular activation state may modulate GS‐X pump activity/expression and that, under appropriate stimuli (e.g. during immune response) macrophages may provide a self‐defense against etectrophilic CP‐PGs by forming GS‐conjugates that can be extruded from cells through the GS‐X pump. ras oncogene expression may be linked with MRP1/GS‐X pump expression/activity, since C2C12 promyoblasts transformed by v‐H‐ras transfection doubled GS‐X pump activity. These results support the proposition that the accumulation of CP‐PGs and the immunosuppression of tumor‐bearing subjects may be attributed to a lack of GS‐X pump activity/expression in lymphocytes.
Gene encoding a human testis sertoli cell component related to LIM domain protein
Authors: Yang J. X. , Miao S. Y. , Wu Y. W. , Zhang Z. , Zong S. D. , Wang L. F. , Koide S. S.
Published: July 2007

The cDNA (HED‐2) encoding a 20 kDa protein found in mammalian epididymal fluid was isolated from a human testis expression library. It is composed of 1908 bp, containing a reading frame of 1479 bp, coding a polypeptide consisting of 493 amino acids, and assigned the accession number: U 15158 by GenBank (Biochem. Mol. Biol. Int. 34, 1131‐1136, 1994). HED‐2 has 99% identity with the zyxin gene in amino acid sequence, a component of cell junction matrix and a member of the LIM domain protein family. Northern blot analysis of RNAs prepared from various human tissues showed that the HED‐2 gene was expressed in all tissues analyzed. Sertoli cells of human testis expressed the gene as determined by an in situ hybridization method. The present study shows that the HED‐2 gene is a member of the LIM domain protein family.
Purification and characterization of sucrose synthase isozymes from etiolated rice seedlings
Authors: Huang Der‐Yi , Wang Ai‐Yu
Published: July 2007

Presence of homo‐ and hetero‐tetrameric rice sucrose synthase (RSS) isoforms in etiolated rice seedlings was demonstrated by immunoprecipitation using monospecific antibodies. Three RSS isozymes with various pI values were purified by ammonium sulfate fractionation, Sepharose CL‐6B gel filtration, DEAE‐Sephacel and Mono‐Q ion exchange chromatographies. They were characterized as heterotetramers composed of RSS1 and RSS2 subunits. All of them used UDP as the best substrate. The presence of divalent metal ions increased the activities of synthesis but inhibited the cleavage of sucrose.
HIV‐1 Nef protein causes death in stressed yeast cells due to determinants near the N‐terminus and elsewhere in Nef
Authors: Macreadie I. , Sankovich S. , Failla P. , Lowe M. , Curtain C. , Castelli L. , Azad A.
Published: July 2007

The Human Immunodeficiency Virus type 1 (HIV‐1) Nef protein is essential for AIDS pathogenesis. In order to determine more about the effects of Nef on basic cellular functions Nef was produced in yeast under a variety of conditions and in multiple cell types. Production of Nef caused cell death in acutely copper‐ or heat‐stressed diploid cells. The N‐terminal melittin‐like region of Nef was involved in toxicity since a Trp5→Ala change within Nef change caused increased toxicity. However, another determinant was also involved in toxicity since production of Nef20‐206 was also still toxic. In each of these Nef‐producing cells there was coincident membrane permeabilisation. These results suggest the possibility of a novel yeast bioassay for Nef inhibitors and that cells producing high levels of Nef may be selectively killed by stress.
Crystallization, preliminary x‐ray analysis and patterson search of a new aspartic protease isolated from human urine
Authors: Canduri Fernanda , Teodoro Lívia G. V. L. , Lorenzi Carla C. B. , Gomes Roseli A. S. , Fontes Marcos R. M. , Arni Raghuvir K. , de Azevedo Walter F.
Published: July 2007

Aspartic protease (EC 3.4.23) make up a widely distributed class of enzymes in animals, plants, microbes and, viruses. In animals these enzymes perform diverse functions, which range from digestion of food proteins to very specific regulatory roles. In contrast the information about the well‐characterized aspartic proteases, very little is known about the corresponding enzyme in urine. A new aspartic protease isolated from human urine has been crystallized and X‐ray diffraction data collected to 2.45Å resolution using a synchrotron radiation source. Crystals belong to the space group P212121. The cell parameters obtained were a=50.99, b=75.56 and c=89.90 Å. Preliminary analysis revealed the presence of one molecule in the asymmetric unit. The structure was determined using the molecular replacement technique and is currently being refined using simulated annealing and conjugate gradient protocols.
The role of proline in the prevention of aggregation during protein folding in vitro
Authors: Kumat T. K. S. , Samuel D. , Jayaraman G. , Srimathi T. , Yu C.
Published: July 2007

Proline effectively inhibits protein aggregation during the refolding of bovine carbonic anhydrase. Other osmolytes used such as glyeine and ethylene glycol fail to exhibit the ‘aggregation‐blockade’ role shown by proline. Results of viscosity and ANS fluorescence (1‐anilino‐8‐naphthalene sulphonic acid) experiments suggest that proline at high concentrations forms an ordered supramolecular assembly. Based on these results, it is proposed that proline behaves as a protein folding chaperone due to the formation of an ordered, amphipathic supramolecular assembly. To our knowledge, this is the first report wherein proline is proposed as a protein folding aid.
Regulation of Na,K‐ATPase activity in renal basolateral membrane of 1‐clip‐1‐kidney hypertensive rat
Authors: Seok Jeong‐Ho , Kim Ju‐Byoung , Hong Jang‐Hee , Sung Ji‐Yeon , Hur Gang‐Min , Lim Kyu , Lee Jae‐Heun
Published: July 2007

The changes of Na,K‐ATPase activity and its regulation have been investigated in the renal cortex of 1‐clip‐1‐kidney hypertensive rat. Ouabain‐sensitive Na,K‐ATPase activity (Emax) and [3H]ouabain‐binding site (Bmax) in the hypertensive rat were slightly increased than those in the control. The levels of Na,K‐ATPase α1‐ and β1‐subunit mRNA of the renal cortex in hypertensive rat were more increased than those in the control. Their increases were repressed by actinomycin‐D, but not altered or more increased by cycloheximide. These results suggest that the increase of Na,K‐ATPase activities and ouabain binding sites in 1‐clip‐1‐kidney hypertensive rat may be correlated with the increases of gene expression in transcription level and/or of mRNA stability of Na,K‐ATPase.
Isolation of the genomic clone of the rhesus monkey beta‐amyloid precursor protein
Authors: Songa Weihong , Lahiri Debomoy K.
Published: July 2007

A change in the regulation of the beta‐amyloid precursor protein (βAPP) gene may be a factor for Alzheimer's disease (AD). We have screened a rhesus monkey genomic library and pulled out a ∼17 kb genomic DNA region which contains the 5′‐flanking region (promoter), first exon and intron of the βAPP gene of the Rhesus monkey (rhβAPP). We have partially characterized the genomic clone by selective restriction enzyme digestion followed by Southern blotting against the βAPP gene‐specific DNA probes. The identity and authenticity of the different bands of the clone were also confirmed by Southern blot analysis of the rhesus monkey genomic DNA. Functional identification of the genomic fragment was determined by a promoter assay using the chloramphenicol acetyltransferase (CAT) enzyme as a reported gene. Our results showed that a 1.2 kb fragment from the 5′‐flanking region of the rhβAPP gene possessed strong promoter activity. The isolation of this genomic clone will enable characterization of the structure and function of the promoter of the rhβAPP gene. Our initial results indicate a high degree of homology between the structure of the rhesus monkey and human βAPP promoter.
Membrane perturbation by the neurotoxic alzheimer amyloid fragment β25‐35 requires aggregation and β‐sheet formation
Authors: Hirakura Yutaka , Satoh Yasushi , Hirashima Naohide , Suzuki Toshiharu , Kagan Bruce L. , Kirino Yutaka
Published: July 2007

The β‐amyloid peptide (βAP) is a major proteinaceous component of senile plaques and cerebrovascular amyloid deposits found in the brain of patients with Alzheimer's disease. βAP is reported to be neurotoxic only when it forms β‐sheet structure and aggregates. In the present study, we report that the neurotoxic core of βAP, βAP‐25‐35 (β25‐35), perturbs liposome membranes, induces membrane current, and exhibits hemolytic activity only in a buffer condition where the peptide forms β‐sheet structure and spontaneously aggregates. In contrast, β25‐35 in its monomeric random coil structure does not perturb lipid membranes significantly, and exhibits no hemolytic activity. Also, the membrane current was inhibited by Congo Red. The ability of β25‐35 to interact with membranes highly correlates with its neurotoxicity reported previously. These results suggest that membrane perturbation by aggregated β25‐35 constitutes the molecular basis of the peptide's neurotoxicity.
The plasma membrane Fe3 ‐reductase activity of Caco‐2 cells is modulated during differentiation
Authors: Ekmekcioglu Cem , Strauss‐Blasche Gerhard , Marktl Wolfgang
Published: July 2007

The aim of the present study was to investigate whether the brush border membrane ferric reductase activity of Caco‐2 cells is modulated during cell differentiation. The ferric reductase activity was determined in whole cells and isolated microvillous membranes at different stages of cell differentiation by measuring the amount of Fe3+ reduced during the incubation time. Our results indicated that the ferric reductase activity decreased in fastly growing cells and reactivated in postconfluent cells in contrast to the alkaline phosphatase and sucrase activities which were progressively expressed during differentiation as conventional indicators of cell maturity. The lowest ferric reductase activity was found in cells at the log phase of proliferation, while freshly seeded or highly differentiated cells had significantly higher enzyme activities. Cells grown under serum‐free conditions had similar ferric iron reduction rates as cells propagated under standard conditions. Reagents or hormones affecting cell metabolism through different pathways had no significant effect on this transplasma membrane redox system.
Inhibition of wheat leaves nitrate reductase activity by Cibacron Blue
Authors: Albassam Badr A.
Published: July 2007

Cibacron Blue F3GA (CB) inhibited the activities of wheat leaves NADH:nitrate reductase and NADH:cytochrome‐c reductase in a time‐independent and concentration dependent manner. The methyl viologen:nitrate reductase activity of the enzyme was unaffected by various CB concentrations used in the experiment. Inhibition of NADH:nitrate reductase was of mixed type (partial competitive and pure noncompetitive) with respect to NADH and noncompetitive with respect to nitrate. The estimated inhibition constant (Ki) values were 1 μM for NADH and 8.4 μM for nitrate. The secondary plots of inhibition with respect to NADH, indicated a dissociation constant (K1) of 8.8 μM for the enzyme‐NADH‐CB complex. This K1 being greater than the Ki suggested that the noncompetitive inhibition is predominant over the competitive inhibition at the NADH binding site.
An inverted cAMP response element mediates the cAMP induction of the ovine β1‐adrenergic receptor gene
Authors: Tseng Yi T. , Stabila Joan , McGonnigal Beth , Nguyen Tien T. , Padbury James F.
Published: July 2007

We identified an inverted, functional cAMP response element (CRE) located at ‐1599 bp relative to the translation start site within the ovine β1‐adrenergic receptor (β1AR) gene promoter. In transfection studies with SK‐N‐MC cells, a 40‐bp oligonucleotide containing the potential CRE, β1AR‐CRE, conferred a 3‐ to 4‐fold increase in luciferase activity mediated by cAMP. The induction was mimicked by co‐transfecting the cells with a vector overexpressing the α‐catalytic subunit of the cAMP‐dependent protein kinase (PKA) without treatment, and was blocked by overexpressing a PKA inhibitor (PKI). In electrophoretic mobility shift assays, a discrete binding pattern was shown in cell nuclear extract probed with the 40 bp β1AR‐CRE. The binding was shown to be specific and supershifted by addition of a CRE binding protein (CREB‐1) antibody. These data demonstrate that cAMP mediates the induction of β1AR gene expression by interacting with an inverted CRE within the promoter region. This is the first reported functional CRE among all β1AR genes.
Cloning of the genomic sequence encoding a processed adenylate kinase 2 pseudogene
Authors: Lee Younghee , Hong Mihui , Kim Yeon Jung , Kim Jae Wha , Kim Cheorl Ho , Lee Kwang Soo , Chang Sung Yeoul , Lim Jong‐Seok , Choe In Seong
Published: July 2007

A chromosomal DNA sequence harboring a processed AK2B pseudogene was isolated from a human genomic library. It was a variant of the AK2B gene sequence including several point mutations, deletions, and insertions. The nuclcotidc sequence of the ORF of the AK2B pseudogene predicted a truncated form of the AK2B mutant suggesting that the processed pseudogene is nonfunctional. A repetitive sequence, AAAAGAGAG, found in the 5′ and 3′ flanking regions of the pseudogene and the poly(A) tract in the 3′ end junction suggest that a mRNA of AK2B may have been converted to the processed pseudogene by retrotransposition events. Previously, it was suggested that an adenylate kinase (AK) 2 related gene on chromosome 2, confirmed by Southern analysis using somatic cell hybrid cell lines, may be a processed pseudogene. It is proposed that the processed pseudogene isolated in this study may be the AK2 related nonfunctional gene localized on human chromosomes 2.
Roles of nitrate, nitrite and ammonium ion in phytochrome regulation of nitrate reductase gene expression in maize
Authors: Raghuram Nandula , Sopory Sudhir K.
Published: July 2007

The influence of nitrate and its metabolites on the nitrate reductase (NR) gene expression and its relationship with phytochrome (Pfr) regulation of NR in etiolated maize leaves is examined. Nitrate induction and Pfr stimulation are brought about by independent signalling phenomena. Phorbol myristate acetate (PMA), a stimulator of protein kinase C (PKC), mimicked the effect of red light but could not replace the nitrate requirement for the induction of NR transcript accumulation. This suggests that while PKC‐type enzymes may be involved in mediating the Pfr signal, nitrate may follow an independent signalling mechanism. Experiments with 5‐hydroxytryptamine (5HT) and lithium ions (Li+), which are known to modulate phosphoinositide (PI) turnover, indicated that in addition to generating Pfr‐induced second messengers for PKC activation, PI cycle may also generate other signals which mediate nitrate induction of NR gene expression in the dark. The products of nitrate reduction ie, nitrite and ammonium ion had inhibitory and stimulatory effects respectively, on NR transcript accumulation. They work mainly at the level of nitrate induction.
High level expression of soluble angiogenin in Escherichia coli
Authors: Yoon Jong Myung , Kim Seung Ho , Kwon Oh Byung , Han Seung Hee , Kim Byong Kak
Published: July 2007

Human angiogenin was genetically engineered and contained the E. coli Omp A signal sequence for secreting soluble angiogenin to the periplasm under tac promoter control. The angiogenin sequence was encoded in a single gene and expressed as a 14.4 kilodalton soluble protein in E. coli. It was purified by CM‐Sepharose ion‐exchange chromatography and by a heparin‐Sepharose affinity chromatography procedure. The biological activity of angiogenin was established by its ability to inhibit mRNA‐dependent rabbit reticulocyte cell‐free translation.
Hepatitis B virus: DNA polymerase activity of deletion mutants
Authors: Kim Younhee , Hong Young Bin , Jung Guhung
Published: July 2007

The hepadnavirus P gene product is a multifunctional protein with priming, DNA‐and RNA‐dependent DNA polymerase, and RNase H activities. Nested N‐ or C‐terminal deletion mutations and deletions of domain(s) in human HBV polymerase have been made. Wild‐type and deletion forms of MBP‐fused HBV polymerase were expressed in E. coli, purified by amylose column chromatography, and the DNA‐dependent DNA polymerase activities of the purified proteins were compared. Deletion of the terminal protein or spacer regions reduced enzyme activity to 70%, respectively. However, deletion of the RNase H domain affected polymerase activity more than that of the terminal protein or spacer region. The polymerase domain alone or the N‐terminal deletion of the polymerase domain still exhibited enzymatic activity. In this report, it is demonstrated that the minimal domain for the polymerizing activity of the HBV polymerase is smaller than the polymerase domain.
Optical biosensor studies on the productive complex formation between the components of cytochrome P450scc dependent monooxygenase system
Authors: Ivanov Yuri D. , Usanov Sergey A. , Archakov Alexander I.
Published: July 2007

The formation of individual complexes between the components of cholesterol side chain cleavage system ‐ cytochrome P450scc, adrenodoxin (Ad) and adrenodoxin reductase (AdR) was kinetically characterized and their association and dissociation rate constants were measured by optical biosensor. The dominant role of interprotein electrostatic interactions in productive complex formation was demonstrated. Despite of the fact that P450scc and AdR compete for the binding with the same or closely placed negatively charged groups on the surface of immobilized Ad, the formation of the AdR/P450scc/Ad ternary complex upon AdR immobilization on dextran was registered. It is shown, that Ad does not bind to AdR immobilized via amino groups AdRim but it is possible only after the preliminary binding of P450scc to AdRim. The life time of such ternary complex, about 15 s, is sufficient for the realization of 5‐8 catalytic cycles.
Age‐dependent change in arachidonic acid metabolic capacity in rat alveolar macrophages
Authors: Chakraborti Tapati , Mandal Malay , Das Sudip , Chakraborti Sajal
Published: July 2007

Arachidonic acid (AA) metabolism was assessed in cultured alveolar macrophages (AM) obtained from newborn (10 days old) and adult (2 months and 4 months old) rats. The AMs were stimulated with the calcium ionophore, A23187 (10μM). The released radiolabelled AA metabolites were measured by thin layer chromatography. The results showed that among different aged rats, the synthesis of 5‐1ipoxygenase (5‐LO) metabolites, LTB4, LTC4, LTD4 and 5‐HETE were increased with age inspite of similar levels of [14C]AA release. In response to A23187, 5‐LO metabolic capacity of 2 and 4 months old adult rat AMs were increased 21‐fold and 34‐fold, respectively, compared with 10 days old rat AMs. As the metabolic capacity increased, the release of prostaglandins and thromboxane B2 tended to decrease markedly. Newborn rats (10 days old) AM, at the initial developmental stage, did not produce a noticeable amount of 5‐LO metabolites which, conceivably, contribute to high susceptibility of neonatal lung to infection.
Glutamate release is involved in PAF‐increased cyclic GMP levels in hippocampus
Authors: Calcerrada M. C. , Catalan R. E. , Martínez A. M.
Published: July 2007

The involvement of glutamate in PAF‐increased cyclic GMP levels was studied. Glutamate treatment caused a dose‐response increase of cyclic GMP levels in hippocampal slices. The presence of 1 mM glutamate did not modify the effect caused by 10‐7M PAF. To elucidate the involvement of glutamate in this action, slices were treated with PAF in the presence of MK‐801, a NMDA receptor antagonist. Results indicate that PAF‐increased cyclic GMP levels were obtained by NMDA receptors activation. Finally, results obtained from the experiments performed with PAF in the presence of riluzole, to inhibit the glutamate release, demonstrated that glutamate release is a stage in the PAF‐induced increase of cyclic GMP levels in hippocampus.
Immunosuppressive effects of the heat shock protein 90‐binding antibiotic geldanamycin
Authors: Sugita Takahisa , Tanaka Sumiko , Murakami Takanori , Miyoshi Hidetaka , Ohnuki Tetsuo
Published: July 2007

Ansamycin antibiotic, geldanamycin has a unique pharmacological effect to bind to heat shock protein 90 (hsp90) and deplete hsp90 substrates. We investigated the immunopharmacological effects of geldanamycin. Geldanamycin depleted cellular Raf‐1 of rat splenic cells without affecting the steady state levels of hsp90 and downstream mitogen activated protein (MAP) kinases, ERK1 and ERK2. In parallel, it inhibited mitogen‐induced nuclear factor‐kappa B (NF‐κB) activation in these cells. Geldanamycin also had a potent suppressive effect on recall antigen‐induced T cell proliferation, with an IC50 value of 1 nM. In vivo, geldanamycin suppressed the progression of adjuvant‐induced arthritis dose‐dependently. These results suggest that geldanamycin exerts an immunosuppressive effect partly through destabilizing Raf‐1, and raise a new strategy for the prevention of inflammatory diseases.
Expression of a novel chimeric protein containing the a chain of tissue‐type plasminogen activator and the B chain of pro‐urokinase in insect cells using the baculovirus system
Authors: Zhao Chun‐mei , Zhang Hong‐tao , Hu Mei‐hao
Published: July 2007

A hybrid cDNA tu‐pa, which contains Serl‐Thr263 of tissue‐type plasminogen activator (t‐PA) and Ser138‐Leu411 of pro‐urokinase (pro‐UK) was constructed and expressed in the Sf9‐AcNPV system. The expression level was approximate 2.5 mg/L. Tu‐PA was purified via one‐step affinity column conjugated with monoclonal antibody against the B chain of pro‐UK, which showed a single band of approximate 60kDa in SDS‐PAGE. The specific activity of the chimeric protein on fibrin plate was 200,000IU/mg protein. Tu‐PA had a higher selectivity for fibrin than UK and pro‐UK. Its activity can be promoted by CNBr degraded fibrin fragments as t‐PA.
RAPD profile based genetic characterization of chemotypic variants of Artemisia annua L.
Authors: Sangwan R. S. , Sangwan N. S. , Jain D. C. , Kumar S. , Ranade S. A.
Published: July 2007

The annual herbaceous plant, Artemisia annua L., belonging to family Asteraceae, is the natural source of the highly potent antimalarial compound, artemisinin, besides producing valuable essential oil. The plant is at present the sole commercial source for artemisinin production since all the chemical syntheses are non‐viable. Therefore, economic and practical considerations dictate that plants with maximum content of artemisinin be found and/or ways to increase their artemisinin content be sought. The key to this selection and breeding is a comprehension of chemical and genetic variability and suitable selection(s) of elites from within the available population. In the present study, RAPD analyses of selected chemotypes from a decade old introduced population in India were carried out using arbitrary primers. The RAPD data clearly indicate the distinction amongst these plants. Further, the detection of highly polymorphic profiles (97 polymorphic markers out of a total of 101 markers) suggests the existence of very high levels of genetic variation in the Indian population despite geographical isolation and opens out a strong possibility of further genetic improvement for superior artemisinin content. UPGMA analyses of RAPD and phytochemical trait data indicate that the wide phytochemical diversity is included within the genetic diversity. These results further support the prospects for selection and breeding of superior artemisinin containing lines.
Denitration of peroxynitrite‐treated proteins by “protein nitratases” from dog prostate
Authors: Kuo W. N. , Kanadia R. N. , Shanbhag V. R.
Published: July 2007

Putative “protein nitratases,” which catalyze denitration of peroxynitrite (PN)‐treated proteins, were detected in the crude extract of dog prostate. Nitratase activity was monitored by the decreased intensity of nitrotyrosine immunoreactive‐bands in Western blot and increased nitrate level in dialysate of incubation mixture, which contained prostate crude extract, protease inhibitors and a PN‐treated substrate, such as treated histone (III‐S), BSA, invertase, or polylysine. Nitratases were activated by preincubation with m‐calpain/Ca2+. Furthermore, after denitration, the activity of PN/DTT‐treated invertase decreased to the similar activity level of DTT‐treated invertase. At least two different types of nitratases may occur: type I, reductant‐dependent, and type II, reductant‐independent.
Peroxynitrite Diminishes Gap Junctional Communication: Protection by Selenite Supplementation
Authors: Sharov Victor S. , Briviba Karlis , Sies Helmut
Published: July 2007

Loss of intercellular communication via gap junctions has been correlated with progression of cells to a malignant phenotype. Here, we show that peroxynitrite, a mediator of toxicity in inflammatory processes, diminishes gap junctional intercellular communication (GJIC) in WB‐F344 rat liver epithelial cells, assayed by the scrapeloading dye‐transfer technique as well as by microinjection of a fluorescent dye into single cells. Exposure of cultured cells to a steady‐state concentration of peroxynitrite of 1.6 muM for 4 min or to 3‐morpholinosydnonimine (SIN‐1) at 0.5 mM strongly diminished GJIC. These concentrations of peroxynitrite or SIN‐1 were not cytotoxic. When cells were grown in a medium supplemented with sodium selenite (0.1‐1 muM) for 72 h, substantial protection was afforded against the decrease in GJIC by peroxynitrite. Thus, peroxynitrite can disrupt GJIC, and selenium‐containing proteins protect.
Glucose Erythrocyte Transporter in Women with Breast Cancer: Changes in Lipid Composition of Erythrocyte Membrane
Authors: Cresp C. , Quevedo S. , Roca P. , Palou A.
Published: July 2007

Women with breast cancer show altered blood glucose compartmentation compared with healthy women, with lower concentrations in plasma and similar concentrations in blood cells. The goal of this paper was to study whether this pattern was the result of changes in the erythrocyte glucose transporter and, if so, to assess the possible changes in lipid environment of the erythrocyte membrane. In 12 women with different degrees of breast cancer and 12 age‐matched healthy women, the lipid composition of erythrocyte membrane and erythrocyte glucose transport were studied. Women with breast cancer showed changes in both the kinetic variables and the lipid environment of the glucose transporter, in keeping with an increase in fluidity of the erythrocyte membrane. The results obtained would account, in part, for the changes in glucose compartmentation.
Regulation of Human eIF4E by 4E‐BP1: Binding Analysis Using Surface Plasmon Resonance
Authors: Youtani Takeshi , Tomoo Koji , Ishida Toshimasa , Miyoshi Hiroshi , Miura Kin‐Ichiro
Published: July 2007

The interaction between recombinant human eukaryotic initiation factor (eIF)4E and recombinant glutathione S‐transferase (GST)‐fused human 4E‐binding protein (BP)1 was analyzed by using the surface plasmon resonance method. The association rate of 4E‐BP1 with eIF4E increased by about two orders of magnitude in the presence of m7GTP (a model compound of mRNA cap structure), but the dissociation rate was scarcely affected, indicating the cap‐dependent binding of 4E‐BP1 to eIF4E. On the other hand, phosphorylation of 4E‐BP1 weakened its interaction with eIF4E whether m7GTP was present or not. However, phosphorylation of 4E‐BP1 already associated with eIF4E or its m7GTP complex did not cause any change of the association, probably because of incomplete phosphorylation. This suggests that the regulation of eIF4E by 4E‐BP phosphorylation is performed at its free state, not when it is in the associated state with eIF4E. Given these results, we discuss how 4E‐BP regulates eIF4E at the first step of translation initiation.
The value of different customer satisfaction and loyalty metrics in predicting customer retention, recommendation, and share‐of‐wallet
Authors: Keiningham Timothy L. , Cooil Bruce , Aksoy Lerzan , Andreassen Tor W. , Weiner Jay
Published: July 2007

Purpose

The purpose of this research is to examine different customer satisfaction and loyalty metrics and test their relationship to customer retention, recommendation and share of wallet using micro (customer) level data.

Design/methodology/approach

The data for this study come from a two‐year longitudinal Internet panel of over 8,000 US customers of firms in one of three industries (retail banking, mass‐merchant retail, and Internet service providers (ISPs)). Correlation analysis, CHAID, and three types of regression analyses (best‐subsets, ordinal logistic, and latent class ordinal logistic regression) were used to test the hypotheses.

Findings

Contrary to Reichheld's assertions, the results indicate that recommend intention alone will not suffice as a single predictor of customers' future loyalty behavior. Use of a multiple indicator instead of a single predictor model performs better in predicting customer recommendations and retention.

Research limitations/implications

The limitation of the paper is that it uses data from only three industries.

Practical implications

The presumption of managers when looking at recommend intention as the primary, even sole gauge of customer loyalty appears to be erroneous. The consequence is potential misallocations of resources due to myopic focus on customers' recommend intentions.

Originality/value

This is the first scientific study that examines recommend intentions and its impact on retention and recommendation on the micro (customer) level.
Effect of body positioning during transcranial Doppler detection of right‐to‐left shunts
Authors: Lao A. Y. , Sharma V. K. , Tsivgoulis G. , Malkoff M. D. , Alexandrov A. V. , Frey J. L.
Published: July 2007

We adopted an expanded transcranial Doppler (TCD) protocol to evaluate if additional injections of agitated saline in different positions would improve shunt detection or grading. We report the safety and feasibility of this expanded contrast TCD protocol. Patients with ischemic stroke were evaluated. The standard protocol for RLS detection was followed and expanded after the initial injection in the supine position to the right lateral decubitus, upright sitting, and sitting with right lateral leaning. Changes in blood pressure, heart rate, and any subjective complaints were noted. Changes in body position and additional agitated saline injections were tolerated. Right‐to‐left shunt (RLS) was detected in 35% of patients (n = 55). If the initial supine testing was negative, all subsequent positions/injections were also negative for RLS. However, if the supine injection was positive for RLS, the change in body positions increased the microbubble (μB) count in eight of 19 (42%) RLS‐positive patients. The mean μB count in RLS‐positive patients was 20 (95% CI: 9–32). The use of three additional body positions increased the μB count to 73 (95% CI: 13–132). The highest μB yield was achieved in the upright sitting position. Our findings support the safety and feasibility of the expanded TCD protocol. If the initial supine Valsalva‐aided contrast TCD test is negative, there may be no need to study the patient in additional positions. However, if μB are detected in the supine position, additional testing for RLS in alternative positions may be found to be worthwhile.
Heme oxygenase‐1 inhibits T cell‐dependent skin inflammation and differentiation and function of antigen‐presenting cells
Authors: Listopad Joanna , Asadullah Khusru , Sievers Claudia , Ritter Thomas , Meisel Christian , Sabat Robert , Döcke Wolf‐Dietrich
Published: July 2007

Abstract:  Heme oxygenase‐1 (HO‐1) is increased in psoriatic skin. We asked for the impact of physiological and pharmacological HO‐1 induction on skin immunity and the mechanisms involved in HO‐1‐induced immunomodulation. We found cutaneous HO‐1 expression upregulated comparable with suppressors of cytokine signalling (SOCS)1 and SOCS3 in psoriasis and atopic eczema and temporarily increased in murine ovalbumin‐induced late phase reaction (LPR) and 2,4‐dinitrofluorobenzene (DNFB)‐induced contact hypersensitivity (CHS). Cutaneous inflammation was enhanced by HO‐1 inhibition and was abrogated by treatment with the HO‐1 inducer cobaltic protoporphyrin (CoPP) both when applied around sensitization or before challenge. HO‐1 inhibition specifically prevented the anti‐inflammatory CoPP effect. CoPP inhibited T cell proliferation in splenocytes of treated mice and in human mixed leukocyte reaction and lymphocyte transformation test. CoPP induced HO‐1 in antigen‐presenting cells and depressed monocytic accessory molecule expression and the differentiation and maturation of monocyte‐derived dendritic cells (MDDC). It decreased tumor necrosis factor (TNF)‐α and interleukin (IL)‐12 production while increasing IL‐10 secretion. The antigen‐presenting capacity was diminished in CoPP‐treated and HO‐1‐transduced MDDC. We demonstrate for the first time the physiological role of HO‐1 in the limitation of skin inflammation and implement pharmacological HO‐1 induction as a therapeutic approach for T cell‐dependent inflammatory dermatoses. Suppression of antigen‐presenting cells may represent a main anti‐inflammatory mechanism of HO‐1.
Cognitive dysfunction and the neurobiology of ageing in cats
Authors: Gunn‐Moore D. , Moffat K. , Christie L.‐A. , Head E.
Published: July 2007

With improvements in nutrition and veterinary medicine the life expectancy of pet cats is increasing. Accompanying this growing geriatric population there are increasing numbers of cats with signs of apparent senility. A recent study suggests that 28 per cent of pet cats aged 11 to 14 years develop at least one geriatric onset behavioural problem, and this increases to over 50 per cent for cats of 15 years of age or older. While behavioural changes may result from systemic illness, organic brain disease or true behavioural problems, the possibility of age‐related cognitive dysfunction is often overlooked. Studies have revealed a number of changes in the brains of geriatric cats that showed signs of cognitive dysfunction, and potential causes include vascular insufficiency leading to hypoxia, increased free radical damage and the deposition of β‐amyloid plaques and/or the modification of other proteins. By recognising the importance of behavioural changes in old cats, investigating them fully for potentially treatable medical conditions, and instigating dietary and environmental modifications to meet their changing needs, we can make the lives of our geriatric cats much more comfortable and rewarding.