bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of the antifungal antibiotic clotrimazole on human cardiac repolarization potassium currents
Authors: Tian Miao , Dong Ming‐Qing , Chiu Shui‐Wha , Lau Chu‐Pak , Li Gui‐Rong 
Published: December 2005

The antifungal antibiotic clotrimazole (CLT) shows therapeutic effects on cancer, sickle cell disease, malaria, etc. by inhibiting membrane intermediate‐conductance Ca2+‐activated K+channels (IKCa). However, it is unclear whether this drug would affect human cardiac K+currents. The present study was therefore designed to investigate the effects of CLT on transient outward K+current (Ito1), and ultra‐rapid delayed rectifier K+current (IKur) in isolated human atrial myocytes, and cloned hERG channel current (IhERG) and recombinant human cardiac KCNQ1/KCNE1 channel current (IKs) expressed in HEK 293 cells.It was found that CLT inhibitedIto1with an IC50of 29.5 μM, acceleratedIto1inactivation, and decreased recovery ofIto1from inactivation. In addition, CLT inhibited human atrialIKurin a concentration‐dependent manner (IC50=7.6 μM).CLT substantially suppressedIhERG(IC50=3.6 μM), and negatively shifted the activation conductance ofIhERG. Moreover, CLT inhibitedIKs(IC50=15.1 μM), and positively shifted the activation conductance of the current.These results indicate that the antifungal antibiotic CLT substantially inhibits human cardiac repolarization K+currents includingIto1,IKur,IhERG, andIKs. However, caution is recommended when correlating the observedin vitroeffects on cardiac ion currents to the clinical relevance.British Journal of Pharmacology(2006)147, 289–297. doi:10.1038/sj.bjp.0706590
Constitutive expression of catABC genes in the aniline-assimilating bacterium Rhodococcus species AN-22: production, purification, characterization and gene analysis of CatA, CatB and CatC
Authors: Matsumura Eitaro , Sakai Masashi , Hayashi Katsuaki , Murakami Shuichiro , Takenaka Shinji , Aoki Kenji 
Published: December 2005

The aniline-assimilating bacterium Rhodococcus sp. AN-22 was found to constitutively synthesize CatB (cis,cis-muconate cycloisomerase) and CatC (muconolactone isomerase) in its cells growing on non-aromatic substrates, in addition to the previously reported CatA (catechol 1,2-dioxygenase). The bacterium maintained the specific activity of the three enzymes at an almost equal level during cultivation on succinate. CatB and CatC were purified to homogeneity and characterized. CatB was a monomer with a molecular mass of 44 kDa. The enzyme was activated by Mn2+, Co2+ and Mg2+. Native CatC was a homo-octamer with a molecular mass of 100 kDa. The enzyme was stable between pH 7.0 and 10.5 and was resistant to heating up to 90 °C. Genes coding for CatA, CatB and CatC were cloned and named catA, catB and catC respectively. The catABC genes were transcribed as one operon. The deduced amino acid sequences of CatA, CatB and CatC showed high identities with those from other Gram-positive micro-organisms. A regulator gene such as catR encoding a regulatory protein was not observed around the cat gene cluster of Rhodococcus sp. AN-22, but a possible relic of catR was found in the upstream region of catA. Reverse transcriptase-PCR and primer extension analyses showed that the transcriptional start site of the cat gene cluster was located 891 bp upstream of the catA initiation codon in the AN-22 strain growing on both aniline and succinate. Based on these data, we concluded that the bacterium constitutively transcribed the catABC genes and translated its mRNA into CatA, CatB and CatC.
Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) transcriptional regulation by Oct-1 in human endothelial cells: implications for atherosclerosis
Authors: Chen Jiawei , Liu Yong , Liu Hongmei , Hermonat Paul L. , Mehta Jawahar L. 
Published: December 2005

LOX-1, a receptor for ox-LDL (oxidized low-density lipoprotein), has recently been determined to play a critical role in the progression of atherosclerosis. LOX-1 expression (mRNA and protein) has been shown to be up-regulated by pro-atherogenic stimuli, such as ox-LDL and Ang II (angiotensin II). However, the molecular mechanisms of these up-regulations are unclear. In the present study, we explored LOX-1 transcriptional promoter activation in response to ox-LDL and Ang II. Under basal states, LOX-1 core promoter (LOX-1 −35/+36) was found to be sufficient for its basal activity in HCAECs (human coronary artery endothelial cells). More importantly, we found that ox-LDL (60 μg/ml for 24 h) induced LOX-1 promoter activity significantly and that a 105 bp fragment (between nt −1599 and −1494) was required for this activation. Within this 106 bp fragment, there is a potential binding motif for the transcription factor Oct-1 (octamer-1). By electrophoretic mobility-shift assay, we observed the activation of Oct-1 by ox-LDL. The critical role of Oct-1 in ox-LDL-induced LOX-1 promoter activation was further confirmed by mutagenesis assay. For comparison, we also examined LOX-1 promoter activation in response to Ang II (1 μmol/l for 24 h). Interestingly, another promoter region, between nt −2336 and −1990, was required for Ang II-induced LOX-1 promoter activation. In conclusion, the present study strongly suggests that ox-LDL, by activating Oct-1, induces LOX-1 promoter activation. Furthermore, this study suggests that while ox-LDL and Ang II both induce LOX-1 expression in HCAECs, the underlying mechanisms of promoter activation are different from each other.
ScOPT1 and AtOPT4 function as proton-coupled oligopeptide transporters with broad but distinct substrate specificities
Authors: Osawa Hiroki , Stacey Gary , Gassmann Walter 
Published: December 2005

A group of OPTs (oligopeptide transporters) exclusively identified in plants and fungi are proposed to transport oligopeptides and derivatives of three to six amino acids in length, but their transport mechanisms and biological functions are poorly understood. We expressed the Saccharomyces cerevisiae (yeast) OPT ScOPT1 and five Arabidopsis thaliana AtOPTs in Xenopus laevis oocytes for two-electrode voltage-clamp studies. ScOPT1 produced inward currents in response to GSH or GSSG, the phytochelatin (PC) PC2 and oligopeptides including the tetrapeptide GGFL, but not KLGL. Inward currents were dependent on the external proton and substrate concentrations, with high affinity for both. This and the inward currents evoked by substrates with net negative charges showed that ScOPT1 is a proton-coupled transporter. ScOPT1 displayed highest apparent affinity for PC2, with small differences in the maximal current among substrates. Glutathione transport by any of the tested AtOPTs, including AtOPT6, was not detected in yeast growth complementation assays. With AtOPT4, initially only small KLGL-dependent currents were recorded in batches of oocytes showing high ScOPT1 expression. AtOPT4 expression was optimized by swapping the 5′-untranslated region with that of ScOPT1. AtOPT4 displayed a higher affinity for KLGL than ScOPT1 did for any peptide. AtOPT4-mediated KLGL transport was detectable at pH 5.0, but not at pH 6.0 or 7.0. Taken together, our results demonstrate that ScOPT1 and AtOPT4 are proton-coupled OPTs with broad but distinct substrate specificities and affinities.
Characterization of the gene encoding glyoxalase II from Leishmania donovani: a potential target for anti-parasite drugs
Authors: Padmanabhan Prasad K. , Mukherjee Angana , Madhubala Rentala 
Published: December 2005

The glyoxalase system is a ubiquitous detoxification pathway that protects against cellular damage caused by highly reactive oxoaldehydes such as methylglyoxal which is mainly formed as a by-product of glycolysis. The gene encoding GLOII (glyoxalase II) has been cloned from Leishmania donovani, a protozoan parasite that causes visceral leishmaniasis. DNA sequence analysis revealed an ORF (open reading frame) of ∼888 bp that encodes a putative 295-amino-acid protein with a calculated molecular mass of 32.5 kDa and a predicted pI of 6.0. The sequence identity between human GLOII and LdGLOII (L. donovani GLOII) is only 35%. The ORF is a single-copy gene on a 0.6-Mb chromosome. A ∼38 kDa protein was obtained by heterologous expression of LdGLOII in Escherichia coli, and homogeneous enzyme was obtained after affinity purification. Recombinant L. donovani GLOII showed a marked substrate specificity for trypanothione hemithioacetal over glutathione hemithioacetal. Antiserum against recombinant LdGLOII protein could detect a band of anticipated size ∼32 kDa in promastigote extracts. By overexpressing the GLOII gene in Leishmania donovani using Leishmania expression vector pspαhygroα, we detected elevated expression of GLOII RNA and protein. Overexpression of the GLOII gene will facilitate studies of gene function and its relevance as a chemotherapeutic target. This is the first report on the molecular characterization of glyoxalase II from Leishmania spp. The difference in the substrate specificity of the human and Leishmania donovani glyoxalase II enzyme could be exploited for structure-based drug design of selective inhibitors against the parasite.
Identification and analysis of the promoter region of the human methionine sulphoxide reductase A gene
Authors: De Luca Antonella , Sacchetta Paolo , Di Ilio Carmine , Favaloro Bartolo 
Published: December 2005

MsrA (methionine sulphoxide reductase A) is an antioxidant repair enzyme that reduces oxidized methionine to methionine. Moreover, the oxidation of methionine residues in proteins is considered to be an important consequence of oxidative damage to cells. To understand mechanisms of human msrA gene expression and regulation, we cloned and characterized the 5′ promoter region of the human msrA gene. Using 5′-RACE (rapid amplification of cDNA ends) analysis of purified mRNA from human cells, we located the transcription initiation site 59 nt upstream of the reference MsrA mRNA sequence, GenBank® accession number BC 054033. The 1.3 kb of sequence located upstream of the first exon of msrA gene was placed upstream of the luciferase reporter gene in a pGL3-Basic vector and transfected into different cell lines. Sequentially smaller fragments of the msrA promoter region were generated by PCR, and expression levels were monitored from these constructs within HEK-293 and MCF7 human cell lines. Analysis of deletion constructs revealed differences in promoter activity in these cell lines. In HEK-293 cells, the promoter activity was constant from the minimal promoter region to the longest fragment obtained. On the other hand, in MCF7 cells we detected a down-regulation in the longest fragment. Mutation of a putative negative regulatory region that is located between −209 and −212 bp (the CCAA box) restored promoter activity in MCF7 cells. The location of the msrA promoter will facilitate analysis of the transcriptional regulation of this gene in a variety of pathological contexts.
Effect of ATP on preadipocyte migration and adipocyte differentiation by activating P2Y receptors in 3T3-L1 cells
Authors: Omatsu-Kanbe Mariko , Inoue Kazuko , Fujii Yusuke , Yamamoto Takefumi , Isono Takahiro , Fujita Norihisa , Matsuura Hiroshi 
Published: December 2005

The effect of extracellular ATP on adipogenesis was investigated using the mouse 3T3-L1 cell line. Incubation of cells with ATP (1–100 μM) for 5 min induced actin filament reorganization and membrane ruffling mediated through P2Y receptors. Enhancement of preadipocyte migration into fat cell clusters is one of the essential processes of adipose tissue development in vivo and cell migration assays revealed that stimulation of P2Y receptors enhanced chemokinesis (migration) in a concentration dependent manner. In this cell line, growth arrest is required before initiation of differentiation and growth-arrested post-confluent cells can be converted into adipocytes by the presence of the adipogenic hormones dexamethasone, 3-isobutyl-1-methylxanthine and insulin. On the other hand, those hormones alone do not trigger differentiation in proliferating cells. ATP did not induce differentiation when applied alone to either proliferating or postconfluent cells. By contrast, proliferating cells (density &lt;50%) preincubated with ATP for 5 min and subsequently given the adipogenic hormones in the continued presence of ATP, underwent adipocyte differentiation mediated through phospholipase C-coupled P2Y receptors. These adipocytes were found to show very similar characteristics, including morphology and intracellular triacylglycerol accumulation compared with adipocytes differentiated from post-confluent preadipocytes with those adipogenic hormones. When proliferating cells were preincubated with ATP before the addition of the adipogenic hormones, gene expression of aP2 (adipose protein 2) was markedly increased within 6 days, whereas without ATP pretreatment the expression level stayed very low. These results suggest that extracellular ATP renders preadipocytes responsive to adipogenic hormones during the growth phase.
The forkhead transcription factor Foxi1 directly activates the AE4 promoter
Authors: Kurth Ingo , Hentschke Moritz , Hentschke Suna , Borgmeyer Uwe , Gal Andreas , Hübner Christian A. 
Published: December 2005

Intercalated cells are highly specialized cells within the renal collecting duct epithelium and play an important role in systemic acid–base homoeostasis. Whereas type A intercalated cells secrete protons via an apically localized H+-ATPase, type B intercalated cells secrete HCO3−. Type B intercalated cells specifically express the HCO3−/Cl− exchanger AE4 (anion exchanger 4), encoded by Slc4a9. Mice with a targeted disruption of the gene for the forkhead transcription factor Foxi1 display renal tubular acidosis due to an intercalated cell-differentiation defect. Collecting duct cells in these mice are characterized by the absence of inter-calated cell markers including AE4. To test whether Slc4a9 is a direct target gene of Foxi1, an AE4 promoter construct was generated for a cell-based reporter gene assay. Co-transfection with the Foxi1 cDNA resulted in an approx. 100-fold activation of the AE4 promoter construct. By truncating the AE4 promoter at the 5′-end, we demonstrate that a fragment of approx. 462 bp upstream of the transcription start point is sufficient to mediate activation by Foxi1. Sequence analysis of this region revealed at least eight potential binding sites for Foxi1 in both sense and antisense orientation. Only one element was bound by recombinant Foxi1 protein in bandshift assays. Mutation of this site abolished both binding in bandshift assays and transcriptional activation by co-transfection of Foxi1 in the reporter gene assay. We thus identify the AE4 promoter as a direct target of Foxi1.
Expression and characterization of a histidine-rich protein, Hpn: potential for Ni2+ storage in Helicobacter pylori
Authors: Ge Ruiguang , Watt Rory M. , Sun Xuesong , Tanner Julian A. , He Qing-Yu , Huang Jian-Dong , Sun Hongzhe 
Published: December 2005

Hpn is a small cytoplasmic protein found in Helicobacter pylori, which binds Ni2+ ions with moderate affinity. Consisting of 60 amino acids, the protein is rich in histidine (28 residues, 46.7%), as well as glutamate, glycine and serine residues (in total 31.7%), and contains short repeating motifs. In the present study, we report the detailed biophysical characterization of the multimeric status and Ni2+-binding properties of purified recombinant Hpn under physiologically relevant conditions. The protein exists as an equilibration of multimeric forms in solution, with 20-mers (approx. 136 kDa) being the predominant species. Using equilibrium dialysis, ICP-MS (inductively coupled plasma MS) and UV/visible spectroscopy, Hpn was found to bind five Ni2+ ions per monomer at pH 7.4, with a dissociation constant (Kd) of 7.1 μM. Importantly, Ni2+ binding to Hpn is reversible: metal is released either in the presence of a chelating ligand such as EDTA, or at a slightly acidic pH (pH for half dissociation, pH1/2 ∼6.3). Ni2+ binding induces conformational changes within the protein, increasing β-sheet and reducing α-helical content, from 22% to 37%, and 20% to 10% respectively. Growth curves of Escherichia coli BL21(DE3) both with and without the hpn gene performed under Ni2+ pressure clearly implied a role for Hpn to protect the cells from higher concentrations of external metal ions. Similarly, the accumulation of Ni2+ in these cells expressing Hpn from a plasmid was approx. 4-fold higher than in uninduced controls or control cultures that lacked the plasmid. Similarly, levels of Ni2+ in wild-type H. pylori 26695 cells were higher than those in H. pylori hpn-deletion mutant strains. Hpn may potentially serve multiple roles inside the bacterium: storage of Ni2+ ions in a ‘reservoir’; donation of Ni2+ to other proteins; and detoxification via sequestration of excess Ni2+.
Fission yeast Dss1 associates with the proteasome and is required for efficient ubiquitin-dependent proteolysis
Authors: Jossé Lyne , Harley Margaret E. , Pires Isabel M. S. , Hughes David A. 
Published: December 2005

Human DSS1 associates with BRCA2, a tumour suppressor protein required for efficient recombinational DNA repair, but the biochemical function of DSS1 is not known. Orthologues of DSS1 are found in organisms such as budding yeast and fission yeast that do not have BRCA2-related proteins, indicating that DSS1 has a physiological role independent of BRCA2. The DSS1 orthologue in Saccharomyces cerevisiae has been shown to associate with the 26 S proteasome and, in the present paper, we report that in the distantly related fission yeast Schizosaccharomyces pombe, Dss1 associates with the 19 S RP (regulatory particle) of the 26 S proteasome. A role for S. pombe Dss1 in proteasome function is supported by three lines of evidence. First, overexpression of two components of the 19 S RP, namely Pad1/Rpn11 and Mts3/Rpn12, rescued the temperature-sensitive growth defect of the dss1 mutant. Secondly, the dss1 mutant showed phenotypes indicative of a defect in proteasome function: growth of the dss1 mutant was inhibited by low concentrations of L-canavanine, an amino acid analogue, and cells of the dss1 mutant accumulated high molecular mass poly-ubiquitylated proteins. Thirdly, synthetic growth defects were found when the dss1 mutation was combined with mutations in other proteasome subunit genes. These findings show that DSS1 has an evolutionarily conserved role as a regulator of proteasome function and suggest that DSS1 may provide a link between BRCA2 and ubiquitin-mediated proteolysis in human cells.
Intracellular signalling pathways activated by leptin
Authors: Frühbeck Gema 
Published: December 2005

Leptin is a versatile 16 kDa peptide hormone, with a tertiary structure resembling that of members of the long-chain helical cytokine family. It is mainly produced by adipocytes in proportion to fat size stores, and was originally thought to act only as a satiety factor. However, the ubiquitous distribution of OB-R leptin receptors in almost all tissues underlies the pleiotropism of leptin. OB-Rs belong to the class I cytokine receptor family, which is known to act through JAKs (Janus kinases) and STATs (signal transducers and activators of transcription). The OB-R gene is alternatively spliced to produce at least five isoforms. The full-length isoform, OB-Rb, contains intracellular motifs required for activation of the JAK/STAT signal transduction pathway, and is considered to be the functional receptor. Considerable evidence for systemic effects of leptin on body mass control, reproduction, angiogenesis, immunity, wound healing, bone remodelling and cardiovascular function, as well as on specific metabolic pathways, indicates that leptin operates both directly and indirectly to orchestrate complex pathophysiological processes. Consistent with leptin's pleiotropic role, its participation in and crosstalk with some of the main signalling pathways, including those involving insulin receptor substrates, phosphoinositide 3-kinase, protein kinase B, protein kinase C, extracellular-signal-regulated kinase, mitogen-activated protein kinases, phosphodiesterase, phospholipase C and nitric oxide, has been observed. The impact of leptin on several equally relevant signalling pathways extends also to Rho family GTPases in relation to the actin cytoskeleton, production of reactive oxygen species, stimulation of prostaglandins, binding to diacylglycerol kinase and catecholamine secretion, among others.
SLPI and elafin: one glove, many fingers
Authors: Williams Steven E. , Brown Thomas I. , Roghanian Ali , Sallenave Jean-Michel 
Published: December 2005

Elafin and SLPI (secretory leucocyte protease inhibitor) have multiple important roles both in normal homoeostasis and at sites of inflammation. These include antiprotease and antimicrobial activity as well as modulation of the response to LPS (lipopolysaccharide) stimulation. Elafin and SLPI are members of larger families of proteins secreted predominantly at mucosal sites, and have been shown to be modulated in multiple pathological conditions. We believe that elafin and SLPI are important molecules in the controlled functioning of the innate immune system, and may have further importance in the integration of this system with the adaptive immune response. Recent interest has focused on the influence of inflamed tissues on the recruitment and phenotypic modulation of cells of the adaptive immune system and, indeed, the local production of elafin and SLPI indicate that they are ideally placed in this regard. Functionally related proteins, such as the defensins and cathelicidins, have been shown to have direct effects upon dendritic cells with potential alteration of their phenotype towards type I or II immune responses. This review addresses the multiple functions of elafin and SLPI in the inflammatory response and discusses further their roles in the development of the adaptive immune response.
English‐language academic journals from China: a great opportunity
Authors: Xian Jia 
Published: December 2005

ABSTRACTThe conditions for developing English‐language academic journals in China are now favourable. Government investment in scientific research is increasing annually, there are many researchers at a high academic level with good English, and many key scientific research laboratories and engineering research centres have been established. There is also a big increase in the number of editors qualified in English. At present both the number and quality of English‐language journals are low, so the Chinese government is acting to support such journals and remedy their defects. There is thus a great opportunity for the development of such journals.
Documentation for institutional repositories
Authors: Probets Steve , Jenkins Celia 
Published: December 2005

ABSTRACTThe documentation of seven academic institutional repositories (IRs) was compared and contrasted. This was followed by semi‐structured interviews with six practitioners experienced in the set‐up, management and maintenance of IRs, including representatives of three JISC FAIR projects. The aim was to identify the requirements of policy documentation provided by IRs. Although many issues were found to be handled differently, several common factors emerged. These included the importance of developing documentation in collaboration with academics, departments and senior management. Policies should be formulated only when the aims of the IR have been clearly defined and the documentation itself should be concise and understandable, with the rights and responsibilities of stakeholders clearly presented.