bluuebunny/crossref_metadata_2025_split huggingface dataset:
Immunization with a recombinant fowlpox virus expressing a hepatitis C virus core–E1 polyprotein variant, protects mice and African green monkeys (Chlorocebus aethiops sabaeus) against challenge with a surrogate vaccinia virus
Authors: Alvarez‐Lajonchere Liz , Amador‐Cañizares Yalena , Frías Roberto , Milian Yoamel , Musacchio Alexis , Guerra Ivis , Acosta‐Rivero Nelson , Martínez Gillian , Castro Jorge , Puentes Pedro , Cosme Karelia , Dueñas‐Carrera Santiago 
Published: May 2008

HCV (hepatitis C virus) is a worldwide health problem nowadays. No preventive vaccine is available against this pathogen, and therapeutic treatments currently in use have important drawbacks, including limited efficacy. In the present work a recombinant fowlpox virus, FPCoE1, expressing a truncated HCV core–E1 polyprotein, was generated. FPCoE1 virus generally failed to elicit a humoral immune response against HCV antigens in BALB/c mice. By contrast, mice inoculated with FPCoE1 elicited a positive interferon‐γ secretion response against HCV core in ex‐vivo ELISPOT (enzyme‐linked immunospot) assays. Remarkably, mice inoculated with FPCoE1 significantly controlled viraemia in a surrogate challenge model with vvRE, a recombinant vaccinia virus expressing HCV structural antigens. In fact, 40% of the mice had no detectable levels of vvRE in their ovaries. Administration of FPCoE1 in vervet monkeys [Chlorocebus (formerly Cercophitecus) aethiops sabaeus] induced lymphoproliferative response against HCV core and E1 proteins in 50% of immunized animals. Monkeys immunized with FPCoE1 had no detectable levels of vvRE in their blood, whereas monkeys inoculated with FP9, the negative control virus, had detectable levels of vvRE in blood up to 7 days after challenge. In conclusion, recombinant fowlpox virus FPCoE1 is able to induce an anti‐HCV immune response in mice and monkeys. This ability could be rationally employed to develop effective strategies against HCV infection by using FPCoE1 in combination with other vaccine candidates or antiviral treatments.
Small‐interfering‐RNA‐mediated silencing of human glutamate dehydrogenase induces apoptosis in neuroblastoma cells
Authors: Choi Myung‐Min , Kim Eun‐A , Huh Jae‐Wan , Choi Soo Young , Cho Sung‐Woo , Yang Seung‐Ju 
Published: May 2008

In the nervous system, GDH (glutamate dehydrogenase) is enriched in astrocytes and is important for recycling glutamate, a major excitatory neurotransmitter. The function of hGDH (human GDH) may be important in neurodegenerative diseases such as Parkinson's disease. To test the effect of decreased hGDH expression, several vector‐based plasmidlinked hGDH siRNAs (small interfering RNAs) were expressed intracellularly in BE(2)C human neuroblastoma cells. Immunoblotting and reverse‐transcription–PCR confirmed that expression of hGDH protein and mRNA was knocked down by co‐transfection with phGDH‐siRNA vectors in BE(2)C human neuroblastoma cells. TUNEL (terminal uridine deoxynucleotidyl transferase dUTP nick‐end labelling) and DNA fragmentation assays 48 h after transfection of phGDH‐siRNAs revealed that inhibition of hGDH expression induced cellular apoptosis and activated phospho‐ERK1/2 (phospho‐extracellular‐signal‐regulated kinase 1/2). These findings show that inhibition of hGDH expression by siRNA is related to apoptosis in neuronal cells.
Transcriptome and proteome analysis of antibody‐producing mouse myeloma NS0 cells cultivated at different cell densities in perfusion culture
Authors: Krampe Britta , Swiderek Halina , Al‐Rubeai Mohamed 
Published: May 2008

A combined gene and protein expression profiling was performed to gain a deeper insight into the intracellular response of the antibody‐producing GS‐NS0 cell line in continuous perfusion culture. Growth rate, production rate, metabolic activity and viability declined with increasing cell density, dilution rate and time. Transcriptome and proteome analyses of cells at three different densities revealed 53 genes and 47 proteins as having significantly altered expression levels at HCD (high cell density). The results showed an increased up‐regulation of genes/proteins involved in cellular energy production with increasing cell density. Furthermore, the intensified process triggered a cellular response to external stress stimuli, revealed by an overexpression of the genes/proteins implicated in cell‐cycle arrest [e.g. Rb1 (retinoblastoma 1 gene) and Cdkn1b (cyclin‐dependent kinase inhibitor 1B gene)] and in the induction of pro‐apoptotic genes/proteins [e.g. Tnfrsf (tumour necrosis factor receptor superfamily gene), Nfκbia (gene coding for nuclear factor‐κB inhibitor), HSP60 (heat‐shock protein of molecular mass 60 kDa) and heterogeneous nuclear ribonucleoprotein K]. Interestingly, we observed a down‐regulation of the transcription factor interferon regulatory factor 4 involved in the unfolded‐protein‐response process and protein disulfide‐isomerase family members responsible for protein folding and assembly. Additionally, subunits of proteasome complex were highly expressed at HCD. Microarray, real‐time quantitative reverse‐transcription PCR and Western‐blot analyses demonstrated a consistent trend of decrease in IgG heavy‐chain level with increasing cell density. HSP60, which inhibits apoptosis by complexing with pro‐apoptotic proteins such as Bax and Bak, was repressed at HCD. Overall, the data suggested that the balance among several factors involved in energy metabolism might be essential for fine‐tuning the cell choice between survival and apoptosis, leaning towards the side of apoptosis at HCD. The results provide significant information for cell‐engineering strategies and solutions to problems that prevail in HCD culture.
A monoclonal‐antibody‐based ELISA for the detection of human FADD (Fas‐associated death domain)
Authors: Ye Jianqiang , Shao Hongxia , Ma Dingyuan , Qin Aijian , Han Bing , Marikar Faiz M. M. T. , Cheng Wei , Huang Xiaofeng , Qiu Yudong , Hu Qingang , Hua Zichun 
Published: May 2008

FADD (Fas‐associated death domain) has been widely expressed in various tissues and its expression has been recently demonstrated to correlate with tumour progression and prognosis. Currently, measurement of FADD expression mainly depends on Western‐blot or immunohistochemical approaches. To develop a conventional sandwich ELISA avenue for the detection of FADD protein to supplement Western blotting or immunohistochemistry, a series of mAbs (monoclonal antibodies) specific for FADD protein, designated 3A3, 3F9, 3G4, 4B9, 4G1, 7A8, 7B8 and 7F4, were produced by fusing mouse s/p20 myeloma cells with the spleen cells of a mouse immunized with the Escherichia coli‐expressed recombinant His6‐FADD protein. On the basis of the characterization of these mAbs, purified 3F9 was selected as the capture antibody and the biotin‐conjugated 3A3 was selected as the detection antibody in sandwich ELISA. The limit of detection for the ELISA was 0.3 ng of purified His6–FADD (FADD tagged with hexahistidine), and it could detect both recombinant and native human FADD protein. Furthermore, the positive reaction of the ELISA could be blocked by rabbit anti‐FADD sera. All of these results indicated that the ELISA developed in the present paper could be a promising tool for detection of FADD protein.
Development of an in vitro reproductive screening assay for novel pharmaceutical compounds
Authors: Edwards Vicki , Markovic Elda , Matisons Janis , Young Fiona 
Published: May 2008

An in vitro reproductive cell‐based toxicity assay was developed using MLTC‐1 (murine Leydig tumour cell line) in order to examine the reproductive toxicity of two novel nanopharmaceutical compounds, namely ethylene glycol mono allyl ether and poly(ethylene glycol) octa‐functionalized polyhedral oligomeric silsesquioxane. Three commonly used cytotoxicity assays, namely the MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H ‐tetrazolium bromide], MTS [3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H ‐tetrazolium] and Crystal Violet assays, were compared, and the MTT assay proved to be the most accurate and reproducible for the MLTC‐1 cell line. The doubling rate of the MLTC‐1 cells was 30±3.5 h and the optimal seeding density for the MTT assay was 20000 cells per well, and the optimized MTT assay utilized a 4 h cell adherence followed by incubation with 0.5 mg/ml MTT for 1 h. The intra‐ and inter‐assay CV (coefficient of variation) values were 12.3 and 11% respectively. MLTC‐1 cells only produce the reproductive hormone progesterone in response to hCG (human chorionic gonadotropin), which stimulated progesterone production dose‐dependently from 0 to 100 m.i.u. (milliinternational units)/ml (2706±1118 ng/ml). H2O2 as a negative control killed 100% of cells at 1000 μg/ml. The two nanopharmaceutical compounds were cytotoxic at concentrations ≥0.1 μg/ml, but hCG decreased cytotoxicity to ≥1000 μg/ml (P <0.001). hCG‐stimulated progesterone synthesis afforded some protection against the cytotoxic effects of the two novel nanotechnology compounds; therefore doses ≤100 μg/ml and an exposure period of 1 h would be recommended for testing in in vivo animal reproductive assays.
Aquaporin‐1 and −9 are differentially regulated by oestrogen in the efferent ductule epithelium and initial segment of the epididymis
Authors: Oliveira Cleida A. , Carnes Kay , França Luiz R. , Hermo Louis , Hess Rex A. 
Published: May 2008

Background information. Efferent ductules reabsorb more than 90% of the rete testis fluid, a process that involves ion transporters and AQP (aquaporin) water channels. Oestrogen has been shown to modulate the expression of the ion transporters involved in this activity, but reports of AQP regulation in the male tract have been confounding. To understand better the regulation of AQP1 and AQP9, we investigated their expression in rat efferent ductules and initial segment of the epididymis after treatment with the pure antioestrogen ICI 182,780 or bilateral efferent duct ligation, or castration, followed by hormone replacement.Results. Results show that AQP9 is modulated by oestrogen in the efferent ductule epithelium, but not in the initial segment of the epididymis. DHT (5α‐dihydrotestosterone) also modulated AQP9 in efferent ductules. AQP9 was down‐regulated by the antioestrogen in efferent ductules on day 45 post‐treatment, which occurred before the non‐ciliated cells had shown significant loss of microvilli. DHT, but not oestradiol, modulated AQP9 expression in the initial segment of the epididymis. In contrast, testosterone, DHT, oestrogen or the antioestrogen did not alter AQP1 staining, indicating constitutive expression of AQP1 in the efferent ductule epithelium. AQP1 expression was induced in peritubular cells of efferent ductules and in the initial segment of the epididymis after castration and long‐term treatment with the antioestrogen. Although peritubular AQP1 staining in efferent ductules was partially reversed by the androgens, it was not reversed after any treatment in the initial segment of the epididymis.Conclusions. These results demonstrate that efferent ductules are unique in requiring both oestrogen and androgen to regulate an important mediator of fluid reabsorption, whereas the initial segment is dependent only on androgen stimulation.
The α‐glucanase Agn1p is required for cell separation in Schizosaccharomyces pombe
Authors: García Ignacio , Jiménez David , Martín Victoria , Durán Angel , Sánchez Yolanda 
Published: May 2008

Background information. In animal cells, cytokinesis occurs by constriction of an actomyosin ring. In fission yeast, ring constriction is followed by deposition of a multilayered division septum that must be cleaved to release the two daughter cells. Although many studies have focused on the actomyosin ring and septum assembly, little is known about the later steps involving the cleavage of the cell wall.Results. We identified a novel gene in Schizosaccharomyces pombe, namely the agn1+ gene that has homology to fungal 1,3‐α‐glucanases (mutanases). Disruption of the agn1+ gene is not lethal to the cells, but does interfere with their separation, whereas overexpression of Agn1p is toxic and causes cell lysis. Agn1p levels reach a peak during septation and the protein localizes to the septum region before cell separation. Moreover, agn1+ is responsible for the 1,3‐α‐glucanase activity, which shows a maximum at the end of septation.Conclusions. Our results clearly suggest the existence of a relationship between agn1+, 1,3‐α‐glucanase activity and the completion of septation in S. pombe. Agn1p could be involved in the cleavage of the cylinder of the old wall that surrounds the primary septum, a region rich in α‐glucans.
Expression and function of semaphorin 7A in bone cells
Authors: Delorme Guillaume , Saltel Frederic , Bonnelye Edith , Jurdic Pierre , Machuca‐Gayet Irma 
Published: May 2008

Background information. Sema‐7A is a glycosylphosphatidylinositol‐anchored semaphorin that was first identified in the immune system. It is a member of a large family of proteins involved in axon guidance signalling. Sema‐7A is expressed in the myeloid and the lymphoid lineage and seems to be involved in cytokine expression and chemotaxy through its receptor Plexin C1. However, it can promote axon outgrowth, acting through a β1 subunit‐containing integrin receptor.Results and conclusions. In the present study, we have investigated its regulation and function in bone cells. Semiquantitative reverse transcriptase—PCR demonstrated that Sema‐7A mRNA is present during all stages of osteoblast differentiation and maturation in mouse calvaria cells and in MC3T3 cell line in vitro. Its expression is also regulated during primary osteoclast differentiation in vitro. We report that Sema‐7A is capable of increasing the migration of MC3T3 cells and that this process is mediated by the mitogen‐activated protein kinase pathway in osteoblasts, probably through the integrin subunit β1. Moreover, the addition of recombinant soluble Sema‐7A to the culture enhances osteoclast fusion. These findings indicate for the first time the possible involvement of Sema‐7A in bone cell differentiation.
Specific plasma membrane aquaporins of the PIP1 subfamily are expressed in sieve elements and guard cells
Authors: Fraysse Laure C. , Wells Brian , McCann Maureen C. , Kjellbom Per 
Published: May 2008

Background information. Transmembrane water flow is aided by water‐specific channel proteins, aquaporins. Plant genomes code for approx. 35 expressed and functional aquaporin isoforms. Plant aquaporins fall into four different subfamilies of which the PIPs (plasma membrane intrinsic proteins) constitute the largest and evolutionarily most conserved subfamily with 13 members in Arabidopsis and maize. Furthermore, the PIPs can be divided into two phylogenetic groups, PIP1 and PIP2, of which the PIP1 isoforms are most tightly conserved, sharing >90% amino acid sequence identity. As the nomenclature implies, the majority of PIPs have been shown to be localized at the plasma membrane. Recently, two highly abundant plasma membrane aquaporins, SoPIP2;1 and SoPIP1;2, have been purified and structurally characterized.Results. We report the cloning of a cDNA encoding SoPIP1;2 and show that there are at least five additional sequences homologous with SoPIP2;1 and SoPIP1;2 in the spinach genome. To understand their role in planta, we have investigated the cellular localization of the aquaporin homologues SoPIP1;2 and SoPIP1;1. By Western‐ and Northern‐blot analyses and by immunocytochemical detection at the light and electron microscopic levels, we show that SoPIP1;2 is highly expressed in phloem sieve elements of leaves, roots and petioles and that SoPIP1;1 is present in stomatal guard cells.Conclusions. Localization of the two abundant aquaporin isoforms suggests roles for specific PIPs of the PIP1 subgroup in phloem loading, transport and unloading, and in stomatal movements.
Proton exclusion by an aquaglyceroprotein: a voltage clamp study
Authors: Saparov Sapar M. , Tsunoda Satoshi P. , Pohl Peter 
Published: May 2008

Background information. In silico both orthodox aquaporins and aquaglyceroporins are shown to exclude protons. Supporting experimental evidence is available only for orthodox aquaporins. In contrast, the subset of the aquaporin water channel family that is permeable to glycerol and certain small, uncharged solutes has not yet been shown to exclude protons. Moreover, different aquaglyceroporins have been reported to conduct ions when reconstituted in planar bilayers.Results. To clarify these discrepancies, we have measured proton permeability through the purified Escherichia coli glycerol facilitator (GlpF). Functional reconstitution into planar lipid bilayers was demonstrated by imposing an osmotic gradient across the membrane and detecting the resulting small changes in ionic concentration close to the membrane surface. The osmotic water flow corresponds to a GlpF single channel water permeability of 0.7×10−14 cm3·subunit−1·s−1. Proton conductivity measurements carried out in the presence of a pH gradient (1 unit) revealed an upper limit of the H+ (OH−) to H2O molecules transport stoichiometry of 2×10−9. A significant GlpF‐mediated ion conductivity was also not detectable.Conclusions. The lack of a physiologically relevant GlpF‐mediated proton conductivity agrees well with predictions made by molecular dynamics simulations.
FLRG, a new ADAM12‐associated protein, modulates osteoclast differentiation
Authors: Bartholin Laurent , Destaing Olivier , Forissier Stéphanie , Martel Sylvie , Maguer‐Satta Véronique , Jurdic Pierre , Rimokh Ruth 
Published: May 2008

Background information. FLRG (follistatin‐related gene) is a secreted glycoprotein that is highly homologous with follistatin. These proteins are involved in the regulation of various biological effects mediated by their binding to TGF‐β (transforming growth factor‐β) superfamily members, activin A and bone morphogenetic proteins. To characterize further the function of FLRG, we used a yeast two‐hybrid screen to look for other possible functional partners.Results. We report a direct interaction between the cysteine‐rich domain of FLRG and ADAM12 (a disintegrin and metalloprotease 12). ADAMs are metalloprotease‐disintegrin proteins that have been implicated in cell adhesion, protein ectodomain shedding, matrix protein degradation and cell fusion. Several studies have reported that ADAM12 protein, as well as activin A, are important regulators of osteoclast differentiation. We observed that the expressions of ADAM12 and activin A are modulated during osteoclast formation, whereas the FLRG expression seemed to remain quite constant. We showed that the FLRG protein inhibits osteoclast differentiation from murine primary spleen cells and macrophage RAW264.7 cells cultured in the presence of RANK‐L (receptor activator of nuclear factor κB ligand) and M‐CSF (macrophage colony‐stimulating factor). Addition of FLRG protein to precursors significantly reduces the number of osteoclasts, as well as the average number of nuclei in each osteoclast.Conclusions. Our study indicates that the FLRG protein may contribute to bone formation by inhibiting osteoclast differentiation.
Purification and functional characterization of aquaporin‐8
Authors: Liu Kun , Nagase Hiroaki , Huang Chunyi George , Calamita Giuseppe , Agre Peter 
Published: May 2008

Background information. Aquaporins (AQPs) are a family of channels permeable to water and some small solutes. In mammals, 13 members (AQP0–AQP12) have been found. AQP8 is widely distributed in many tissues and organs. Previous studies in frog oocytes suggested that AQP8 was permeable to water, urea and ammonium, but no direct characterization had yet been reported.Results. We expressed recombinant rAQP8, hAQP8 and mAQP8 (rat, human and mouse AQP8 respectively) in yeast, purified the proteins to homogeneity and reconstituted them into proteoliposomes. Although showing high sequence similarity, AQP8 proteins from the three species had to be purified with different detergents prior to reconstitution. In stopped‐flow studies, all three AQP8 proteoliposomes showed water permeability, which was inhibited by mercuric chloride and rescued by 2‐mercaptoethanol. rAQP8 and hAQP8 proteoliposomes did not transport glycerol or urea but were permeable to formamide, which was also inhibited by mercuric chloride. In the oocyte transport assay, hAQP8‐injected oocytes showed significantly higher [14C]methylammonium uptake than water‐injected oocytes.Conclusions. In the present study, we successfully purified rAQP8, hAQP8 and mAQP8 proteins and characterized their biochemical and biophysical properties. All three AQP8 proteins transport water. rAQP8 and hAQP8 are not permeable to urea or glycerol. Moreover, hAQP8 is permeable to ammonium analogues (formamide and methylammonium). Our results suggest that AQP8 may transport ammonium in vivo and physiologically contribute to the acid—base equilibrium.
β‐Oestradiol rescues ΔF508CFTR functional expression in human cystic fibrosis airway CFBE41o− cells through the up‐regulation of NHERF1
Authors: Fanelli Teresa , Cardone Rosa Angela , Favia Maria , Guerra Lorenzo , Zaccolo Manuela , Monterisi Stefania , De Santis Teresa , Riccardi Stefania Maria , Reshkin Stephan Joel , Casavola Valeria 
Published: May 2008

Background information. CF (cystic fibrosis) is a disease caused by mutations within the CFTR (CF transmembrane conductance regulator) gene. The most common mutation, ΔF508 (deletion of Phe‐508), results in a protein that is defective in folding and trafficking to the cell surface but is functional if properly localized in the plasma membrane. We have recently demonstrated that overexpression of the PDZ protein NHERF1 (Na+/H+‐exchanger regulatory factor 1) in CF airway cells induced both a redistribution of ΔF508CFTR from the cytoplasm to the apical membrane and the PKA (protein kinase A)‐dependent activation of ΔF508CFTR‐dependent chloride secretion. In view of the potential importance of the targeted up‐regulation of NHERF1 in a therapeutic context, and since it has been demonstrated that oestrogen treatment increases endogenous NHERF1 expression, we tested the hypothesis that oestrogen treatment can increase NHERF1 expression in a human bronchiolar epithelial CF cell line, CFBE41o−, with subsequent rescue of apical ΔF508CFTR chloride transport activity.Results. We found that CFBE41o− cells do express ERs (oestrogen receptors) in the nuclear fraction and that β‐oestradiol treatment was able to significantly rescue ΔF508CFTR‐dependent chloride secretion in CFBE41o− cell monolayers with a peak between 6 and 12 h of treatment, demonstrating that the ΔF508CFTR translocated to the apical membrane can function as a cAMP‐responsive channel, with a significant increase in chloride secretion noted at 1 nM β‐oestradiol and a maximal effect observed at 10 nM. Importantly, knock‐down of NHERF1 expression by transfection with siRNA (small interfering RNA) for NHERF1 inhibited the β‐oestradiol‐dependent increase in ΔF508CFTR protein expression levels and completely prevented the β‐oestradiol‐dependent rescue of ΔF508CFTR transport activity.Conclusions. These results demonstrate that β‐oestradiol‐dependent up‐regulation of NHERF1 significantly increases ΔF508CFTR functional expression in CFBE41o− cells.