bluuebunny/crossref_metadata_2025 huggingface dataset:
Application of chitin and chitosan as elicitors of coumarins and furoquinolone alkaloids in Ruta graveolens L. (common rue)
Authors: Orlita Aleksandra , Sidwa‐Gorycka Matylda , Paszkiewicz Monika , Malinski Edmund , Kumirska Jolanta , Siedlecka Ewa M. , Łojkowska Ewa , Stepnowski Piotr
Published: May 2008

Common rue (Ruta gra veolens L.) accumulates various types of secondary metabolites, such as coumarins furanocoumarins, acridone and quinolone alkaloids and flavonoids. Elicitation is a tool extensively used for enhancing secondary‐metabolite yields. Chitin and chitosan are examples of elicitors inducing phytoalexin accumulation in plant tissue. The present paper describes the application of chitin and chitosan as potential elicitors of secondary‐metabolite accumulation in R. graveolens shoots cultivated in vitro . The simple coumarins, linear furanocoumarins, dihydrofuranocoumarins and furoquinolone alkaloids biosynthesized in the presence of chitin and chitosan were isolated, separated and identified. There was a significant increase in the growth rate of R. graveolens shoots in the presence of either chitin or chitosan. Moreover, the results of the elicitation of coumarins and alkaloids accumulated by R. graveolens shoots in the presence of chitin and chitosan show that both compounds induced a significant increase in the concentrations of nearly all the metabolites. Adding 0.01% chitin caused the increase in the quantity (μg/g dry weight) of coumarins (pinnarin up to 116.7, rutacultin up to 287.0, bergapten up to 904.3, isopimpinelin up to 490.0, psoralen up to 522.2, xanhotoxin up to 1531.5 and rutamarin up to 133.7). The higher concentration of chitosan (0.1%) induced production of simple coumarins (pinnarin up to 116.7 and rutacultin up to 287.0), furanocoumarins (bergapten up to 904.3, isopimpinelin up to 490.0, psoralen up to 522.2, xanhotoxin up to 1531.5) and dihydrofuranocoumarins (chalepin up to 18 and rutamarin up to 133.7). Such a dramatic increase in the production of nearly all metabolites suggests that these compounds may be participating in the natural resistance mechanisms of R. graveolens . The application of chitin‐ and chitosan‐containing media may be considered a promising prospect in the biotechnological production of xanthotoxin, isopimpinelin, psoralen, chalepin or methoxylated dictamnine derivatives.
Complexation of retroviruses with polymers significantly increases the number of genes transferred to murine embryonic stem cells, but does not raise transgene expression levels
Authors: Chilton Jamie M. , Le Doux Joseph M.
Published: May 2008

Embryonic stem cells efficiently silence retrovirus transgene expression. To help to solve this problem, retroviruses have been developed that are more resistant to silencing, such as retroviruses derived from the MSCV (murine‐stem‐cell virus). A complementary approach to increasing transgene expression might be to increase the number of integrated transgenes. To test this approach, we formed polymer complexes with MSCV‐derived ecotropic retroviruses, concentrated them up to 40‐fold and transduced two different murine embryonic stem cell lines, with a mouse fibroblast cell line as a control. The number of integrated transgenes increased more than 50‐fold in the embryonic stem cell lines, yet, surprisingly, transgene expression did not increase. Interestingly, the embryonic stem cells had significantly fewer integrated transgenes than the mouse fibroblasts, even though transduction conditions were identical, which suggests that embryonic stem cells may restrict a post‐binding step of retrovirus transduction.
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.
Clinical update on renal aquaporins
Authors: Chen Yung‐Chang , Cadnapaphornchai Melissa A. , Schrier Robert W.
Published: May 2008

Following the discovery of the aquaporin‐1 water channel over a decade ago, molecular techniques have been developed to examine the role of renal aquaporin water channels under numerous physiological and pathological conditions. The present article reviews current knowledge regarding the function and dysfunction of renal aquaporins in disorders of water metabolism.
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.
Aquaporins from pathogenic protozoan parasites: structure, function and potential for chemotherapy
Authors: Beitz Eric
Published: May 2008

Infectious diseases, caused by protozoa, such as malaria, sleeping sickness, Chagas' disease or leishmaniasis, are a global threat. The increase in the number of affected individuals and the rapid spread of drug‐resistant strains call for specific novel strategies to combat human pathogenic parasites. In the search for novel drug targets, transport proteins for nutrients and metabolites of the parasite—host interface are getting into focus. The present review summarizes and discusses the currently available results on protozoan aquaporins. Various genes coding for aquaporin water and solute channels have been identified in the protozoan genomes and they are probable elements of the parasite's cell membrane. Phylogenetic analysis reveals that individual aquaporin genes are of bacterial or plant origin. So far, six protozoan aquaporins have been cloned and functionally characterized. Typically, these are bifunctional channels and pass water at intermediate to high rates as well as uncharged solutes. In the present review, amino acid compositions of the individual pore entries are compared and permeability properties are attributed to specific protein features. Furthermore, possible physiological roles in osmotic protection and metabolism are discussed. Finally, the potential of protozoan aquaporins for use as a target or entry pathway for chemotherapeutic compounds is reviewed.
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.
Apo‐Hsp90 coexists in two open conformational states in solution
Authors: Bron Patrick , Giudice Emmanuel , Rolland Jean‐Paul , Buey Rubén M. , Barbier Pascale , Díaz J. Fernando , Peyrot Vincent , Thomas Daniel , Garnier Cyrille
Published: May 2008

Background information. Hsp90 (90 kDa heat‐shock protein) plays a key role in the folding and activation of many client proteins involved in signal transduction and cell cycle control. The cycle of Hsp90 has been intimately associated with large conformational rearrangements, which are nucleotide‐binding‐dependent. However, up to now, our understanding of Hsp90 conformational changes derives from structural information, which refers to the crystal states of either recombinant Hsp90 constructs or the prokaryotic homologue HtpG (Hsp90 prokaryotic homologue).

Results and discussion. Here, we present the first nucleotide‐free structures of the entire eukaryotic Hsp90 (apo‐Hsp90) obtained by small‐angle X‐ray scattering and single‐particle cryo‐EM (cryo‐electron microscopy). We show that, in solution, apo‐Hsp90 is in a conformational equilibrium between two open states that have never been described previously. By comparing our cryo‐EM maps with HtpG and known Hsp90 structures, we establish that the structural changes involved in switching between the two Hsp90 apo‐forms require large movements of the NTD (N‐terminal domain) and MD (middle domain) around two flexible hinge regions.

Conclusions. The present study shows, for the first time, the structure of the entire eukaryotic apo‐Hsp90, along with its intrinsic flexibility. Although large structural rearrangements, leading to partial closure of the Hsp90 dimer, were previously attributed to the binding of nucleotides, our results reveal that they are in fact mainly due to the intrinsic flexibility of Hsp90 dimer. Taking into account the preponderant role of the dynamic nature of the structure of Hsp90, we reconsider the Hsp90 ATPase cycle.
Toxoplasma gondii Hsp20 is a stripe‐arranged chaperone‐like protein associated with the outer leaflet of the inner membrane complex
Authors: de Miguel Natalia , Lebrun Maryse , Heaslip Aoife , Hu Ke , Beckers Con J. , Matrajt Mariana , Dubremetz Jean F. , Angel Sergio O.
Published: May 2008

Background information. Toxoplasma gondii is among the most successful parasites, with nearly half of the human population chronically infected. T. gondii has five sHsps [small Hsps (heat‐shock proteins)] located in different subcellular compartments. Among them, Hsp20 showed to be localized at the periphery of the parasite body. sHsps are widespread, constituting the most poorly conserved family of molecular chaperones. The presence of sHsps in membrane structures is unusual.

Results. The localization of Hsp20 was further analysed using high‐resolution fluorescent light microscopy as well as electron microscopy, which revealed that Hsp20 is associated with the outer surface of the IMC (inner membrane complex), in a set of discontinuous stripes following the same spiralling trajectories as the subpellicular microtubules. The detergent extraction profile of Hsp20 was similar to that of GAP45 [45 kDa GAP (gliding‐associated protein)], a glideosome protein associated with the IMC, but was different from that of IMC1 protein. Although we were unable to detect interacting protein partners of Hsp20 either in normal or stressed tachyzoites, an interaction of Hsp20 with phosphatidylinositol 4‐phosphate and phosphatidylinositol 4,5‐bisphosphate phospholipids could be observed.

Conclusions. Hsp20 was shown to be associated with a specialized membranous structure of the parasite, the IMC. This discontinuous striped‐arrangement is unique in T. gondii, indicating that the topology of the outer leaflet of the IMC is not homogeneous.
Oxidative stress regulates IGF1R expression in vascular smooth-muscle cells via p53 and HDAC recruitment
Authors: Kavurma Mary M. , Figg Nichola , Bennett Martin R. , Mercer John , Khachigian Levon M. , Littlewood Trevor D.
Published: May 2008

Apoptosis of VSMCs (vascular smooth-muscle cells) leads to features of atherosclerotic plaque instability. We have demonstrated previously that plaque-derived VSMCs have reduced IGF1 (insulin-like growth factor 1) signalling, resulting from a decrease in the expression of IGF1R (IGF1 receptor) compared with normal aortic VSMCs [Patel, Zhang, Siddle, Soos, Goddard, Weissberg and Bennett (2001) Circ. Res. 88, 895–902]. In the present study, we show that apoptosis induced by oxidative stress is inhibited by ectopic expression of IGF1R. Oxidative stress repressed IGF1R expression at multiple levels, and this was also blocked by mutant p53. Oxidative stress also induced p53 phosphorylation and apoptosis in VSMCs. p53 negatively regulated IGF1R promoter activity and expression and, consistent with this, p53−/− VSMCs demonstrated increased IGF1R expression, both in vitro and in advanced atherosclerotic plaques in vivo. Oxidative-stress-induced interaction of endogenous p53 with TBP (TATA-box-binding protein) was dependent on p53 phosphorylation. Oxidative stress also increased the association of p53 with HDAC1 (histone deacetylase 1). Trichostatin A, a specific HDAC inhibitor, or p300 overexpression relieved the repression of IGF1R following oxidative stress. Furthermore, acetylated histone-4 association with the IGF1R promoter was reduced in cells subjected to oxidative stress. These results suggest that oxidative-stress-induced repression of IGF1R is mediated by the association of phosphorylated p53 with the IGF1R promoter via TBP, and by the subsequent recruitment of chromatin-modifying proteins, such as HDAC1, to the IGF1R promoter–TBP–p53 complex.
Comparable potency of IFNα2 and IFNβ on immediate JAK/STAT activation but differential down-regulation of IFNAR2
Authors: Marijanovic Zrinka , Ragimbeau Josiane , van der Heyden José , Uzé Gilles , Pellegrini Sandra
Published: May 2008

Type I IFNs (interferons) (IFNα/β) form a family of related cytokines that control a variety of cellular functions through binding to a receptor composed of IFNAR (IFNα receptor subunit) 1 and 2. Among type I IFNs, the α2 and β subtypes exhibit a large difference in their binding affinities to IFNAR1, and it was suggested that high concentrations of IFNAR1 may compensate for its low intrinsic binding affinity for IFNα2. We tested whether receptor-proximal signalling events are sensitive to IFNAR1 surface concentration by investigating the relationship between relative IFNAR1/IFNAR2 surface levels and IFNα2 and IFNβ signalling potencies in several cell lines. For this, we monitored the activation profile of JAK (Janus kinase)/STAT (signal transducer and activator of transcription) proteins, measured basal and ligand-induced surface decay of each receptor subunit and tested the effect of variable IFNAR1 levels on IFNα2 signalling potency. Our data show that the cell-surface IFNAR1 level is indeed a limiting factor for assembly of the functional complex, but an increased concentration of it does not translate into an IFNα/β differential JAK/STAT signalling nor does it change the dynamics of the engaged receptor. Importantly, however, our data highlight a differential effect upon routing of IFNAR2. Following binding of IFNα2, IFNAR2 is internalized, but, instead of being routed towards degradation as it is when complexed to IFNβ, it recycles back to the cell surface. These observations suggest strongly that the stability and the intracellular lifetime of the ternary complex account for the differential control of IFNAR2. Moreover, the present study opens up the attractive possibility that endosomal-initiated signalling may contribute to IFNα/β differential bioactivities.
Effect of branched-chain amino acids on muscle atrophy in cancer cachexia
Authors: Eley Helen L. , Russell Steven T. , Tisdale Michael J.
Published: May 2008

In the present study, the BCAAs (branched-chain amino acids) leucine and valine caused a significant suppression in the loss of body weight in mice bearing a cachexia-inducing tumour (MAC16), producing a significant increase in skeletal muscle wet weight, through an increase in protein synthesis and a decrease in degradation. Leucine attenuated the increased phosphorylation of PKR (double-stranded-RNA-dependent protein kinase) and eIF2α (eukaryotic initiation factor 2α) in skeletal muscle of mice bearing the MAC16 tumour, due to an increased expression of PP1 (protein phosphatase 1). Weight loss in mice bearing the MAC16 tumour was associated with an increased amount of eIF4E bound to its binding protein 4E-BP1 (eIF4E-binding protein 1), and a progressive decrease in the active eIF4G–eIF4E complex due to hypophosphorylation of 4E-BP1. This may be due to a reduction in the phosphorylation of mTOR (mammalian target of rapamycin), which may also be responsible for the decreased phosphorylation of p70S6k (70 kDa ribosomal S6 kinase). There was also a 5-fold increase in the phosphorylation of eEF2 (eukaryotic elongation factor 2), which would also decrease protein synthesis through a decrease in translation elongation. Treatment with leucine increased phosphorylation of mTOR and p70S6k, caused hyperphosphorylation of 4E-BP1, reduced the amount of 4E-BP1 associated with eIF4E and caused an increase in the eIF4G–eIF4E complex, together with a reduction in phosphorylation of eEF2. These changes would be expected to increase protein synthesis, whereas a reduction in the activation of PKR would be expected to attenuate the increased protein degradation.
Human copper transporter 2 is localized in late endosomes and lysosomes and facilitates cellular copper uptake
Authors: van den Berghe Peter V. E. , Folmer Dineke E. , Malingré Helga E. M. , van Beurden Ellen , Klomp Adriana E. M. , van de Sluis Bart , Merkx Maarten , Berger Ruud , Klomp Leo W. J.
Published: May 2008

High-affinity cellular copper uptake is mediated by the CTR (copper transporter) 1 family of proteins. The highly homologous hCTR (human CTR) 2 protein has been identified, but its function in copper uptake is currently unknown. To characterize the role of hCTR2 in copper homoeostasis, epitope-tagged hCTR2 was transiently expressed in different cell lines. hCTR2–vsvG (vesicular-stomatitis-virus glycoprotein) predominantly migrated as a 17 kDa protein after imunoblot analysis, consistent with its predicted molecular mass. Chemical cross-linking resulted in the detection of higher-molecular-mass complexes containing hCTR2–vsvG. Furthermore, hCTR2–vsvG was co-immunoprecipitated with hCTR2–FLAG, suggesting that hCTR2 can form multimers, like hCTR1. Transiently transfected hCTR2–eGFP (enhanced green fluorescent protein) was localized exclusively to late endosomes and lysosomes, and was not detected at the plasma membrane. To functionally address the role of hCTR2 in copper metabolism, a novel transcription-based copper sensor was developed. This MRE (metal-responsive element)–luciferase reporter contained four MREs from the mouse metallothionein 1A promoter upstream of the firefly luciferase open reading frame. Thus the MRE–luciferase reporter measured bioavailable cytosolic copper. Expression of hCTR1 resulted in strong activation of the reporter, with maximal induction at 1 μM CuCl2, consistent with the Km of hCTR1. Interestingly, expression of hCTR2 significantly induced MRE–luciferase reporter activation in a copper-dependent manner at 40 and 100 μM CuCl2. Taken together, these results identify hCTR2 as an oligomeric membrane protein localized in lysosomes, which stimulates copper delivery to the cytosol of human cells at relatively high copper concentrations. This work suggests a role for endosomal and lysosomal copper pools in the maintenance of cellular copper homoeostasis.
Two independent routes of de novo vitamin B6 biosynthesis: not that different after all
Authors: Fitzpatrick Teresa B. , Amrhein Nikolaus , Kappes Barbara , Macheroux Peter , Tews Ivo , Raschle Thomas
Published: May 2008

Vitamin B6 is well known in its biochemically active form as pyridoxal 5′-phosphate, an essential cofactor of numerous metabolic enzymes. The vitamin is also implicated in numerous human body functions ranging from modulation of hormone function to its recent discovery as a potent antioxidant. Its de novo biosynthesis occurs only in bacteria, fungi and plants, making it an essential nutrient in the human diet. Despite its paramount importance, its biosynthesis was predominantly investigated in Escherichia coli, where it is synthesized from the condensation of deoxyxylulose 5-phosphate and 4-phosphohydroxy-L-threonine catalysed by the concerted action of PdxA and PdxJ. However, it has now become clear that the majority of organisms capable of producing this vitamin do so via a different route, involving precursors from glycolysis and the pentose phosphate pathway. This alternative pathway is characterized by the presence of two genes, Pdx1 and Pdx2. Their discovery has sparked renewed interest in vitamin B6, and numerous studies have been conducted over the last few years to characterize the new biosynthesis pathway. Indeed, enormous progress has been made in defining the nature of the enzymes involved in both pathways, and important insights have been provided into their mechanisms of action. In the present review, we summarize the recent advances in our knowledge of the biosynthesis of this versatile molecule and compare the two independent routes to the biosynthesis of vitamin B6. Surprisingly, this comparison reveals that the key biosynthetic enzymes of both pathways are, in fact, very similar both structurally and mechanistically.
Profiling constitutive proteolytic events in vivo
Authors: Timmer John C. , Enoksson Mari , Wildfang Eric , Zhu Wenhong , Igarashi Yoshinobu , Denault Jean-Benard , Ma Yuliang , Dummitt Benjamin , Chang Yie-Hwa , Mast Alan E. , Eroshkin Alexey , Smith Jeffrey W. , Tao W. Andy , Salvesen Guy S.
Published: May 2008

Most known organisms encode proteases that are crucial for constitutive proteolytic events. In the present paper, we describe a method to define these events in proteomes from Escherichia coli to humans. The method takes advantage of specific N-terminal biotinylation of protein samples, followed by affinity enrichment and conventional LC (liquid chromatography)–MS/MS (tandem mass spectrometry) analysis. The method is simple, uses conventional and easily obtainable reagents, and is applicable to most proteomics facilities. As proof of principle, we demonstrate profiles of proteolytic events that reveal exquisite in vivo specificity of methionine aminopeptidase in E. coli and unexpected processing of mitochondrial transit peptides in yeast, mouse and human samples. Taken together, our results demonstrate how to rapidly distinguish real proteolysis that occurs in vivo from the predictions based on in vitro experiments.
Enzymes of glycerol and glyceraldehyde metabolism in mouse liver: effects of caloric restriction and age on activities
Authors: Hagopian Kevork , Ramsey Jon J. , Weindruch Richard
Published: May 2008

The influence of caloric restriction on hepatic glyceraldehyde- and glycerol-metabolizing enzyme activities of young and old mice were studied. Glycerol kinase and cytoplasmic glycerol-3-phosphate dehydrogenase activities were increased in both young and old CR (calorie-restricted) mice when compared with controls, whereas triokinase increased only in old CR mice. Aldehyde dehydrogenase and aldehyde reductase activities in both young and old CR mice were unchanged by caloric restriction. Mitochondrial glycerol-3-phosphate dehydrogenase showed a trend towards an increased activity in old CR mice, whereas a trend towards a decreased activity in alcohol dehydrogenase was observed in both young and old CR mice. Serum glycerol levels decreased in young and old CR mice. Therefore increases in glycerol kinase and glycerol-3-phosphate dehydrogenase were associated with a decrease in fasting blood glycerol levels in CR animals. A prominent role for triokinase in glyceraldehyde metabolism with CR was also observed. The results indicate that long-term caloric restriction induces sustained increases in the capacity for gluconeogenesis from glycerol.
Structure and function of window glass and Pyrex
Authors: Phillips J. C. , Kerner R.
Published: May 2008

Window glass is a ternary mixture, while Pyrex (after window glass, the most common form of commercial glass) is a quaternary. Here, we investigate the chemical, physical, and mathematical factors that determine the compositions of these optimized glasses. Previously, we succeeded in deriving exactly the composition of window glass (sodium calcium silicate) without adjustable parameters. Borosilicates are a much more challenging problem, and Pyrex (sodium aluminum borosilicate) requires a different approach. Our analysis shows that mean-field (or global) models (networks without significant clustering) are sufficient for window glass and probably most other commercial silicate glasses. However, it appears that the most important property of pyrex, its ability to resist mechanical shocks, requires a cluster model (large medium range order). We propose such a model, and argue that it also follows from hierarchical principles. Our model is strongly supported by specific experiments, and we suggest further experiments to test the principles underlying commercial glasses.
Ion heating during reconnection in the Madison Symmetric Torus reversed field pinch
Authors: Gangadhara S. , Craig D. , Ennis D. A. , Den Hartog D. J. , Fiksel G. , Prager S. C.
Published: May 2008

Measurements of localized ion heating during magnetic reconnection in the Madison Symmetric Torus reversed field pinch [R. N. Dexter, D. W. Kerst, T. W. Lovell, S. C. Prager, and J. C. Sprott, Fusion Technol. 19, 131 (1991)] are presented using two beam-based diagnostics: Charge exchange recombination spectroscopy and Rutherford scattering. Data have been collected from three types of impulsive reconnection event, in which the resistive tearing mode activity associated with reconnection is present either in the edge plasma, the core plasma, or throughout the plasma volume. A drop in the stored magnetic energy is required for ion heating to be observed during magnetic reconnection, and when this occurs, heating is concentrated in regions where reconnection is taking place. The magnitude of the observed temperature rise during reconnection varies with ion species, suggesting that the heating mechanism has a mass and/or charge dependence. Both the magnitude and spatial structure of the observed temperature rise also depend on the plasma current and density. Nonetheless, the fraction of released magnetic energy converted to ion thermal energy remains roughly constant over a range of plasma conditions.
An extensively valid and stable method for derivation of all parameters of a solar cell from a single current-voltage characteristic
Authors: Ishibashi Ken-ichi , Kimura Yasuo , Niwano Michio
Published: May 2008

It is important to precisely estimate values of all solar cell parameters in order to analyze the performance of a solar cell. We have proposed a stable method for the derivation of solar cell parameters from a single I-V curve under only the valid assumption for a general solar cell. Our method was applied to experimental I-V curves of a selection of solar cells, comprising of silicon, organic, and dye-sensitized solar cells, which were previously reported. Each I-V curve was calculated using the values of parameters derived from our method, and was found to be in good agreement with the experimental I-V curves, compared to previous work. Our extensively valid and stable method can be applied to for the analysis of all kinds of solar cells showing various characteristics that follow the single diode model and is very useful for improving the performance of developing solar cells.
Unexpected molecular weight dependence of shish-kebab structure in the oriented linear low density polyethylene/high density polyethylene blends
Authors: Liang Si , Wang Ke , Tang Changyu , Zhang Qin , Du Rongni , Fu Qiang
Published: May 2008

In this study, highly oriented shish-kebab structure was achieved via imposing oscillatory shear on the melts of linear low density polyethylene (LLDPE)/high density polyethylene (HDPE) blends during the packing stage of injection molding. To investigate the effect of molecular weight of HDPE on the formation of shish-kebab structure, two kinds HDPE with large melt flow index (low molecular weight) and small melt flow index (high molecular weight) were added into LLDPE matrix. The structural characteristics of LLDPE/HDPE blends were systematically elucidated through two-dimensional wide-angle x-ray scattering, scanning electron microscopy, and differential scanning calorimetry. Interestingly, an unexpected molecular weight dependence of shish-kebab structure of the prepared samples was found that the addition of HDPE with low molecular weight resulted in an higher degree of orientation, better regularity of lamellar arrangement, thicker lamellar size, and higher crystal melting temperature than that adding HDPE with high molecular weight. Correspondingly, the blend containing low molecular weight HDPE had better tensile strength. A possible mechanism was suggested to elucidate the role of HDPE molecular weight on the formation of shish-kebab structure in the oriented blends, considering the change of chain mobility and entanglement density with change of molecular weight.
On the nature of the high-frequency relaxation in a molecular glass former: A joint study of glycerol by field cycling NMR, dielectric spectroscopy, and light scattering
Authors: Gainaru C. , Lips O. , Troshagina A. , Kahlau R. , Brodin A. , Fujara F. , Rössler E. A.
Published: May 2008

Fast field cycling H1 NMR relaxometry is applied to determine the dispersion of spin-lattice relaxation time T1(ω) of the glass former glycerol in broad temperature (75–360K) and frequency (10kHz–30MHz) ranges. The relaxation data are analyzed in terms of a susceptibility χ″(ω)∝ω∕T1(ω), related to the second rank (l=2) molecular orientational correlation function. Broadband dielectric spectroscopic results suggest the validity of frequency temperature superposition above the glass transition temperature Tg. This allows to combine NMR data of different temperatures into a single master curve χ″(ωτα) that extends over 15 decades in reduced frequency ωτα, where τα is the structural α-relaxation time. This master curve is compared with the corresponding ones from dielectric spectroscopy (l=1) and depolarized light scattering (l=2). At ωτα<1, NMR susceptibility is significantly different from both the dielectric and light scattering results. At ωτα>1, there rather appears a difference between the susceptibilities of rank l=1 and l=2. Specifically, at ωτα⪢1, where the susceptibility is dominated by the so-called excess wing, the NMR and light scattering spectra (both l=2) rather coincide with each other and are about three times more intense than the dielectric (l=1) spectrum. This is explained by assuming that the high frequency dynamics correspond to only small-angle excursions. Below Tg, dielectric and NMR susceptibility compare well and exhibit an exponential temperature dependence.
Thermal and electrical conductivities of water-based nanofluids prepared with long multiwalled carbon nanotubes
Authors: Glory J. , Bonetti M. , Helezen M. , Mayne-L’Hermite M. , Reynaud C.
Published: May 2008

Thermal and electrical conductivities of suspensions of multiwalled carbon nanotubes (MWCNT) in water were measured as a function of temperature, nanotube weight content, and nanotube length. Nanotubes were dispersed in water by using gum Arabic as surfactant. The thermal conductivity was measured by the steady-state method by using a coaxial-cylinder cell that allows the sample temperature to be varied from 15to75°C. Our measurements show that the thermal conductivity enhancement as compared to water linearly increases when the MWCNT weight content increases from 0.01to3wt%, reaching 64% for the MWCNT weight content of 3wt%. The thermal conductivity enhancement is found to be temperature independent up to MWCNT weight content of 2wt%. The average length of the nanotubes appears to be a very sensitive parameter. The thermal conductivity enhancement as compared to water increases by a factor of 3 when the nanotube average length increases in the 0.5–5μm range. Electrical conductivity measurements show that the electrical properties do not follow the same trend as a function of MWCNT weight content, as compared to thermal properties. The electrical conductivity is mainly constant in the studied range, but undergoes a drop when the weight content decreases to about 0.1wt%, which suggests that the MWCNT network in the base fluid might be percolating at this very low value. By comparison, the thermal conductivity does not show any percolation threshold.