bluuebunny/crossref_metadata_2025 huggingface dataset:
Autoproteolytic activation of human aspartylglucosaminidase
Authors: SAARELA Jani , OINONEN Carita , JALANKO Anu , ROUVINEN Juha , PELTONEN Leena
Published: February 2004

Aspartylglucosaminidase (AGA) belongs to the N-terminal nucleophile (Ntn) hydrolase superfamily characterized by an N-terminal nucleophile as the catalytic residue. Three-dimensional structures of the Ntn hydrolases reveal a common folding pattern and equivalent stereochemistry at the active site. The activation of the precursor polypeptide occurs autocatalytically, and for some amidohydrolases of prokaryotes, the precursor structure is known and activation mechanisms are suggested. In humans, the deficient AGA activity results in a lysosomal storage disease, aspartylglucosaminuria (AGU) resulting in progressive neurodegeneration. Most of the disease-causing mutations lead to defective molecular maturation of AGA, and, to understand the structure–function relationship better, in the present study, we have analysed the effects of targeted amino acid substitutions on the activation process of human AGA. We have evaluated the effect of the previously published mutations and, in addition, nine novel mutations were generated. We could identify one novel amino acid, Gly258, with an important structural role on the autocatalytic activation of human AGA, and present the molecular mechanism for the autoproteolytic activation of the eukaryotic enzyme. Based on the results of the present study, and by comparing the available information on the activation of the Ntn-hydrolases, the autocatalytic processes of the prokaryotic and eukaryotic enzymes share common features. First, the critical nucleophile functions both as the catalytic and autocatalytic residue; secondly, the side chain of this nucleophile is oriented towards the scissile peptide bond; thirdly, conformational strain exists in the precursor at the cleavage site; finally, water molecules are utilized in the activation process.
Crystal structure of a platelet-agglutinating factor isolated from the venom of Taiwan habu (Trimeresurus mucrosquamatus)
Authors: HUANG Kai-Fa , KO Tzu-Ping , HUNG Chin-Chun , CHU John , WANG Andrew H.-J. , CHIOU Shyh-Horng
Published: February 2004

Platelet glycoprotein Ib (GPIb)-binding proteins (GPIb-BPs) from snake venoms are usually C-type lectins, which target specific sites of GPIbα and elicit distinct effects on platelets. In the present paper, we report a tetrameric platelet-agglutinating factor (molecular mass 121.1 kDa), termed mucrocetin, purified from the venom of Taiwan habu (Trimeresurus mucrosquamatus). Mucrocetin is a GPIbα agonist with a binding site distinct from that of flavocetin-A (a snake venom GPIbα antagonist) on GPIbα, in spite of the high sequence identity (94.6%) between the two venom lectins. The crystal structure of mucrocetin was solved and refined to 2.8 Å (1 Å=0.1 nm) resolution, which shows an interesting crystal packing of six-layer cylinders of doughnut-shaped molecules. The four αβ heterodimers are arranged in an unusual square-shaped ring stabilized by four interdimer ‘head-to-tail’ disulphide bridges. Detailed structural comparison between mucrocetin and flavocetin-A suggests that their disparate platelet effects are probably attributable to different charge distributions on the putative concave binding surface. A unique positively charged patch on the binding surface of mucrocetin, formed by Lys102, Lys108, Lys109 and Arg123 in the α-subunit coupled with Lys22, Lys102, Lys116 and Arg117 in the β-subunit, appears to be the primary determinant of its platelet-agglutinating activity. Conceivably, this interesting venom factor may provide a useful tool to study platelet agglutination by binding to the GPIb–IX–V complex.
Expression and characterization of cathepsin P
Authors: MASON Robert W. , BERGMAN Carolyn A. , LU Guizhen , HOLBROOK Jennifer FRENCK , SOL-CHURCH Katia
Published: February 2004

The mouse genome contains a family of clan C1A proteases that appear to be restricted to rodents within Eutherian (placental) mammals. mRNA analysis has shown that these genes are expressed exclusively in placenta. Sequence analysis predicts that the expressed proteins will be functional and consequently it was proposed that this family of proteases may have evolved to perform subspecialized functions of the closely related protease, cathepsin L, that is expressed in placental tissues of all mammalian species. In the present study, it was shown that cathepsin P can be expressed in Pichia pastoris as an inactive zymogen that can be activated with proteinase K, chymotrypsin or pancreatic elastase at neutral pH. Unlike other mammalian cathepsins, cathepsin P could also be autoactivated at neutral pH, but not at acidic pH. The activated enzyme was capable of hydrolysing peptidyl substrates and the protein substrates azocasein and transferrin, with optimal activity at pH 6.5–7.5. Little activity could be detected at pH 5.0 and below. Salts such as Na2SO4 and hyaluronate stimulated the activity of the protease against peptidyl substrates. The properties of cathepsin P appear to be quite distinct from those of cathepsin L, indicating that the duplication that gave rise to cathepsin P has probably not yielded an enzyme that provides a subfunction of cathepsin L in rodents. It seems probable that cathepsin P has evolved to perform a function that is performed by an evolutionarily unrelated protease in other mammalian species.
Cy5 maleimide labelling for sensitive detection of free thiols in native protein extracts: identification of seed proteins targeted by barley thioredoxin h isoforms
Authors: MAEDA Kenji , FINNIE Christine , SVENSSON Birte
Published: February 2004

Barley thioredoxin h isoforms HvTrxh1 and HvTrxh2 differ in temporal and spatial distribution and in kinetic properties. Target proteins of HvTrxh1 and HvTrxh2 were identified in mature seeds and in seeds after 72 h of germination. Improvement of the established method for identification of thioredoxin-targeted proteins based on two-dimensional electrophoresis and fluorescence labelling of thiol groups was achieved by application of a highly sensitive Cy5 maleimide dye and large-format two-dimensional gels, resulting in a 10-fold increase in the observed number of labelled protein spots. The technique also provided information about accessible thiol groups in the proteins identified in the barley seed proteome. In total, 16 different putative target proteins were identified from 26 spots using tryptic in-gel digestion, matrix-assisted laser-desorption ionization–time-of-flight MS and database search. HvTrxh1 and HvTrxh2 were shown to have similar target specificity. Barley α-amylase/subtilisin inhibitor, previously demonstrated to be reduced by both HvTrxh1 and HvTrxh2, was among the identified target proteins, confirming the suitability of the method. Several α-amylase/trypsin inhibitors, some of which are already known as target proteins of thioredoxin h, and cyclophilin known as a target protein of m-type thioredoxin were also identified. Lipid transfer protein, embryospecific protein, three chitinase isoenzymes, a single-domain glyoxalase-like protein and superoxide dismutase were novel identifications of putative target proteins, suggesting new physiological roles of thioredoxin h in barley seeds.
Lipid rafts: heterogeneity on the high seas
Authors: PIKE Linda J.
Published: February 2004

Lipid rafts are membrane microdomains that are enriched in cholesterol and glycosphingolipids. They have been implicated in processes as diverse as signal transduction, endocytosis and cholesterol trafficking. Recent evidence suggests that this diversity of function is accompanied by a diversity in the composition of lipid rafts. The rafts in cells appear to be heterogeneous both in terms of their protein and their lipid content, and can be localized to different regions of the cell. This review summarizes the data supporting the concept of heterogeneity among lipid rafts and outlines the evidence for cross-talk between raft components. Based on differences in the ways in which proteins interact with rafts, the Induced-Fit Model of Raft Heterogeneity is proposed to explain the establishment and maintenance of heterogeneity within raft populations.
Probing Zn2+-binding effects on the zinc-ribbon domain of human general transcription factor TFIIB
Authors: GHOSH Mahua , ELSBY Laura M. , MAL Tapas K. , GOODING Jane M. , ROBERTS Stefan G. E. , IKURA Mitsuhiko
Published: February 2004

The general transcription factor, TFIIB, plays an important role in the assembly of the pre-initiation complex. The N-terminal domain (NTD) of TFIIB contains a zinc-ribbon motif, which is responsible for the recruitment of RNA polymerase II and TFIIF to the core promoter region. Although zinc-ribbon motif structures of eukaryotic and archaeal TFIIBs have been reported previously, the structural role of Zn2+ binding to TFIIB remains to be determined. In the present paper, we report NMR and biochemical studies of human TFIIB NTD, which characterize the structure and dynamics of the TFIIB Zn2+-binding domain in both Zn2+-bound and -free states. The NMR data show that, whereas the backbone fold of NTD is pre-formed in the apo state, Zn2+ binding reduces backbone mobility in the β-turn (Arg28–Gly30), induces enhanced structural rigidity of the charged-cluster domain in the central linker region of TFIIB and appends a positive surface charge within the Zn2+-binding site. V8 protease-sensitivity assays of full-length TFIIB support the Zn2+-dependent structural changes. These structural effects of Zn2+ binding on TFIIB may have a critical role in interactions with its binding partners, such as the Rpb1 subunit of RNA polymerase II.
Expression and localization of tumour necrosis factor-α and its converting enzyme in human abdominal aortic aneurysm
Authors: SATOH Hidetoshi , NAKAMURA Motoyuki , SATOH Mamoru , NAKAJIMA Takayuki , IZUMOTO Hiroshi , MAESAWA Chihaya , KAWAZOE Kouhei , MASUDA Tomoyuki , HIRAMORI Katsuhiko
Published: February 2004

Abdominal aortic aneurysm (AAA) is characterized by chronic aortic wall inflammation and loss of matrix components. Proinflammatory cytokines such as tumour necrosis factor-α (TNF-α) are thought to be involved in this inflammatory process and, therefore, to play an important role in the pathogenesis of human AAA. TNF-α-converting enzyme (TACE) has recently been purified and cloned as a disintegrin and metalloproteinase that converts TNF-α precursor into its mature form. The aim of the present study was to determine whether TNF-α and TACE were expressed and localized in aortic tissues in human AAA. Infrarenal aortic tissues were obtained from AAA patients (n=19) undergoing elective aneurysm reconstruction and from autopsy cases without cardiovascular disorders as normal controls (n=5). Internal thoracic artery samples were also obtained from patients with coronary artery disease undergoing coronary artery bypass grafting to represent biopsied conduit vessels (n=5). The AAA specimens were taken from the mid-portion of the aneurysm and from the longitudinal transition zone between the non-dilated aorta and the proximal aspect of the aneurysm. TNF-α and TACE mRNA levels were determined by real-time quantitative reverse transcriptase–PCR. Expression levels of both TNF-α mRNA and TACE mRNA were significantly greater in the transition zone than in the mid-portion (both P<0.05). Expression levels of both forms of mRNA were significantly higher in AAA samples than in control aortas or atherosclerotic arteries. There was a significant correlation between the expression of TNF-α mRNA with that of TACE mRNA in AAA (r=0.54, P<0.005). Immunostaining was positive for both TNF-α and TACE in CD68-positive macrophages in the media and adventitia obtained from the transition zone in AAA, whereas neither TNF-α nor TACE was expressed in control vessels. In conclusion, the concomitant activation and localization of TNF-α and TACE in the media and adventitia of the transition zone in human AAA underlines the importance of this system in the pathogenesis of this disorder.
Effects of oxidative stress on endothelial function after a high-fat meal
Authors: TSAI Wei-Chuan , LI Yi-Heng , LIN Chih-Chan , CHAO Ting-Hsing , CHEN Jyh-Hong
Published: February 2004

Postprandial lipaemia is known to cause endothelial dysfunction, but its underlying mechanism is still under debate. The present study was undertaken to investigate the effects of postprandial lipaemia on endothelial dysfunction and oxidative stress. We measured plasma glutathione peroxidase (GSH-Px), an antioxidant enzyme, and the urinary excretion of 8-epi-prostaglandin F2α (8-PGF2α), a free radical-catalysed product from the oxidative modification of arachidonic acid, in 16 healthy subjects (mean age, 30±5 years) without major coronary risk factors. Plasma high-sensitive C-reactive protein, soluble intercellular cell-adhesion molecule-1 and vascular cell-adhesion molecule-1 were also measured. High-resolution ultrasound was used to assess the flow-mediated vasodilatation (FMD) of the brachial artery. Blood and urine samples were collected before and 2, 4 and 6 h after a standard high-fat meal (3677 J, containing 50 g of fat). Serum triacylglycerol (triglyceride) increased and FMD decreased significantly after a high-fat meal. Plasma GSH-Px significantly decreased from 27.2±12.3 µg/ml to 25.7±11.8 µg/ml (P=0.022) 2 h after the meal, and urinary excretion of 8-PGF2α significantly increased from 1286±1401 pg/mg of creatinine to 2197±1343 pg/mg of creatinine (P=0.014) at 4 h after the meal. However, there were no significant changes in the levels of high-sensitive C-reactive protein and adhesion molecules after a high-fat meal. In conclusion, endothelial dysfunction was observed after consuming a high-fat meal and is associated with augmented oxidative stress manifested by the depletion of serum antioxidant enzymes and increased excretion of oxidative modification products.
Distribution and elimination of crystalloid fluid in pre-eclampsia
Authors: DROBIN Dan , HAHN Robert G.
Published: February 2004

Pre-eclampsia (PE) is a disease of pregnancy associated with peripheral oedema and hypovolaemia, but few details are known about how women with PE handle a volume load of crystalloid fluid compared with healthy pregnant women. To study this issue, Ringer's acetate solution (12.5 ml/kg of body weight) was given by intravenous infusion over 30 min to eight women with PE and to eight healthy pregnant women matched with respect to gestational week (mean, 34 weeks). Venous blood was sampled and excreted urine was collected over 90 min to study the time course of the volume expansion by means of volume kinetic analysis. The results show that the size of the central body fluid space expanded by the infused fluid was smaller in PE (mean, 2940 ml compared with 4240 ml respectively; P<0.04), and the clearance constants for distribution (100 ml/min compared with 43 ml/min; P<0.04) and elimination (125 ml/min compared with 36 ml/min; P<0.02) were higher in the women with PE than in the controls. Less excess volume accumulated in the central body fluid space in the presence of PE, whereas the rates of distribution and elimination were higher during and for 15 min after the infusion. It is concluded that Ringer's acetate solution fluid is both distributed and eliminated faster in women with PE than in matched pregnant controls.
Antioxidant defence mechanisms: new players in the pathogenesis of non-alcoholic steatohepatitis?
Authors: LECLERCQ Isabelle A.
Published: February 2004

This comment discusses the study by Videla and colleagues in this issue of Clinical Science in which they have analysed surgical liver biopsies from obese patients with non-alcoholic fatty liver disease and demonstrate that fatty liver is associated with a state of oxidative stress and a decreased antioxidant activity. As the disease progresses to steatohepatitis, the defect in antioxidant systems increases. Thus efficiency of antioxidant defence mechanisms, by setting the threshold at which pro-oxidants would become injurious, might represent a determining factor for disease progression from stable steatosis to steatohepatitis.
Frequency-modulation transient absorption spectrum of the HCCl Ã1A′′(0,0,0)←X̃1A′(0,0,0) transition
Authors: Chang Bor-Chen , Sears Trevor J.
Published: February 2004

The rotationally resolved spectrum of the HCCl Ã1A′′(0,0,0)←X̃1A′(0,0,0) transition between 12 230 and 12 410 cm-1 was obtained for the first time at Doppler-limited resolution using a frequency-modulation (FM) transient absorption technique. Five major subbands (Ka=0←1, 1←0, 1 ←2, 2←1, and 2←3) of HC35Cl and one subband (Ka=0←1) of HC37Cl were observed and assigned. In addition, the forbidden subband (Ka=0←0) of HC35Cl was also observed. Thus far, we have assigned most (more than 70%) of the strong lines. The analysis of these subbands determines the geometry for the HCCl Ã(0,0,0) level: ∠HCCl=134.7° and RC-Cl=1.623 Å while RC-H is fixed at the corresponding value of CH2. The height of the barrier to linearity was found to be approximately 2170 cm-1 above the vibrationless level of the Ã state. The rotational structure of this spectrum shows irregular perturbations. Both random and J-dependent (anomalous K-type doubling) effects were observed. As in methylene, the perturbations most likely arise from Renner-Teller effect between the two singlet states in combination with spin–orbit coupling between the singlet and triplet states.
The broad host range pathogen
Pseudomonas aeruginosa
strain PA14 carries two pathogenicity islands harboring plant and animal virulence genes
Authors: He Jianxin , Baldini Regina L. , Déziel Eric , Saucier Maude , Zhang Qunhao , Liberati Nicole T. , Lee Daniel , Urbach Jonathan , Goodman Howard M. , Rahme Laurence G.
Published: February 2004

The ubiquitous bacterium
Pseudomonas aeruginosa
is the quintessential opportunistic pathogen. Certain isolates infect a broad range of host organisms, from plants to humans. The pathogenic promiscuity of particular variants may reflect an increased virulence gene repertoire beyond the core
P. aeruginosa
genome. We have identified and characterized two
P. aeruginosa
pathogenicity islands (PAPI-1 and PAPI-2) in the genome of PA14, a highly virulent clinical isolate. The 108-kb PAPI-1 and 11-kb PAPI-2, which are absent from the less virulent reference strain PAO1, exhibit highly modular structures, revealing their complex derivations from a wide array of bacterial species and mobile elements. Most of the genes within these islands that are homologous to known genes occur in other human and plant bacterial pathogens. For example, PAPI-1 carries a complete gene cluster predicted to encode a type IV group B pilus, a well known adhesin absent from strain PAO1. However, >80% of the PAPI-1 DNA sequence is unique, and 75 of its 115 predicted ORF products are unrelated to any known proteins or functional domains. Significantly, many PAPI-1 ORFs also occur in several
P. aeruginosa
cystic fibrosis isolates. Twenty-three PAPI ORFs were mutated, and 19 were found to be necessary for full plant or animal virulence, with 11 required for both. The large set of “extra” virulence functions encoded by both PAPIs may contribute to the increased promiscuity of highly virulent
P. aeruginosa
strains, by directing additional pathogenic functions.
Mmm2p, a mitochondrial outer membrane protein required for yeast mitochondrial shape and maintenance of mtDNA nucleoids
Authors: Youngman Matthew J. , Hobbs Alyson E. Aiken , Burgess Shawn M. , Srinivasan Maithreyan , Jensen Robert E.
Published: February 2004

The mitochondrial outer membrane protein, Mmm1p, is required for normal mitochondrial shape in yeast. To identify new morphology proteins, we isolated mutations incompatible with the mmm1-1 mutant. One of these mutants, mmm2-1, is defective in a novel outer membrane protein. Lack of Mmm2p causes a defect in mitochondrial shape and loss of mitochondrial DNA (mtDNA) nucleoids. Like the Mmm1 protein (Aiken Hobbs, A.E., M. Srinivasan, J.M. McCaffery, and R.E. Jensen. 2001. J. Cell Biol. 152:401–410.), Mmm2p is located in dot-like particles on the mitochondrial surface, many of which are adjacent to mtDNA nucleoids. While some of the Mmm2p-containing spots colocalize with those containing Mmm1p, at least some of Mmm2p is separate from Mmm1p. Moreover, while Mmm2p and Mmm1p both appear to be part of large complexes, we find that Mmm2p and Mmm1p do not stably interact and appear to be members of two different structures. We speculate that Mmm2p and Mmm1p are components of independent machinery, whose dynamic interactions are required to maintain mitochondrial shape and mtDNA structure.
Mammalian PAR-1 determines epithelial lumen polarity by organizing the microtubule cytoskeleton
Authors: Cohen David , Brennwald Patrick J. , Rodriguez-Boulan Enrique , Müsch Anne
Published: February 2004

Epithelial differentiation involves the generation of luminal surfaces and of a noncentrosomal microtubule (MT) network aligned along the polarity axis. Columnar epithelia (e.g., kidney, intestine, and Madin-Darby canine kidney [MDCK] cells) generate apical lumina and orient MT vertically, whereas liver epithelial cells (hepatocytes and WIFB9 cells) generate lumina at cell–cell contact sites (bile canaliculi) and orient MTs horizontally. We report that knockdown or inhibition of the mammalian orthologue of Caenorhabditis elegans Par-1 (EMK1 and MARK2) during polarization of cultured MDCK and WIFB9 cells prevented development of their characteristic lumen and nonradial MT networks. Conversely, EMK1 overexpression induced the appearance of intercellular lumina and horizontal MT arrays in MDCK cells, making EMK1 the first known candidate to regulate the developmental branching decision between hepatic and columnar epithelial cells. Our experiments suggest that EMK1 primarily promotes reorganization of the MT network, consistent with the MT-regulating role of this gene product in other systems, which in turn controls lumen formation and position.
Differential functions of G protein and Baz–aPKC signaling pathways in Drosophila neuroblast asymmetric division
Authors: Izumi Yasushi , Ohta Nao , Itoh-Furuya Asako , Fuse Naoyuki , Matsuzaki Fumio
Published: February 2004

Drosophila melanogaster neuroblasts (NBs) undergo asymmetric divisions during which cell-fate determinants localize asymmetrically, mitotic spindles orient along the apical–basal axis, and unequal-sized daughter cells appear. We identified here the first Drosophila mutant in the Gγ1 subunit of heterotrimeric G protein, which produces Gγ1 lacking its membrane anchor site and exhibits phenotypes identical to those of Gβ13F, including abnormal spindle asymmetry and spindle orientation in NB divisions. This mutant fails to bind Gβ13F to the membrane, indicating an essential role of cortical Gγ1–Gβ13F signaling in asymmetric divisions. In Gγ1 and Gβ13F mutant NBs, Pins–Gαi, which normally localize in the apical cortex, no longer distribute asymmetrically. However, the other apical components, Bazooka–atypical PKC–Par6–Inscuteable, still remain polarized and responsible for asymmetric Miranda localization, suggesting their dominant role in localizing cell-fate determinants. Further analysis of Gβγ and other mutants indicates a predominant role of Partner of Inscuteable–Gαi in spindle orientation. We thus suggest that the two apical signaling pathways have overlapping but different roles in asymmetric NB division.
Human telomerase RNA and box H/ACA scaRNAs share a common Cajal body–specific localization signal
Authors: Jády Beáta E. , Bertrand Edouard , Kiss Tamás
Published: February 2004

Telomerase is a ribonucleoprotein reverse transcriptase that uses its RNA component as a template for synthesis of telomeric DNA repeats at the ends of linear eukaryotic chromosomes. Here, fluorescence in situ hybridization demonstrates that in HeLa cancer cells, human telomerase RNA (hTR) accumulates in the nucleoplasmic Cajal bodies (CBs). Localization of transiently expressed hTR to CBs is supported by a short sequence motif (411-UGAG-414) that is located in the 3′-terminal box H/ACA RNA-like domain of hTR and that is structurally and functionally indistinguishable from the CB-specific localization signal of box H/ACA small CB-specific RNAs. In synchronized HeLa cells, hTR shows the most efficient accumulation in CBs during S phase, when telomeres are most likely synthesized. CBs may function in post-transcriptional maturation (e.g., cap hypermethylation of hTR), but they may also play a role in the assembly and/or function of telomerase holoenzyme.
Spred-2 Suppresses Aorta-Gonad-Mesonephros Hematopoiesis by Inhibiting MAP Kinase Activation
Authors: Nobuhisa Ikuo , Kato Reiko , Inoue Hirofumi , Takizawa Makiko , Okita Keisuke , Yoshimura Akihiko , Taga Tetsuya
Published: February 2004

In midgestation mouse embryos, the aorta-gonad-mesonephros (AGM) region generates hematopoietic stem cells and definitive hematopoiesis is regulated by cell–cell interaction and signaling molecules. We showed that a Ras/mitogen-activated protein (MAP) kinase signaling-specific inhibitor and a dominant negative mutant Ras blocked the production of CD45+ hematopoietic cells in embryonic day 11.5 AGM culture, indicating an essential role for the MAP kinase pathway in AGM hematopoiesis. Overexpression of the Ras/MAP kinase pathway regulator, Spred-2, in the AGM culture significantly reduced the number of CD45+ cells. In contrast, production of CD45+ cells from the AGM region of Spred-2–null mice was up-regulated as compared with wild-type littermates. Furthermore, Spred-2–deficient mice exhibited elevated hematopoietic colony formation from vascular endothelial-cadherin+ cells. These data indicate that Spred-2 functions as a negative regulator of AGM hematopoiesis by inhibiting hematopoietic cytokine signaling.
Spontaneous Development of Psoriasis in a New Animal Model Shows an Essential Role for Resident T Cells and Tumor Necrosis Factor-α
Authors: Boyman Onur , Hefti Hans Peter , Conrad Curdin , Nickoloff Brian J. , Suter Mark , Nestle Frank O.
Published: February 2004

Psoriasis is a common T cell–mediated autoimmune disorder where primary onset of skin lesions is followed by chronic relapses. Progress in defining the mechanism for initiation of pathological events has been hampered by the lack of a relevant experimental model in which psoriasis develops spontaneously. We present a new animal model in which skin lesions spontaneously developed when symptomless prepsoriatic human skin was engrafted onto AGR129 mice, deficient in type I and type II interferon receptors and for the recombination activating gene 2. Upon engraftment, resident human T cells in prepsoriatic skin underwent local proliferation. T cell proliferation was crucial for development of a psoriatic phenotype because blocking of T cells led to inhibition of psoriasis development. Tumor necrosis factor-α was a key regulator of local T cell proliferation and subsequent disease development. Our observations highlight the importance of resident T cells in the context of lesional tumor necrosis factor-α production during development of a psoriatic lesion. These findings underline the importance of resident immune cells in psoriasis and will have implications for new therapeutic strategies for psoriasis and other T cell–mediated diseases.
Essential Role for OspA/B in the Life Cycle of the Lyme Disease Spirochete
Authors: Yang Xiaofeng F. , Pal Utpal , Alani Sophie M. , Fikrig Erol , Norgard Michael V.
Published: February 2004

The molecular basis of how Borrelia burgdorferi (Bb), the Lyme disease spirochete, maintains itself in nature via a complex life cycle in ticks and mammals is poorly understood. Outer surface (lipo)protein A (OspA) of Bb has been the most intensively studied of all borrelial molecular constituents, and hence, much has been speculated about the potential role(s) of OspA in the life cycle of Bb. However, the precise function of OspA (along with that of its close relative and operonic partner, outer surface [lipo]protein B [OspB]) heretofore has not been directly determined, due primarily to the inability to generate an OspA/B-deficient mutant from a virulent strain of Bb. In this study, we created an OspA/B-deficient mutant of an infectious human isolate of Bb (strain 297) and found that OspA/B function was not required for either Bb infection of mice or accompanying tissue pathology. However, OspA/B function was essential for Bb colonization of and survival within tick midguts, events crucial for sustaining Bb in its natural enzootic life cycle.
Transcriptional Activation of Metalloid Tolerance Genes inSaccharomyces cerevisiaeRequires the AP-1–like Proteins Yap1p and Yap8p
Authors: Wysocki Robert , Fortier Pierre-Karl , Maciaszczyk Ewa , Thorsen Michael , Leduc Anick , Odhagen Åsa , Owsianik Grzegorz , Ulaszewski Stanislaw , Ramotar Dindial , Tamás Markus J.
Published: February 2004

All organisms are equipped with systems for detoxification of the metalloids arsenic and antimony. Here, we show that two parallel pathways involving the AP-1–like proteins Yap1p and Yap8p are required for acquisition of metalloid tolerance in the budding yeast S. cerevisiae. Yap8p is demonstrated to reside in the nucleus where it mediates enhanced expression of the arsenic detoxification genes ACR2 and ACR3. Using chromatin immunoprecipitation assays, we show that Yap8p is associated with the ACR3 promoter in untreated as well as arsenic-exposed cells. Like for Yap1p, specific cysteine residues are critical for Yap8p function. We further show that metalloid exposure triggers nuclear accumulation of Yap1p and stimulates expression of antioxidant genes. Yap1p mutants that are unable to accumulate in the nucleus during H2O2treatment showed nearly normal nuclear retention in response to metalloid exposure. Thus, our data are the first to demonstrate that Yap1p is being regulated by metalloid stress and to indicate that this activation of Yap1p operates in a manner distinct from stress caused by chemical oxidants. We conclude that Yap1p and Yap8p mediate tolerance by controlling separate subsets of detoxification genes and propose that the two AP-1–like proteins respond to metalloids through distinct mechanisms.
The Tetraspan Protein EMP2 Modulates the Surface Expression of Caveolins and Glycosylphosphatidyl Inositol-linked Proteins
Authors: Wadehra Madhuri , Goodglick Lee , Braun Jonathan
Published: February 2004

Caveolae are a subset of lipid rafts enriched in glycosphingolipids and cholesterol-rich domains, but selectively lacking glycosylphosphatidyl inositol-anchored proteins (GPI-APs). Caveolin proteins are the organizing component of caveolae, but the corresponding proteins for other classes of lipid rafts are poorly defined. Epithelial membrane protein-2 (EMP2), a member of the four-transmembrane superfamily, facilitates plasma membrane delivery of certain integrins. In this study, we found by laser confocal microscopy that EMP2 was associated with GPI-APs (detected by the GPI-AP binding bacterial toxin proaerolysin). Biochemical membrane fractionation and methyl-β-cyclodextrin treatment demonstrated that this association occurred within lipid rafts. EMP2 did not associate with caveolin-bearing membrane structures, and recombinant overexpression of EMP2 in NIH3T3 cells decreased caveolin-1 and caveolin-2 protein levels while increasing the surface expression of GPI-APs. Conversely, a ribozyme construct that specifically cleaves the EMP2 transcript reduced surface GPI-APs and increased caveolin protein expression. These findings suggest that EMP2 facilitates the formation and surface trafficking of lipid rafts bearing GPI-APs, and reduces caveolin expression, resulting in impaired formation of caveolae.
The Role of Rab27a in the Regulation of Melanosome Distribution within Retinal Pigment Epithelial Cells
Authors: Futter Clare E. , Ramalho José S. , Jaissle Gesine B. , Seeliger Mathias W. , Seabra Miguel C.
Published: February 2004

Melanosomes within the retinal pigment epithelium (RPE) of mammals have long been thought to exhibit no movement in response to light, unlike fish and amphibian RPE. Here we show that the distribution of melanosomes within the mouse RPE undergoes modest but significant changes with the light cycle. Two hours after light onset, there is a threefold increase in the number of melanosomes in the apical processes that surround adjacent photoreceptors. In skin melanocytes, melanosomes are motile and evenly distributed throughout the cell periphery. This distribution is due to the interaction with the cortical actin cytoskeleton mediated by a tripartite complex of Rab27a, melanophilin, and myosin Va. In ashen (Rab27a null) mice RPE, melanosomes are unable to move beyond the adherens junction axis and do not enter apical processes, suggesting that Rab27a regulates melanosome distribution in the RPE. Unlike skin melanocytes, the effects of Rab27a are mediated through myosin VIIa in the RPE, as evidenced by the similar melanosome distribution phenotype observed in shaker-1 mice, defective in myosin VIIa. Rab27a and myosin VIIa are likely to be required for association with and movement through the apical actin cytoskeleton, which is a prerequisite for entry into the apical processes.
A Tektin Homologue Is Decreased inChlamydomonasMutants Lacking an Axonemal Inner-Arm Dynein
Authors: Yanagisawa Haru-aki , Kamiya Ritsu
Published: February 2004

In ciliary and flagellar axonemes, various discrete structures such as inner and outer dynein arms are regularly arranged on the outer doublet microtubules. Little is known about the basis for their regular arrangement. In this study, proteins involved in the attachment of inner-arm dyneins were searched by a microtubule overlay assay on Chlamydomonas mutant axonemes. A 58-kDa protein (p58) was found ∼80% diminished in the mutants ida6 and pf3, both lacking one (species e) of the seven inner-arm species (a–g). Analysis of its cDNA indicated that p58 is homologous to tektin, a protein that was originally found in sea urchin and thought to be crucial for the longitudinal periodicity of the doublet microtubule. Unlike sea urchin tektin, which is a component of protofilament ribbons that occur after Sarkosyl treatment of axonemes, p58 was not contained in similar Sarkosyl-resistant ribbons from Chlamydomonas axonemes. Immunofluorescence microscopy showed that p58 was localized uniformly along the axoneme and on the basal body. The p58 signal was reduced in ida6 and pf3. These results suggest that a reduced amount of p58 is sufficient for the production of outer doublets, whereas an additional amount of it is involved in inner-arm dynein attachment.
Separate Roles and Different Routing of Calnexin and ERp57 in Endoplasmic Reticulum Quality Control Revealed by Interactions with Asialoglycoprotein Receptor Chains
Authors: Frenkel Zehavit , Shenkman Marina , Kondratyev Maria , Lederkremer Gerardo Z.
Published: February 2004

The thiol oxidoreductase endoplasmic reticulum (ER)p57 interacts with newly synthesized glycoproteins through ternary complexes with the chaperones/lectins calnexin or calreticulin. On proteasomal inhibition calnexin and calreticulin concentrate in the pericentriolar endoplasmic reticulum-derived quality control compartment that we recently described. Surprisingly, ERp57 remained in an endoplasmic reticulum pattern. Using asialoglycoprotein receptor H2a and H2b as models, we determined in pulse-chase experiments that both glycoproteins initially bind to calnexin and ERp57. However, H2b, which will exit to the Golgi, dissociated from calnexin and remained bound for a longer period to ERp57, whereas the opposite was true for the endoplasmic reticulum-associated degradation substrate H2a that will go to the endoplasmic reticulum-derived quality control compartment. At 15°C, ERp57 colocalized with H2b adjacent to an endoplasmic reticulum-Golgi intermediate compartment marker. Posttranslational inhibition of glucose excision prolonged association of H2a precursor to calnexin but not to ERp57. Preincubation with a low concentration (15 μg/ml) of the glucosidase inhibitor castanospermine prevented the association of H2a to ERp57 but not to calnexin. This low concentration of castanospermine accelerated the degradation of H2a, suggesting that ERp57 protects the glycoprotein from degradation and not calnexin. Our results suggest an early chaperone-mediated sorting event with calnexin being involved in the quality control retention of molecules bound for endoplasmic reticulum-associated degradation and ERp57 giving initial protection from degradation and later assisting the maturation of molecules that will exit to the Golgi.
The promoter of the Chinese hamster ovary dihydrofolate reductase gene regulates the activity of the local origin and helps define its boundaries
Authors: Saha Swati , Shan Yujie , Mesner Larry D. , Hamlin Joyce L.
Published: February 2004

The dihydrofolate reductase (DHFR) and 2BE2121 genes in the Chinese hamster are convergently transcribed in late G1 and early S phase, and bracket an early-firing origin of replication that consists of a 55-kb zone of potential initiation sites. To test whether transcription through the DHFR gene is required to activate this origin in early S phase, we examined the two-dimension (2D) gel patterns of replication intermediates from several variants in which parts or all of the DHFR promoter had been deleted. In those variants in which transcription was undetectable, initiation in the intergenic spacer was markedly suppressed (but not eliminated) in early S phase. Furthermore, replication of the locus required virtually the entire S period, as opposed to the usual 3–4 h. However, restoration of transcription with either the wild-type Chinese hamster promoter or a Drosophila-based construct restored origin activity to the wild-type pattern. Surprisingly, 2D gel analysis of promoterless variants revealed that initiation occurs at a low level in early S phase not only in the intergenic region, but also in the body of the DHFR gene. The latter phenomenon has never been observed in the wild-type locus. These studies suggest that transcription through the gene normally increases the efficiency of origin firing in early S phase, but also suppresses initiation in the body of the gene, thus helping to define the boundaries of the downstream origin.
Nuclear protein phosphatases with Kelch-repeat domains modulate the response to brassinosteroids in Arabidopsis
Authors: Mora-García Santiago , Vert Grégory , Yin Yanhai , Caño-Delgado Ana , Cheong Hyeonsook , Chory Joanne
Published: February 2004

Perception of the plant steroid hormone brassinolide (BL) by the membrane-associated receptor kinase BRI1 triggers the dephosphorylation and accumulation in the nucleus of the transcriptional modulators BES1 and BZR1. We identified bsu1-1D as a dominant suppressor of bri1 in Arabidopsis. BSU1 encodes a nuclear-localized serine–threonine protein phosphatase with an N-terminal Kelch-repeat domain, and is preferentially expressed in elongating cells. BSU1 is able to modulate the phosphorylation state of BES1, counteracting the action of the glycogen synthase kinase-3 BIN2, and leading to increased steady-state levels of dephosphorylated BES1. BSU1 belongs to a small gene family; loss-of-function analyses unravel the extent of functional overlap among members of the family and confirm the role of these phosphatases in the control of cell elongation by BL. Our data indicate that BES1 is subject to antagonistic phosphorylation and dephosphorylation reactions in the nucleus, which fine-tune the amplitude of the response to BL.
Collaborative digital reference: an Ask a Librarian (UK) overview
Authors: Berube Linda
Published: February 2004

Since its inception in 1997, Ask a Librarian (UK) has been a model for best practice in collaborative digital reference in public libraries. Innovative development currently includes trialling the integration of virtual reference with the standard Web‐form service. In addition to a description of the Ask a Librarian service, this paper provides an overview of digital reference practice, procedures, and issues, with particular emphasis on collaboration.
Optimisation of the utilisation of cashew apple in yogurt production
Authors: Aroyeun S.O.
Published: February 2004

Cashew apple was used as a nutritional additive in the production of yogurt. The yogurt so produced (CAY‐A) had a higher vitamin C content of 53.70g/100ml than both samples B and C with values 14.1/100ml and 19.2g/100ml of vitamin C respectively. The cashew apple fortified yogurt was evaluated for physicochemical parameters like pH, refractive index, dpecific gravity, titratable acidity, ash, moisture, protein and fat and the values 4.10 (pH), 20° Brix soluble solid content, specific gravity 1.023, titratable acidity (0.78g/100ml lactic acid), 0.84 per cent ash, 77.0 per cent moisture, 3.22 per cent protein and 3.2 per cent fat were obtained. The three yogurts produced were presented to ten regular tasters of yogurt for sensory evaluations using Fan Milk yogurt as the reference sample C. Assessors were asked to identify the odd sample. This method was used to determine if ingredient substitution or some other change in the manufacturing process could result in a detectable difference in products. The result obtained indicated that the yogurt into which cashew apple had been added compared favorably with the reference sample in all the attributes evaluated and there was no significant difference at p ≤ 0.05.
Legumes and grains are important sources of selenium in the Mauritian vegetarian diet
Authors: Subratty A.H. , Seebhujun A. , Khadaroo N. , Fakira‐Jhurry A. , Reesaul C. , Gunny F.B.H.
Published: February 2004

This article focuses on the selenium (Se) content of 29 legumes and grains available in Mauritius. Selenium was determined using an ultraviolet/visible spectrophotometric method. Results showed that there was considerable variation in the Se content of the legumes and grains that varied from 0.009 ± 0.003μg/g in Dhall Mung (Phaseolus areus) to 1.380 ± 0.003μg/g in Gram Noir (Cicer arietinum). The overall mean Se content of the legumes and grains analysed was 0.231 ± 0.00μg/g. In conclusion, the present study tends to show that legumes and grains are important vehicles of Se intake in the Mauritian diet.
Flexible tactile sensor technology: bringing haptics to life
Authors: Lowe Mark , King Alison , Lovett Elizabeth , Papakostas Thomas
Published: February 2004

The continued evolution of computer technology requires us now more than ever to investigate and understand man‐machine interfaces. Physical interface peripherals such as touch‐screens and force feedback systems demand a comprehension of the tactile forces involved. To accomplish this, flexible, easy‐to‐install, minimally intrusive sensors are essential. Thanks to the development of such sensors, many doors have been opened for innovative haptic applications in a variety of fields including medicine, manufacturing, and entertainment.
Force‐based impedance control of a haptic master system for teleoperation
Authors: Frisoli A. , Sotgiu E. , Avizzano C.A. , Checcacci D. , Bergamasco M.
Published: February 2004

This paper analyzes the design of a force‐based impedance control for a haptic interface system characterized by a parallel kinematics. By exploiting the features of parallel mechanisms, which perform better than the serial ones in terms of dynamic performance, stiffness and position accuracy, and by implementing a closed‐loop force control, the transparency of a haptic master system and the fidelity of resultant force feedback can be consistently improved. Issues for design and control as well as aspects of performance evaluation of haptic interfaces are treated within the paper and some results of the experimental characterization of a haptic interface are presented.
Magnetorheological fluids based haptic device
Authors: Li W.H. , Du H. , Guo N.Q. , Kosasih P.B.
Published: February 2004

This paper presents the development of a haptic device working with magnetorheological (MR) fluids. The computer screen is portioned in various segments and the controllable software monitors the attributed values of the voltage to the screen. The motion of a cursor on the computer screen is controlled by the MR actuator. A resistance force, the strength of which depends on the cursor position, would be generated and perceived by the user. The performance of the device is tested and verified with finite element analysis.
Theoretical and experimental analysis of a piezoelectric tactile sensor for use in endoscopic surgery
Authors: Dargahi Javad , Najarian Siamak
Published: February 2004

This paper describes design, theoretical, and experimental analysis of a polyvinylidene fluoride (PVDF) tactile sensor, which could be integrated with an endoscopic grasper. The sensor exhibited high force sensitivity and linearity. Finite element analysis was employed to study the structural analysis of the tactile sensor with various load application and the results of this modelling are presented as the shear stress distribution and deformation contours. A comparison was made between the theoretical modeling and the experimental results.
A mobile‐agent platform and a game application specifications using M‐UML
Authors: Saleh Kassem , El Morr Christo , Mourtada Aref , Morad Yahya
Published: February 2004

This paper provides the specification of a platform for the management of mobile agents, and describes, using Mobile Unified Modeling Language (M‐UML), a mobile game application that uses the specified mobile platform. The specified platform considers the architectural guidelines described by the Foundation for Intelligent Physical Agents that allows the interoperability between agents running on different agent platforms. Our platform specifications provide the application developer with an application programming interface (API) that allows the launching of mobile agents with two optional features, security and fault‐tolerance. Using the platform’s APIs, new mobile games can then be efficiently developed and deployed on the Internet.
Development of a distributed multi‐player computer game for scientific experimentation of team training protocols
Authors: Cao Richard Sen , Volz Richard A. , Johnson Jamison , Whetzel Jonathan , Xu Dianxiang , Nanjanath Maitreyi
Published: February 2004

To support research on using intelligent agents in team training, a distributed multi‐player game in Java that executes in real time was developed. Basing development on the game Space Fortress, which has been widely used by cognitive psychologists for studying training protocols, typical experiments involved in excess of 100 trials of the game under different training conditions. Describes the design of this game to achieve these features and real‐time performance in the Java environment.
Real Tournament – mobile context‐aware gaming for the next generation
Authors: Wu Maomao , Mitchell Keith , McCaffery Duncan , Finney Joe , Friday Adrian
Published: February 2004

With the recent advances in mobile networking, context‐aware computing, and sensor‐based computing, researchers and game designers are able to explore the potential of combining these new technologies to develop mobile, networked, context‐aware, augmented reality multiplayer games. As part of new research collaboration between Lancaster University, Cisco Systems, Microsoft Research and Orange – MIPv6 Systems Research Lab, such a mobile context‐aware multiplayer game is proposed and explored. The proposed game, Real Tournament, gathers real‐time contextual information, e.g. physical location and orientation, from the players and injects them into the game engine to generate game events.
Taxonomy of online game security
Authors: Ki Junbaek , Hee Cheon Jung , Kang Jeong‐Uk , Kim Dogyun
Published: February 2004

Presents a classification of known attacks in online games and provide security solutions against them. While previous works just presented attacks and solutions case by case, this paper newly classifies attacks by objectives and methods. Moreover, presents attacks in each of four layers: client, server, network, and environment. Through this systematic classification, solutions can be provided more efficiently against even unknown future attacks.