bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of a delivery vehicle for intracellular transport of botulinum neurotoxin antagonists1
Authors: Goodnough Michael C , Oyler George , Fishman Paul S , Johnson Eric A , Neale Elaine A , Keller James E , Tepp William H , Clark Mike , Hartz Stephanie , Adler Michael
Published: October 2002

A targeted delivery vehicle (DV) was developed for intracellular transport of emerging botulinum neurotoxin (BoNT) antagonists. The DV consisted of the isolated heavy chain (HC) of BoNT/A coupled to a 10‐kDa amino dextran via the heterobifunctional linker 3‐(2‐pyridylthio)‐propionyl hydrazide. The HC served to target BoNT‐sensitive cells and promote internalization of the complex, while the dextran served as a platform to deliver model therapeutic molecules to the targeted cells. To determine the ability of this chimeric glycoprotein to enter neurons, dextran and HC were labeled independently with the fluorescent dyes Oregon green 488 and Cy3, respectively. Internalization of DV was monitored in primary cortical cells using laser confocal microscopy. Incubation of cells for 24 h with DV resulted in discrete punctate labeling of both soma and processes. The Cy3 and Oregon green 488 signals were generally co‐localized, suggesting that the complex remained in the same intracellular compartment during the initial 24 h. The DV‐associated fluorescence was reduced progressively by co‐application of increasing concentrations of unlabeled BoNT/A holotoxin. The results suggest that the BoNT/A HC is able to mediate internalization of a coupled dextran, even though the latter bears no resemblance to the BoNT/A light chain (LC). The HC of BoNT/A thus offers promise as a selective carrier to deliver BoNT antagonists to the nerve terminal cytoplasm for inhibiting the proteolytic activity of internalized BoNT/A LC.
Temperature effects on the presteady‐state and transport‐associated currents of GABA cotransporter rGAT1
Authors: Binda Francesca , Bossi Elena , Giovannardi Stefano , Forlani Greta , Peres Antonio
Published: October 2002

The effects of temperature on the γ‐aminobutyric acid (GABA) uptake and on the presteady‐state and transport‐associated currents of the GABA cotransporter, rat γ‐aminobutyric acid transporter 1 (rGAT1), have been studied using heterologous oocyte expression and voltage‐clamp. Increasing temperature from 15 to 30°C increased GABA uptake, diminished the maximal value of the relaxation time constant of the presteady‐state currents and increased the amplitude of the current associated with the transport of GABA. The curve of the presteady‐state charge versus voltage was shifted toward negative potentials by increasing the temperature, while the maximal amount of charge (Q
max) remained constant; the τ versus V curve was also negatively shifted by increasing temperatures. Analysis of the outward (α) and inward (β) rate constants as functions of temperature showed that they are affected differently, with a Q
10=3.4 for α and Q
10=1.5 for β. The different temperature coefficients of the rate constants account for the observed shifts. These observations are consistent with a charge moving mechanism based on a conformational change of the protein; the weaker temperature sensitivity of the inward rate constant suggests a rate‐limiting diffusional component on this process.
Aggregation of LDL with chondroitin‐4‐sulfate makes LDL oxidizable in the presence of water‐soluble antioxidants
Authors: Abuja Peter M
Published: October 2002

The content of plasma and arterial interstitial fluid in water‐soluble antioxidants makes it unlikely for low‐density lipoprotein (LDL) to oxidize by the oxidation mechanisms most frequently discussed. By aggregation of LDL in the presence of chondroitin‐4‐sulfate (C‐4‐S), but not with chondroitin‐6‐sulfate or sphingomyelinase, a complex arises which can oxidize in the presence of 20 μM ascorbate and 300 μM urate. This oxidation sensitivity even persists after the gel‐filtration of an LDL/C‐4‐S/Cu2+ complex, indicating entrapment of Cu2+ within. This corresponds well to the known ability of C‐4‐S to bind copper ions and is a potential mechanism by which LDL oxidation in the arterial intima is facilitated after prolonged retention by the extracellular matrix.
Caspase 3 regulates phosphatidylserine externalization and phagocytosis of oxidatively stressed erythrocytes
Authors: Mandal Debabrata , Moitra Prasun K , Saha Samiran , Basu Joyoti
Published: October 2002

The appearance of phosphatidylserine (PS) on the outer surface of red cells is an important signal for their uptake by macrophages. We report for the first time that procaspase 3 present in the anucleated mature human erythrocyte is activated under oxidative stress induced by t‐butylhydroperoxide leading to impairment of the aminophospholipid translocase, PS externalization and increased erythrophagocytosis. This is the first report linking caspase 3 activation to inhibition of flippase activity and uptake of red cells by macrophages.
Glucose induces and leptin decreases expression of uncoupling protein‐2 mRNA in human islets
Authors: Brown James E.P , Thomas Steven , Digby Janet E , Dunmore Simon J
Published: October 2002

Elevated islet uncoupling protein‐2 (UCP‐2) impairs β‐cell function and UCP‐2 may be increased in clinical obesity and diabetes. We investigated the effects of glucose and leptin on UCP‐2 expression in isolated human islets. Human islets were incubated for 24 h with glucose (5.5–22 mmol/l)±leptin (0–10 nmol/l). Some islet batches were incubated at high (22 mmol/l), and subsequently lower (5.5 mmol/l), glucose to assess reversibility of effects. Leptin effects on insulin release were also measured. Glucose dose‐dependently increased UCP‐2 expression in all islet batches, maximally by three‐fold. This was not fully reversed by subsequently reduced glucose levels. Leptin decreased UCP‐2 expression by up to 75%, and maximally inhibited insulin release by 47%, at 22 mmol/l glucose. This is the first report of UCP‐2 expression in human islets and provides novel evidence of its role in the loss of β‐cell function in diabetes.
Purification and characterisation of the BIOH protein from the biotin biosynthetic pathway
Authors: Tomczyk Nicholas H , Nettleship Joanne E , Baxter Robert L , Crichton Hilary J , Webster Scott P , Campopiano Dominic J
Published: October 2002

Conversion of pimeloyl‐coenzyme A (CoA) to biotin in Escherichia coli requires at least four enzymes encoded by genes in the bio operon. One gene, bioH, which is not present in the bioABFCD operon, is required for the synthesis of pimeloyl‐CoA but its exact role in formation of this intermediate is unknown. To investigate this further, we have overexpressed and purified the bioH gene products from both E. coli (BIOH EC) and Neisseria meningitis (BIOH NM) in E. coli. When purified BIOH was incubated with excess CoA and analysed by electrospray mass spectrometry a species of mass corresponding to a BIOH:CoA complex was observed. Mutation of a conserved serine residue to alanine (BIOH EC S82A) did not prevent CoA binding. This is the first report of the purification of BIOH and the observation of a small molecule bound to the protein provides clues to its role in pimeloyl‐CoA synthesis.
The identification of the acid–base catalyst of α‐arabinofuranosidase from Geobacillus stearothermophilus T‐6, a family 51 glycoside hydrolase
Authors: Shallom Dalia , Belakhov Valery , Solomon Dmitry , Gilead-Gropper Sara , Baasov Timor , Shoham Gil , Shoham Yuval
Published: October 2002

The α‐L‐arabinofuranosidase from Geobacillus stearothermophilus T‐6 (AbfA T‐6) belongs to the retaining family 51 glycoside hydrolases. The conserved Glu175 was proposed to be the acid–base catalytic residue. AbfA T‐6 exhibits residual activity towards aryl β‐D‐xylopyranosides. This phenomenon was used to examine the catalytic properties of the putative acid–base mutant E175A. Data from kinetic experiments, pH profiles, azide rescue, and the identification of the xylopyranosyl azide product provide firm support to the assignment of Glu175 as the acid–base catalyst of AbfA T‐6.
Chromatin remodeling during spermiogenesis: a possible role for the transition proteins in DNA strand break repair
Authors: Boissonneault Guylain
Published: October 2002

An important chromatin remodeling process is taking place during spermiogenesis in mammals and DNA strand breaks must be produced to allow the accompanying change in DNA topology. Endogenous DNA strand breaks are indeed detected at mid‐spermiogenesis steps but are no longer present in mature sperm. Both in vitro and in vivo evidence suggests that the DNA‐binding and condensing activities of a set of basic nuclear ‘transition proteins’ may be crucial to the integrity of the chromatin remodeling process. We propose that these proteins are necessary for the repair of the strand breaks so that DNA fragmentation is minimized in the mature sperm.
The gift of Gab
Authors: Liu Yan , Rohrschneider Larry R
Published: October 2002

Gab proteins, including mammalian Gab1, Gab2, Gab3, Drosophila DOS and Caenorhabditis elegans Soc1, comprise a growing family of scaffolding/docking molecules involved in multiple signaling pathways mediated by receptor tyrosine kinases (RTKs) and non‐RTK receptors. This paper reviews the structure/function relationships of Gab proteins and their biological roles during normal growth, differentiation and development programs.
The hepoxilin analog PBT‐3 inhibits heparin‐activated platelet aggregation evoked by ADP
Authors: Reynaud D. , Hinek Alexander , Pace-Asciak C.R.
Published: October 2002

We have previously shown that PBT‐3, a stable synthetic analog of hepoxilins, inhibits the aggregation of human platelets in vitro evoked by collagen through inhibition of thromboxane A2 formation and action on the TP receptor. We now show that PBT‐3 is capable of potently inhibiting the second phase of aggregation evoked by ADP in both washed human platelets and platelet‐rich plasma (PRP), a phase associated with thromboxane formation. Aspirin blocks this second phase as well; so does the thromboxane receptor antagonist SQ 29,548. When ADP‐evoked aggregation in PRP is activated by heparin through an enhancement of thromboxane formation, PBT‐3, aspirin as well as SQ 29,548 block this activation through different mechanisms. These data confirm the inhibitory action of PBT‐3 on aggregation of human platelets through inhibition of both thromboxane formation and blockade of thromboxane receptor action and suggest that this family of compounds may be useful in the treatment of thrombotic disorders in combination with heparin.
The chicken–egg scenario of protein folding revisited
Authors: Uversky Vladimir N. , Fink Anthony L.
Published: October 2002

What is the first step in protein folding – hydrophobic collapse (compaction) or secondary structure formation? It is still not clear if the major driving force in protein folding is hydrogen bonding or hydrophobic interactions or both. We analyzed data on the conformational characteristics of 41 globular proteins in native and partially folded conformational states. Our analysis shows that a good correlation exists between relative decrease in hydrodynamic volume and increase in secondary structure content. No compact equilibrium intermediates lacking secondary structure, or highly ordered non‐compact species, were found. This correlation provides experimental support for the hypothesis that hydrophobic collapse occurs simultaneously with formation of secondary structure in the early stages of the protein folding.
Differential activation of nuclear factor‐κB by tumour necrosis factor receptor subtypes. TNFR1 predominates whereas TNFR2 activates transcription poorly
Authors: McFarlane Shona M , Pashmi Ghazaleh , Connell Michelle C , Littlejohn Alison F , Tucker Steven J , Vandenabeele Peter , MacEwan David J
Published: October 2002

Tumour necrosis factor‐α (TNF‐α) signals though two receptors, TNFR1 and TNFR2. TNFR1 has a role in cytotoxicity, whereas TNFR2 regulates death responses or proliferation. TNF activates pro‐inflammatory transcription factor nuclear factor‐κB (NF‐κB) by uncertain signalling mechanisms. Here we report the contribution of each TNFR towards the NF‐κB activation processes. In human cells expressing endogenous or exogenous TNFR2, in addition to TNFR1, we found both TNFRs capable of activating NF‐κB, as measured by IκBα (inhibitor of NF‐κB) degradation, electrophoretic mobility shift assay and NF‐κB gene reporter assays. TNFR2 activation did not degrade IκBβ. However, TNF‐effects on NF‐κB activation occurred predominantly through TNFR1, with TNFR2 activating the transcription factor poorly.
Selected BTB/POZ‐kelch proteins bind ATP
Authors: T'Jampens Davy , Devriendt Liesbeth , De Corte Veerle , Vandekerckhove Joël , Gettemans Jan
Published: October 2002

Proteins with a bric‐à‐brac, tramtrack, broad‐complex/Poxvirus zinc fingers (BTB/POZ) domain are implicated in a broad variety of biological processes, including DNA binding, regulation of gene transcription and organization of macromolecular structures. Kelch domain containing BTB/POZ proteins like Mayven and Keap1 display limited sequence similarity with the actin‐fragmin kinase from Physarum, a protein kinase with a kelch domain. We show that mouse Keap1, a Caenorhabditis elegans protein that we named CKR, and human Mayven bind 5′‐p‐fluorosulfonyl‐benzoyl‐adenosine (FSBA), a covalently modifying ATP analogue. Binding with 2‐azido‐ATP or ATP‐Sepharose is also demonstrated. In contrast to Mayven, FSBA binding by CKR and Keap1 was specifically inhibited by excess ATP. The ATP binding pocket is located in the N‐terminal half of Keap1. Our findings indicate that several, but not all, BTB/POZ‐kelch domain proteins possess an inconspicuous ATP binding cassette.
Autotaxin promotes motility via G protein‐coupled phosphoinositide 3‐kinase γ in human melanoma cells
Authors: Lee Hoi Young , Bae Gyu-Un , Jung In Duk , Lee Jang-Soon , Kim Yong Kee , Noh Sung Hoon , Stracke Mary L , Park Chang Gyo , Lee Hyang Woo , Han Jeung-Whan
Published: October 2002

Autotaxin (ATX), an exo‐nucleotide pyrophosphatase and phosphodiesterase, stimulates tumor cell motility at sub‐nanomolar levels and augments invasiveness and angiogenesis. We investigated the role of G protein‐coupled phosphoinositide 3‐kinase γ (PI3Kγ) in ATX‐mediated tumor cell motility stimulation. Pretreatment of human melanoma cell line A2058 with wortmannin or LY294002 inhibited ATX‐induced motility. ATX increased the PI3K activity in p110γ, but not p85, immunoprecipitates. This effect was abrogated by PI3K inhibitors or inhibited by pertussis toxin. Furthermore, stimulation of tumor cell motility by ATX was inhibited by catalytically inactive form of PI3Kγ, strongly indicating the crucial role of PI3Kγ for ATX‐mediated motility in human melanoma cells
Mild heat shock induces cyclin D1 synthesis through multiple Ras signal pathways
Authors: Han Song Iy , Oh Su Young , Jeon Won Je , Kim Jung Mo , Lee Jun Hyuk , Chung Hae Young , Choi Yung Hyun , Yoo Mi Ae , Kim Han Do , Kang Ho Sung
Published: October 2002

Hyperthermia such as that occurring during fever may improve cell survival during infection, although its mechanism of action is largely unknown. Here we show that acute exposure to mild, but not severe, heat shock induces the synthesis of cyclin D1 that plays a critical role(s) in G1 progression of the cell cycle. This induction seemed to be regulated through multiple Ras signal pathways involving extracellular signal‐regulated kinase, phosphatidylinositol 3‐kinase, and Rac1/NADPH oxidase, all of which have well been documented to be responsible for growth factor‐induced cyclin D1 expression. In a physiological sense, mild heat shock may regulate cell proliferation through inducing cyclin D1 along with growth factors.
Substrate interferes with dimerisation of outer membrane phospholipase A
Authors: Kingma Roelie L , Egmond Maarten R
Published: October 2002

Outer membrane phospholipase A (OMPLA) activity is regulated by reversible dimerisation with the dimer being the active species. Observed lag phases in activity indicated that dimerisation may be slow relative to turnover. A covalent OMPLA dimer indeed did not display lag phase behaviour. A model for OMPLA kinetics was proposed accounting for a slow dimerisation step. Preincubation conditions determined the initial amount of monomer and influenced both lag times and final activities. Under the conditions used, substrate concentrations higher than 50 mol% inhibited OMPLA activity and increased lag times. Our results may shed more light on mechanisms controlling OMPLA activity in vivo.
Excess noise in modified conductance states following the interaction of ryanoids with cardiac ryanodine receptor channels
Authors: Tanna Bhavna , Welch William , Ruest Luc , Sutko John L , Williams Alan J
Published: October 2002

The interaction of ryanodine with the ryanodine receptor (RyR) produces profound changes in channel function. Open probability increases dramatically and conductance is reduced. In this report we describe differences in the properties of reduced conductance states produced by the interaction of ryanodine derivatives with RyR channels. Some reduced conductance states are considerably noisier than the normal open state of the RyR channel. Inspection and analysis of these events reveals that the excess noise arises from transitions between two conductance states. Following the interaction of certain ryanodine derivatives, RyR channels undergo transitions between two conformations with slightly different ion‐handling properties.
Bcl‐2 overexpression prevents apoptosis induced by ceramidase inhibitors in malignant melanoma and HaCaT keratinocytes
Authors: Raisova Monika , Goltz Gerit , Bektas Meryem , Bielawska Alicja , Riebeling Christian , Hossini Amir M , Eberle Jürgen , Hannun Yusuf A , Orfanos Constantin E , Geilen Christoph C
Published: October 2002

We examined the biological effects of the ceramide analogues (1S,2R)‐2‐N‐myristoylamino‐1‐phenyl‐1‐propanol (D‐e‐MAPP) and (1R,2R)‐2‐N‐myristoylamino‐1‐(4‐nitrophenyl)‐1,3‐propandiol (D‐NMAPPD) on human HaCaT keratinocytes and human melanoma cells. We could demonstrate that D‐e‐MAPP and D‐NMAPPD are able to suppress acid ceramidase activity. The elevation of the endogenous level of ceramide is followed by induction of apoptosis and suppression of proliferation in HaCaT keratinocytes. Moreover, we recently identified a group of human melanoma cell populations which are heterogeneously susceptible to C2‐ceramide‐mediated apoptosis. Studies with these melanoma cells revealed correlation between ceramide‐mediated apoptosis and D‐NMAPPD‐induced apoptosis, confirming the effect of this inhibitor on ceramide signaling in human melanoma cells. These findings suggest ceramidase inhibitors as a potential new therapeutical class of antiproliferative and cytostatic drugs.
Kinetics of precursor cleavage at the dibasic sites
Authors: Glandieres Jean-Marie , Hertzog Maud , Lazar Noureddine , Brakch Noureddine , Cohen Paul , Alpert Bernard , Rholam Mohamed
Published: October 2002

The presence in the P′1 position relative to the LysArg doublet of either Phe, Tyr or Trp residues affects only pro‐OT/Np(7–15) flexibility. This has a measurable effect on the dynamics of the peptide. Since the same modifications have a major influence on the K
m and V
max values of the peptide cleavage, these kinetic parameters should depend on the peptide substrate motions. Therefore, the primary kinetic contribution of substrate cleavage should arise from substrate dynamics rather than from the enzyme.
Sequence and electron paramagnetic resonance analyses of nitrate reductase NarGH from a denitrifying halophilic euryarchaeote Haloarcula marismortui
Authors: Yoshimatsu Katsuhiko , Iwasaki Toshio , Fujiwara Taketomo
Published: October 2002

Genes encoding the NarG and NarH subunits of the molybdo–iron–sulfur enzyme, a nitrate reductase from a denitrifying halophilic euryarchaeota Haloarcula marismortui, were cloned and sequenced. An incomplete cysteine motif reminiscent of that for a [4Fe–4S] cluster binding was found in the NarG subunit, and complete cysteine arrangements for binding one [3Fe–4S] cluster and three [4Fe–4S] clusters were found in the NarH subunit. In conjunction with chemical, electron paramagnetic resonance, and subcellular localization analyses, we firmly establish that the H. marismortui enzyme is a new archaeal member of the known membrane‐bound nitrate reductases whose homologs are found in the bacterial domain.
Distal regulatory regions restrict the expression of cis‐linked genes to the tapetal cells
Authors: Franco Luciana O. , de O. Manes Carmem-Lara , Hamdi Said , Sachetto-Martins Gilberto , de Oliveira Dulce E.
Published: October 2002

The oleosin glycine‐rich protein genes Atgrp‐6, Atgrp‐7, and Atgrp‐8 occur in clusters in the Arabidopsis genome and are expressed specifically in the tapetum cells. The cis‐regulatory regions involved in the tissue‐specific gene expression were investigated by fusing different segments of the gene cluster to the uidA reporter gene. Common distal regulatory regions were identified that coordinate expression of the sequential genes. At least two of these genes were regulated spatially by proximal and distal sequences. The cis‐acting elements (122 bp upstream of the transcriptional start point) drive the uidA expression to floral tissues, whereas distal 5′ upstream regions restrict the gene activity to tapetal cells.
False positive non‐synonymous polymorphisms of G‐protein coupled receptor genes
Authors: Small Kersten M. , Seman Carrie A. , Castator Alex , Brown Kari M. , Liggett Stephen B.
Published: October 2002

Polymorphisms of G‐protein coupled receptor (GPCR) genes are associated with disease risk and modification, and the response to receptor‐directed therapy. Genomic sequencing (∼1700 automated runs) from as many as 120 chromosomes from 60 multiethnic individuals was performed to confirm non‐synonymous coding polymorphisms reported in the dbSNP database from 25 randomly selected GPCR genes. These polymorphisms were in regions of the receptors responsible for structural integrity, ligand binding, G‐protein coupling and phosphoregulation. However, most of these putative polymorphisms could not be confirmed (false positive rate of 68%). Based on these results, we suggest that the variability of the superfamily is not well defined, and we caution against exclusive reliance on databases for selection of candidate GPCR polymorphisms for disease association and pharmacogenetic studies.
Phytanic acid, but not pristanic acid, mediates the positive effects of phytol derivatives on brown adipocyte differentiation
Authors: Schluter Agatha , Giralt Marta , Iglesias Roser , Villarroya Francesc
Published: October 2002

The phytol derivatives phytanic acid and pristanic acid may activate nuclear hormone receptors and influence gene expression and cell differentiation. Phytanic acid induces brown adipocyte differentiation. It was determined that brown fat and brown adipocytes are sites of high gene expression of phytanoyl‐CoA hydroxylase, the enzyme required for initiation of peroxisomal α‐oxidation of phytanic acid. However, the effects of phytanic acid were not mediated by its α‐oxidation product pristanic acid, which did not promote brown adipocyte differentiation or stimulate transcription of the uncoupling protein‐1 gene. Moreover, acute cold exposure of mice caused a dramatic mobilization of the phytanic acid stores in brown adipose tissue thus suggesting that a high local exposure to phytanic acid in brown fat may contribute to signalling adaptive changes in the tissue in response to thermogenic activation.
Aerobic and anaerobic NAD+ metabolism in Saccharomyces cerevisiae
Authors: Panozzo Cristina , Nawara Magdalena , Suski Catherine , Kucharczyk Roza , Skoneczny Marek , Bécam Anne-Marie , Rytka Joanna , Herbert Christopher J
Published: October 2002

In Saccharomyces cerevisiae the nicotinic acid moiety of NAD+ can be synthesized from tryptophan using the kynurenine pathway or incorporated directly using nicotinate phosphoribosyl transferase (NPT1). We have identified the genes that encode the enzymes of the kynurenine pathway and for BNA5 (YLR231c) and BNA6 (YFR047c) confirmed that they encode kynureninase and quinolinate phosphoribosyl transferase respectively. We show that deletion of genes encoding kynurenine pathway enzymes are co‐lethal with the Δnpt1, demonstrating that no other pathway for the synthesis of nicotinic acid exists in S. cerevisiae. Also, we show that under anaerobic conditions S. cerevisiae is a nicotinic acid auxotroph.
Inter‐subunit cross‐linking of methylamine dehydrogenase by cyclopropylamine requires residue αPhe55
Authors: Sun Dapeng , Davidson Victor L
Published: October 2002

Cyclopropylamine is a mechanism‐based inhibitor of the quinoprotein methylamine dehydrogenase (MADH) from Paracoccus denitrificans. The resulting inactivation is accompanied by the formation of a covalent cross‐link between the α and β subunits of MADH. The results of site‐directed mutagenesis studies indicate that Phe55 on the α subunit is required for this process. No cross‐linking is seen with αF55A or αF55I MADH mutants. In contrast, with αF55E MADH cross‐linking of subunits is observed. These results suggest a novel mechanistic role for a phenylalanine residue and the possible importance of protein dynamics in this enzyme mechanism.
PKC regulation of the human equilibrative nucleoside transporter, hENT1
Authors: Coe Imogen , Zhang Ying , McKenzie Tamara , Naydenova Zlatina
Published: October 2002

Regulation of nucleoside transporters is poorly understood. We show that acute stimulation of protein kinase C (PKC) causes a rapid increase in S‐(4‐nitrobenzyl)‐6‐thioinosine‐sensitive (human equilibrative nucleoside transporter 1, hENT1) nucleoside uptake, in human cultured cells, which is not due to increased metabolism and which can be blocked by PKC inhibitors. Use of isoform‐specific inhibitors indicates that PKC δ and/or ϵ (but not α, β or γ) are responsible for the acute effects. Down‐regulation of PKC decreases hENT1‐dependent uridine uptake. These are the first data to show rapid PKC δ/ϵ‐dependent stimulation of hENT1 transport by a mechanism that may involve activation of transporters at the membrane possibly by post‐translational modification of the protein.
Metabolic control analysis of anaerobic glycolysis in human hibernating myocardium replaces traditional concepts of flux control
Authors: Vogt Achim M. , Nef Holger , Schaper Jutta , Poolman Mark , Fell David A. , Kübler Wolfgang , Elsässer Albrecht
Published: October 2002

Myocardial hibernation represents an adaptation to sustained ischemia to maintain tissue vitality during severe supply–demand imbalance which is characterized by an increased glucose uptake. To elucidate this adaptive protective mechanism, the regulation of anaerobic glycolysis was investigated using human biopsies. In hibernating myocardium showing an increase in anaerobic glycolytic flux metabolizing exogenous glucose, the adjustment of flux through this pathway was analyzed by flux:metabolite co‐responses. By this means, a previously unknown pattern of regulation using multisite modulation was found which largely differs from traditional concepts of metabolic control of the Embden–Meyerhof pathway in normal and diseased myocardium.
Involvement of TIRAP/MAL in signaling for the activation of interferon regulatory factor 3 by lipopolysaccharide
Authors: Shinobu Noriaki , Iwamura Tomokatsu , Yoneyama Mitsutoshi , Yamaguchi Kazumi , Suhara Wakako , Fukuhara Yukiko , Amano Fumio , Fujita Takashi
Published: October 2002

Infections of bacteria and viruses induce host defense reactions known as innate responses that include the production of cytokines and chemokines. The production of type I interferon (IFN) is known to be induced by viral double‐stranded (ds) RNA or bacterial lipopolysaccharide (LPS). Although important functions for the transcription factors NF‐κB and interferon regulatory factor‐3 (IRF‐3) are indicated, the molecular signals leading to the activation of IFN genes have yet to be elucidated. We provide several lines of evidence that LPS and dsRNA trigger distinct intracellular signals upstream. Notably, our investigation revealed a critical function for TIRAP/MAL, a signaling adapter for Toll‐like receptor (TLR) 4, in LPS‐induced but not dsRNA‐induced activation of IRF‐3. These results highlight cross‐talk between TLR‐mediated and virus/dsRNA‐induced signals resulting in activation of the IFN system.
Ca‐dependent binding of actin to gelsolin
Authors: Khaitlina Sofia , Hinssen Horst
Published: October 2002

Ca2+ of 0.3–1.0 μM induces both the exposure of tryptic cleavage sites within the gelsolin molecule inaccessible in the Ca‐free conformation, and binding of one actin monomer to the N‐terminal half of gelsolin. On the other hand, gelsolin‐induced enhancement of pyrene actin fluorescence was observed only above 50 μM Ca2+, and a ternary actin/gelsolin complex preformed in 200 μM Ca2+ was stable only above 30 μM Ca2+. These results provide direct evidence for Ca‐induced transitions from closed to open conformation of the gelsolin molecule in the range of 3×10‐7 to 10−6 M Ca2+. They also suggest that Ca2+>10−5 M is required to stabilize actin–actin contacts in the 2:1 actin/gelsolin complex.
Identification of a CTL4/Neu1 fusion transcript in a sialidosis patient
Authors: Uhl Johannes , Penzel Roland , Sergi Consolato , Kopitz Jürgen , Otto Herwart F , Cantz Michael
Published: October 2002

The deficiency of the lysosomal neuraminidase (NEU1; sialidase) causes the storage disorder sialidosis with symptoms ranging from eye abnormalities and neurological disturbances to skeletal malformations, mental retardation and early death. Sialidosis patients encompassing a wide spectrum of clinical symptoms were screened for mutations in neu1. We identified the same homozygous interstitial deletion (11 kb) in two patients causing the fusion of exon 10 of CTL4 (New Gene 22; NG22) with the 3′‐UTR of neu1. In one patient we found the resulting CTL4/Neu1 fusion transcript, in the other we detected an alternatively spliced CTL4 transcript (retention of intron 9).
Estrogens prevent calcium‐induced release of cytochrome c from heart mitochondria
Authors: Morkuniene Ramune , Jekabsone Aiste , Borutaite Vilmante
Published: October 2002

We investigated the effect of estrogens on heart mitochondrial functions and whether estrogens can prevent calcium‐induced release of cytochrome c from mitochondria. 10 nM–10 μM 17β‐estradiol or 4‐hydroxytamoxifen did not affect mitochondrial respiration rate and membrane potential in state 3 and state 4. Higher concentrations of both agents decreased state 3 respiration rate and membrane potential. 100 nM 17β‐estradiol and 4‐hydroxytamoxifen blocked high calcium‐induced cytochrome c release from mitochondria but not mitochondrial swelling. Thus, at physiological concentrations estrogens do not affect mitochondrial respiratory functions but protect heart mitochondria from high calcium‐induced release of cytochrome c.
Two novel antifungal peptides distinct with a five‐disulfide motif from the bark of Eucommia ulmoides Oliv
Authors: Huang Ren-Huai , Xiang Ye , Liu Xiao-Zhu , Zhang Ying , Hu Zhong , Wang Da-Cheng
Published: October 2002

Two antifungal peptides, named EAFP1 and EAFP2, have been purified from the bark of Eucommia ulmoides Oliv. Each of the sequences consists of 41 residues with a N‐terminal blockage by pyroglutamic acid determined by automated Edman degradation in combination with the tandem mass spectroscopy and the C‐terminal ladder sequencing analysis. The primary structurs all contain 10 cysteines, which are cross‐linked to form five disulfide bridges with a pairing pattern (C1–C5, C2–C9, C3–C6, C4–C7, C8–C10). This is the first finding of a plant antifungal peptide with a five‐disulfide motif. EAFP1 and EAFP2 show characteristics of hevein domain and exhibit chitin‐binding properties similar to the previously identified hevein‐like peptides. They exhibit relatively broad spectra of antifungal activities against eight pathogenic fungi from cotton, wheat, potato, tomato and tobacco. The inhibition activity of EAFP1 and EAFP2 can be effective on both chitin‐containing and chitin‐free fungi. The values of IC50 range from 35 to 155 μg/ml for EAFP1 and 18 to 109 μg/ml for EAFP2. Their antifungal effects are strongly antagonized by calcium ions.
Identifying genes related to drug anticancer mechanisms using support vector machine
Authors: Bao Lei , Sun Zhirong
Published: October 2002

In an effort to identify genes related to the cell line chemosensitivity and to evaluate the functional relationships between genes and anticancer drugs acting by the same mechanism, a supervised machine learning approach called support vector machine was used to label genes into any of the five predefined anticancer drug mechanistic categories. Among dozens of unequivocally categorized genes, many were known to be causally related to the drug mechanisms. For example, a few genes were found to be involved in the biological process triggered by the drugs (e.g. DNA polymerase epsilon was the direct target for the drugs from DNA antimetabolites category). DNA repair‐related genes were found to be enriched for about eight‐fold in the resulting gene set relative to the entire gene set. Some uncharacterized transcripts might be of interest in future studies. This method of correlating the drugs and genes provides a strategy for finding novel biologically significant relationships for molecular pharmacology.
2′,3′‐Dialdehydo‐UDP‐N‐acetylglucosamine inhibits UDP‐N‐acetylglucosamine 2‐epimerase, the key enzyme of sialic acid biosynthesis
Authors: Blume Astrid , Chen Hao , Reutter Werner , Schmidt Richard R , Hinderlich Stephan
Published: October 2002

Sialic acids comprise a family of terminal sugars essential for a variety of biological recognition systems. UDP‐N‐acetylglucosamine 2‐epimerase catalyzes the first step of their biosynthesis. Periodate‐oxidized UDP‐N‐acetylglucosamine, namely 2′,3′‐dialdehydo‐UDP‐α‐D‐N‐acetylglucosamine, was found to be an effective inhibitor of this enzyme, compared with the periodate oxidation products of compounds such as UDP, uridine or methyl riboside. It bound covalently to amino acids in the active site causing an irreversible inhibition. This compound may therefore represent a basis for the synthesis of potent inhibitors of UDP‐N‐acetylglucosamine 2‐epimerase and, as a consequence, of the biosynthesis of sialic acids.
Critical role of protein kinase C α and calcium in growth factor induced activation of the Na+/H+ exchanger NHE1
Authors: Maly Karl , Strese Kukka , Kampfer Sonja , Ueberall Florian , Baier Gottfried , Ghaffari-Tabrizi Nassim , Grunicke Hans H , Leitges Michael
Published: October 2002

The ubiquitously expressed Na+/H+ exchanger (NHE1) plays an important role in the regulation of the intracellular pH. Induction of NHE activity by phorbol esters and inhibition of growth factor‐mediated stimulation of the NHE by protein kinase C (PKC) inhibitors suggest an implication of PKCs in the regulation of the NHE. Expression of PKC isotype‐specific dominant negative and constitutively active mutants or downregulation of PKC by isotype‐specific antisense oligonucleotides revealed that stimulation by epidermal growth factor (EGF) or phorbol ester of the NHE in NIH3T3 cells is a PKCα‐specific effect. Elevation of cytoplasmic calcium by a Ca2+ ionophore or thapsigargin causes a growth factor‐independent stimulation of the NHE predominantly mediated by calcium/calmodulin kinase II. It is concluded that in NIH3T3 cells overexpressing the EGF receptor (EGFR6 cells), EGF requires cPKCα for the activation of the NHE, while calcium/calmodulin‐dependent kinases are essential in thapsigargin induced stimulation of the NHE.
An attempt to promote neo‐vascularization by employing a newly synthesized inhibitor of protein tyrosine phosphatase
Authors: Soeda Shinji , Shimada Takuji , Koyanagi Satoru , Yokomatsu Tsutomu , Murano Tetsuo , Shibuya Shiroshi , Shimeno Hiroshi
Published: October 2002

Vascular endothelial growth factor (VEGF) and its receptors play a key role in angiogenesis. VEGF receptor‐2 (VEGFR‐2) has a tyrosine kinase domain, and, once activated, induces the phosphorylation of cytoplasmic signaling proteins. The phosphorylated VEGFR‐2 may be a substrate for intracellular protein tyrosine phosphatases (PTPs) which prevent VEGF signaling. We synthesized a series of α,α‐difluoro(phenyl)methylphosphonic acids (DFPMPAs) which inhibit the action of PTP. In this study, we test their effects on VEGF‐induced angiogenesis. DFPMPA‐3, the most effective inhibitor of human PTP‐1B, promoted tube formation by human umbilical vein endothelial cells (HUVEC) on Matrigel more effectively than any other DFPMPAs. The inhibitor promoted the VEGF‐induced proliferation and migration of HUVEC by inhibiting the dephosphorylation of VEGFR‐2. Its effectiveness was proven through neo‐vascularization in mice. The present findings suggest that targeting PTP to promote therapeutic neo‐vascularization may be a potential strategy.
Sphingosine‐induced c‐jun expression: differences between sphingosine‐ and C2‐ceramide‐mediated signaling pathways
Authors: Sawai Hirofumi , Okazaki Toshiro , Domae Naochika
Published: October 2002

Sphingolipids such as ceramide and sphingosine are putative intracellular signal mediators in cell differentiation, growth inhibition and apoptosis. Previously, we reported that C2‐ceramide induced c‐jun expression in apoptosis of human leukemia HL‐60 cells. Here we report that sphingosine also induced c‐jun expression in apoptosis of HL‐60 cells. Sphingosine‐induced c‐jun expression was stimulated by H‐89, a protein kinase A inhibitor, whereas C2‐ceramide‐induced c‐jun expression was inhibited by protein kinase C inhibitors. Furthermore, H‐89 potentiated sphingosine‐induced but not C2‐ceramide‐induced growth inhibition. These results suggest that sphingosine and C2‐ceramide might induce c‐jun expression and apoptosis in distinct signaling pathways.
Deficiency of triad formation in developing skeletal muscle cells lacking junctophilin type 1
Authors: Komazaki Shinji , Ito Koichi , Takeshima Hiroshi , Nakamura Hiroaki
Published: October 2002

Junctophilins (JP‐1, JP‐2, and JP‐3) are transmembrane proteins expressed in the junctional membrane complexes in excitable cells. Both JP‐1 and JP‐2 are co‐expressed in the triads of skeletal muscle, but only JP‐2 is expressed in cardiac muscle. We analyzed the roles played by JP‐1 and JP‐2 in triad formation in skeletal muscle by comparing developing skeletal muscles in wild‐type and JP‐1‐knockout (KO) mice (both before and after birth). In the skeletal muscles of embryos, most of the couplings between sarcoplasmic reticulum (SR) and transverse tubule (T‐tubule) were diads, with triads being very scarce. The number of triads increased markedly after birth in wild‐type mice. However, there was no increase in the number of triads in the neonates of JP‐1‐KO mice, and they died within 1 day after birth. JP‐2 expression was constant before and after birth, while expression of JP‐1 increased with birth. Quantitative and morphological differences were not seen between wild‐type and JP‐1‐KO mice in the formation of diads in the period just before the JP‐1‐KO mice died. The SR swelled and developed large vacuoles in skeletal muscle cells just before the JP‐1‐KO mice died. The present results strongly suggest that JP‐1 and JP‐2 play important roles in the formation of triads and diads, respectively, during the development of skeletal muscle in mouse.
NMR structure determination and investigation using a reduced proton (REDPRO) labeling strategy for proteins
Authors: Shekhtman Alexander , Ghose Ranajeet , Goger Michael , Cowburn David
Published: October 2002

We present here a stable isotope labeling technique for proteins, which seeks the appropriate compromise between the advantages of (a) random isotope labeling, with its large number of protons available for structure determination, and (b) selective labeling to generate isolated proton spins decreasing spectral complexity and improving relaxation properties of NMR experiments. The described reduced proton (REDPRO) procedure results in side‐chain specific protonation of overexpressed proteins, which is highly selective. The REDPRO labeling scheme provides a sufficient number of NOE constraints for structure calculation. Dramatically improved relaxation properties of the heteronuclear magnetization transfer coupled with TROSY advantages make the proposed labeling scheme an attractive approach for study of high molecular weight protein targets, their ligand sites, and interdomain interactions.
Ring up the curtain on DING proteins
Authors: Berna Anne , Bernier François , Scott Ken , Stuhlmüller Bruno
Published: October 2002

DING proteins have a characteristic DINGGG‐ or closely related N‐terminal sequence. One is found in human synovial fluid, and may be associated with rheumatoid arthritis. Other examples have receptor or signalling roles in various human and animal cells, or are involved in biomineralisation, and several of them bind to phytochemicals. As plant DING proteins have recently been discovered, we hypothesise that the DING protein‐phytochemical association may represent one aspect of a ubiquitous receptor‐linked signalling system. Several microbial proteins related to DING proteins have phosphatase activity, which may relate to biomineralisation in eukaryotic systems. Plant DING proteins and their microbial relatives may elicit allergic responses leading to arthritic disease.
Clinical value of early cleavage embryo
Authors: Tsai Y.C , Chung M.T , Sung Y.H , Tsai T.F , Tsai Y.T , Lin L.Y
Published: October 2002

Objective: This study was undertaken to see if embryo transfer containing early cleavage embryos resulted in better clinical pregnancy rate. Method: The treatment outcomes of IVF‐ET were retrospectively reviewed. Out of 258 transfer cycles, 160 cycles contained no early cleavage embryos (Group I) and 98 cycles contained at least one early cleavage embryo (Group II). The definition of early cleavage embryo is the presence of two blastomeres 24–26 h after insemination. The implantation rate, clinical pregnancy rate were compared between two groups. Student's t‐test and the Mann–Whitney U‐test were used for continuous variables, and the Chi‐squared (χ2) test was used for binary variables. Differences were considered statistically significant at P<0.05. Result: The implantation rate and clinical pregnancy rate were 11.6% and 25.6% in Group I, 18.6% and 38.8% in Group II (P<0.05). Conclusion: Early cleavage embryos possess greater implantation potential. Embryo transfer containing early cleavage embryos had a better clinical pregnancy rate.
Interleukin‐6 and interleukin‐1 receptor antagonist in amniotic fluid and cord blood in patients with pre‐term, premature rupture of the membranes
Authors: Fukuda H. , Masuzaki H. , Ishimaru T.
Published: October 2002

Objective: We investigated the role of interleukin (IL)‐6 and IL‐1 receptor antagonist (IL‐1ra) in pre‐term premature rupture of the membranes (PROM). Method: Amniotic fluid samples were collected from 10 patients with pre‐term PROM (group 1a), 13 patients undergoing genetic amniocentesis (group 2), seven patients with normal vaginal delivery (group 3a), and 11 patients with elective cesarean section (group 4a). Umbilical venous blood was collected from nine cases of pre‐term PROM (group 1b), 19 cases of normal delivery (group 3b) and nine cases of elective cesarean section (group 4b). Results: The concentration of IL‐6 in the amniotic fluid in group 1a was significantly higher than in group 2 (P<0.001), and that in group 3a was significantly higher than in group 4a (P<0.001). The concentration of IL‐1ra in amniotic fluid in group 1a was significantly higher than in group 2 (P<0.001). The concentrations of IL‐6 in umbilical venous blood were not significant among the groups. The concentration of IL‐1ra in umbilical venous blood in group 1b was significantly higher than in group 3b (P<0.05) and group 4b (P<0.05). Conclusions: High concentrations of amniotic fluid IL‐6 present in pre‐term PROM, are indicative of intrauterine inflammation, probably due to sub‐clinical infection. The high concentrations of IL‐1ra in amniotic fluid and umbilical venous blood suggest the possibility that the presence of this anti‐inflammatory cytokine serves to prevent the development of overt inflammation.
An exploratory analysis of new competencies: a resource based view perspective
Authors: Coates Theresa Taylor , McDermott Christopher M
Published: October 2002

The resource based view (RBV) theory of the firm widely acknowledges that capabilities that are unique and are important for achieving sustained competitive advantage. It has been argued in the manufacturing literature that manufacturing facilities, technology and policies are important to gain performance. However, little has been done to examine the capabilities from a resource based perspective. Nor has there been an exploration in how firms develop and leverage new capabilities. The literature also tends to neglect how complex and often intertwined these capabilities are within the firms. This study addresses this gap in the literature by examining the successful development of new resources and competencies that were created when Analog Devices developed an emerging technology. The paper is exploratory and identifies the new competencies and Analog Devices’ ability to capitalize upon them.
Cellular manufacturing for small businesses: key cultural factors that impact the conversion process
Authors: Yauch Charlene A , Steudel Harold J
Published: October 2002

Companies converting to cellular manufacturing (CM) often struggle with implementation and achieve results that are less than anticipated. The existing body of CM research, with its strong emphasis on technical aspects, does not provide practitioners with the assistance needed for successful implementation. Since organizational culture is a strong barrier to change, this research aimed to reveal how culture impacts the CM conversion process. Two exploratory case studies were conducted at small manufacturing companies leading to the identification of eight key cultural factors that impact CM conversion.
Inheritance of symmetry for positive solutions of semilinear elliptic boundary value problems
Authors: Kawohl Bernd , Sweers Guido
Published: October 2002

The paper addresses symmetry results for positive solutions of semilinear elliptic differential equations on a class of non-convex symmetrical domains. An example in two dimensions is the star of David. The moving plane method just shows that solutions coincide on three alternate corners of the star. We will show that the solution is symmetric with respect to remaining reflections, that is, the solution will have the full symmetry. To obtain such type of result, even for domains in higher dimensions, we use a variant of the sliding-method and the maximum principle for domains with small measure.

Des résultats concernant la symétrie des solutions positives d' équations elliptiques semilinéaires sur une classe de domaines symétriques nonconvexes sont établis. Un exemple en dimension deux est l' étoile de David. La méthode des plans mobiles dans sa forme habituelle permet de montrer le symétrie de telles solutions seulement par rapport à trois axes. Nous sommes en mesure de prouver la symétrie par rapport au centre et donc la symétrie totale. Pour obtenir ce type de résultat, même pour des domaines en dimension supérieure à deux, nous utilisons la méthode des balayages et le principe du maximum pour les domaines de mesure petite.