bluuebunny/crossref_metadata_2025 huggingface dataset:
Progastrin processing differs in 7B2 and PC2 knockout animals: a role for 7B2 independent of action on PC2
Authors: Rehfeld Jens F. , Lindberg Iris , Friis-Hansen Lennart
Published: October 2002

Cellular synthesis of neuroendocrine peptides requires prohormone convertases (PCs). In order to determine the role of PC2 for gastrin synthesis, we examined antral extracts from mice lacking PC2 or its chaperone, 7B2. The overall concentrations of precursors and α‐amidated gastrins were similar in all mice. Chromatography, however, revealed that while the K53–K54 site was almost fully cleaved in controls and half cleaved in PC2 null mice, only 23% was cleaved in 7B2 null mice. The results show that PC2 and 7B2 both are required for synthesis of the main form of gastrin (gastrin‐17), and that 7B2 exhibits effects beyond PC2‐mediated cleavages.
Glucosamine modulates IL‐1‐induced activation of rat chondrocytes at a receptor level, and by inhibiting the NF‐κB pathway
Authors: Gouze J.N. , Bianchi A. , Bécuwe P. , Dauça M. , Netter P. , Magdalou J. , Terlain B. , Bordji K.
Published: October 2002

We recently reported that glucosamine reversed the decrease in proteoglycan synthesis and in UDP‐glucuronosyltransferase I mRNA expression induced by interleukin‐1β (IL‐1β) [Arthritis Rheum. 44 (2001) 351–360]. In the present work, we show that glucosamine does not exert the same effects when chondrocytes were stimulated with reactive oxygen species (ROS). In order to better understand its mechanism of action, we determined if glucosamine could prevent the binding of IL‐1β to its cellular receptors or could interfere with its signaling pathway at a post‐receptor level. Addition of glucosamine to rat chondrocytes treated with IL‐1β or with ROS decreased the activation of the nuclear factor κB, but not the activator protein‐1. After treatment with IL‐1β, glucosamine increased the expression of mRNA encoding the type II IL‐1β receptor. These results emphasize the potential role of two regulating proteins of the IL‐1β signaling pathway that could account for the beneficial effect of glucosamine in osteoarthritis.
Components required for membrane assembly of newly synthesized K+ channel KcsA
Authors: van Dalen Annemieke , van der Laan Martin , Driessen Arnold J.M. , Killian J.Antoinette , de Kruijff Ben
Published: October 2002

An Escherichia coli in vitro transcription–translation system was used to study the components involved in the biogenesis of the homotetrameric potassium channel KcsA. We show that a functional signal recognition particle pathway is essential for tetramer formation, probably to direct correct monomer insertion in the membrane. In the absence of YidC or at reduced SecYEG levels, KcsA assembly occurs with lower efficiency. Strikingly, the highest efficiency of tetramerization was observed when transcription–translation was carried out in the presence of pure lipid vesicles, demonstrating that a phospholipid bilayer is the minimal membrane requirement to form the KcsA tetramer. It is concluded that SecYEG and YidC are not required for the formation of tetrameric KcsA in vitro.
Tyrosine kinase participates in vasoconstriction through a Ca2+‐ and myosin light chain phosphorylation‐independent pathway
Authors: Fang Lian-Hua , Kwon Seong-Chun , Zhang Yong-He , Ahn Hee-Yul
Published: October 2002

This study was undertaken to determine the role of tyrosine kinase on intracellular Ca2+ ([Ca2+]i), myosin light chain (MLC) phosphorylation, and contraction caused by norepinephrine (NE) in rat aorta. NE induced a sustained contraction with an increase of [Ca2+]i. On the other hand, NE increased the phosphorylation of the 20 kDa MLC transiently. Pretreatment with genistein and tyrphostin 25, tyrosine kinase inhibitors, significantly inhibited NE‐induced contraction, but did not affect the increase of [Ca2+]i and MLC phosphorylation. These results suggest that tyrosine kinase may regulate the NE‐mediated contraction without altering [Ca2+]i and MLC phosphorylation in rat aorta.
ADP‐evoked phospholipase C stimulation and adenylyl cyclase inhibition in glioma C6 cells occur through two distinct nucleotide receptors, P2Y1 and P2Y12
Authors: Czajkowski Rafal̵ , Lei Lingsheng , Sabal̵a Pawel̵ , Barańska Jolanta
Published: October 2002

In this study we characterized the subtypes of nucleotide P2Y receptors that respond to ADP in glioma C6 cells. Direct visualization of phosphatidylinositol 4,5‐bisphosphate at the cell surface revealed that extracellular ADP activates phospholipase C (PLC). Knock‐down of P2Y1 receptor with antisense oligonucleotide, as well as treatment with MRS2179 and pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (P2Y1 antagonists), attenuates receptor‐mediated PLC activity. Adenylyl cyclase inhibition by ADP remains unchanged under these conditions. Reverse transcription‐PCR analysis showed that P2Y12 receptor is expressed in C6 cells. We therefore conclude that, in glioma C6 cells, two P2Y receptor subtypes are present: P2Y1, coupled to PLC, and P2Y12, negatively coupled to adenylyl cyclase.
Heterologous expression and topography of the main intrinsic protein (MIP) from rat lens
Authors: Drake K.Dawn , Schuette Diana , Chepelinsky Ana B , Crabbe M.James C
Published: October 2002

Wild type rat lens main intrinsic protein (MIP) and MIP mutated (F73I, F75L) to resemble the glycerol facilitator of Escherichia coli in the region of the NPA1 box were used to investigate the topology of MIP in the membrane of Spodoptera frugiperda (Sf21) cells using the baculovirus expression system and expression in mouse erythroid leukaemia cells (MEL C88). Differential fixation for staining was used, with paraformaldehyde for externally exposed antigenic sites, and acetone for both externally and internally exposed protein antigenic sites. Immunofluorescence using antibodies to synthetic MIP peptides showed that wild type MIP had a six transmembrane topography. The N‐ and C‐termini were intracellular in both expression systems, and both NPA boxes were found to be extracellular. These results show that residues around the NPA1 box can influence the folding of the MIP in the membrane, and provide structural evidence for the poor water transport properties of MIP, as the NPA boxes lie outside the plane of the membrane.
Imaging of the fluorescence spectrum of a single fluorescent molecule by prism‐based spectroscopy
Authors: Suzuki Yoshikazu , Tani Tomomi , Sutoh Kazuo , Kamimura Shinji
Published: October 2002

We have devised a novel method to visualize the fluorescence spectrum of a single fluorescent molecule using prism‐based spectroscopy. Equiping a total internal reflection microscope with a newly designed wedge prism, we obtained a spectral image of a single rhodamine red molecule attached to an essential light chain of myosin. We also obtained a spectral image of single‐pair fluorescence resonance energy transfer between rhodamine red and Cy5 in a double‐labeled myosin motor domain. This method could become a useful tool to investigate the dynamic processes of biomolecules at the single‐molecule level.
ACTH secretion by mouse corticotroph AtT20 cells is negatively modulated by the intracellular level of 7B2
Authors: Bergeron François , Sirois Francine , Mbikay Majambu
Published: October 2002

7B2 is a pan‐neuroendocrine protein known to facilitate the trafficking and activation of the prohormone proprotein convertase‐2 (PC2). 7B2‐null mice not only lack PC2 activity, but they also develop an adrenocorticotropic hormone (ACTH) hypersecretion syndrome, suggesting that 7B2 may regulate hormone secretion. To verify this possibility, we introduced into mouse corticotroph AtT20 cells a retroviral vector carrying either a sense or an antisense 7B2 transgene to induce higher and lower 7B2 expression, respectively. Relative to control AtT20 cells, 7B2‐overexpressing cells released less ACTH following KCl‐induced membrane depolarization, whereas cells expressing lower levels of 7B2 released relatively more, suggesting that 7B2‐related peptides modulate regulated secretion in neuroendocrine cells.
Decreased requirement for 4.5S RNA in 16S and 23S rRNA mutants of Escherichia coli
Authors: Brunelli Carleen A , O'Connor Michael , Dahlberg Albert E
Published: October 2002

4.5S RNA is the bacterial homolog of the mammalian signal recognition particle (SRP) RNA that targets ribosome‐bound nascent peptides to the endoplasmic reticulum. To explore the interaction of bacterial SRP with the ribosome, we have isolated rRNA suppressor mutations in Escherichia coli that decrease the requirement for 4.5S RNA. Mutations at C732 in 16S rRNA and at A1668 and G1423 in 23S rRNA altered the cellular responses to decreases in both Ffh (the bacterial homolog of SRP54) and 4.5S RNA levels, while the C1066U mutation in 16S rRNA and G424A mutation in 23S rRNA affected the requirement for 4.5S RNA only. These data are consistent with a dual role for 4.5S RNA, one involving co‐translational protein secretion by a 4.5S–Ffh complex, the other involving free 4.5S RNA.
Positioning of the mRNA stop signal with respect to polypeptide chain release factors and ribosomal proteins in 80S ribosomes
Authors: Bulygin Konstantin N , Repkova Marina N , Ven'yaminova Aliya G , Graifer Dmitri M , Karpova Galina G , Frolova Ludmila Yu , Kisselev Lev L
Published: October 2002

To study positioning of the mRNA stop signal with respect to polypeptide chain release factors (RFs) and ribosomal components within human 80S ribosomes, photoreactive mRNA analogs were applied. Derivatives of the UUCUAAA heptaribonucleotide containing the UUC codon for Phe and the stop signal UAAA, which bore a perfluoroaryl azido group at either the fourth nucleotide or the 3′‐terminal phosphate, were synthesized. The UUC codon was directed to the ribosomal P site by the cognate tRNAPhe, targeting the UAA stop codon to the A site. Mild UV irradiation of the ternary complexes consisting of the 80S ribosome, the mRNA analog and tRNA resulted in tRNA‐dependent crosslinking of the mRNA analogs to the 40S ribosomal proteins and the 18S rRNA. mRNA analogs with the photoreactive group at the fourth uridine (the first base of the stop codon) crosslinked mainly to protein S15 (and much less to S2). For the 3′‐modified mRNA analog, the major crosslinking target was protein S2, while protein S15 was much less crosslinked. Crosslinking of eukaryotic (e) RF1 was entirely dependent on the presence of a stop signal in the mRNA analog. eRF3 in the presence of eRF1 did not crosslink, but decreased the yield of eRF1 crosslinking. We conclude that (i) proteins S15 and S2 of the 40S ribosomal subunit are located near the A site‐bound codon; (ii) eRF1 can induce spatial rearrangement of the 80S ribosome leading to movement of protein L4 of the 60S ribosomal subunit closer to the codon located at the A site; (iii) within the 80S ribosome, eRF3 in the presence of eRF1 does not contact the stop codon at the A site and is probably located mostly (if not entirely) on the 60S subunit.
Ribosome as a molecular machine
Authors: Spirin Alexander S
Published: October 2002

General principles of structure and function of the ribosome are surveyed, and the translating ribosome is regarded as a molecular conveying machine. Two coupled conveying processes, the passing of compact tRNA globules and the drawing of linear mRNA chain through intraribosomal channel, are considered driven by discrete acts of translocation during translation. Instead of mechanical transmission mechanisms and power‐stroke ‘motors’, thermal motion and chemically induced changes in affinities of ribosomal binding sites for their ligands (tRNAs, mRNA, elongation factors) are proposed to underlie all the directional movements within the ribosomal complex. The GTP‐dependent catalysis of conformational transitions by elongation factors during translation is also discussed.
Solution structure of polyglutamine tracts in GST‐polyglutamine fusion proteins
Authors: Masino Laura , Kelly Geoff , Leonard Kevin , Trottier Yvon , Pastore Annalisa
Published: October 2002

Aggregation of expanded polyglutamine (polyQ) seems to be the cause of various genetic neurodegenerative diseases. Relatively little is known as yet about the polyQ structure and the mechanism that induces aggregation. We have characterised the solution structure of polyQ in a proteic context using a model system based on glutathione S‐transferase fusion proteins. A wide range of biophysical techniques was applied. For the first time, nuclear magnetic resonance was used to observe directly and selectively the conformation of polyQ in the pathological range. We demonstrate that, in solution, polyQs are in a random coil conformation. However, under destabilising conditions, their aggregation behaviour is determined by the polyQ length.
Cell‐cell signaling by direct contact increases cell proliferation via a PI3K‐dependent signal
Authors: Nelson Celeste M , Chen Christopher S
Published: October 2002

We report a novel mechanism of cellular growth control. Increasing the density of endothelial or smooth muscle cells in culture increased cell‐cell contact and decreased cell spreading, leading to growth arrest. Using a new method to independently control cell‐cell contact and cell spreading, we found that introducing cell‐cell contact positively regulates proliferation, but that contact‐mediated proliferation can be masked by changes in cell spreading: Round cells with many contacts proliferated less than spread cells with none. Physically blocking cell‐cell contact or inhibiting PI3K signaling abrogated cell‐cell induced proliferation, but inhibiting diffusible paracrine signaling did not. Thus, direct cell‐cell contact induces proliferation in these cells.
A rapid fluorometric assay for the proteolytic activity of SKI‐1/S1P based on the surface glycoprotein of the hemorrhagic fever Lassa virus
Authors: Basak Ajoy , Chrétien Michel , Seidah Nabil G.
Published: October 2002

The subtilase subtilisin kexin isozyme‐1 (SKI‐1)/site 1 protease (S1P), has been implicated in the processing of Lassa virus glycoprotein C (GP‐C) precursor into GP1 and GP2 that are responsible for viral fusion with the host cell membrane. Here, we studied in vitro the kinetics of this cleavage by hSKI‐1 using an intramolecularly quenched fluorogenic (IQF) peptide, Q‐GPC251–263 [Abz‐251Asp‐Ile‐Tyr‐Ile‐Ser‐Arg‐Arg‐Leu‐Leu↓Gly‐Thr‐Phe‐Thr263‐3‐NitroTyr‐Ala‐CONH2], containing the identified site. The measured V
max (app)/K
m (app) was compared to those for other IQF SKI‐substrates. Q‐GPC251–263 is cleaved 10‐fold more efficiently than the previously known best SKI‐substrate, Q‐hproSKI134–142. This study confirmed the role of SKI‐1 in GP‐C processing and provides a novel, rapid and efficient enzymatic assay of SKI‐1.
The interferon‐inducible gene, Ifi204, acquires malignant transformation capability upon mutation at the Rb‐binding sites
Authors: De Andrea Marco , Ravotto Monica , Noris Emanuela , Ying Guo-Guang , Gioia Daniela , Azzimonti Barbara , Gariglio Marisa , Landolfo Santo
Published: October 2002

p204 overexpression in retinoblastoma (Rb)−/− mouse embryo fibroblasts or transfection of p204 mutated at both Rb‐binding sites confer growth advantages, resulting in a significantly higher number of foci in a cell focus assay. To investigate the possibility that mutated p204 acquires malignant transformation capability, NIH3T3 cells were stably transfected with the expression vector pRcRSV204 double‐mutant (p204dm) harboring both the C‐terminal deletion up to amino acid 568 and the point mutation from glutamic acid to lysine at position 427, and analyzed for markers typical of cell immortalization and transformation. We detected a greater abundance of cell colonies in soft agar with p204dm‐expressing cells than vector control cells. The p204dm‐transfected cells also displayed two other characteristics associated with malignant transformation, i.e. growth under low‐serum conditions and formation of tumors in athymic nude mice. Moreover, their telomerase activity was significantly higher than in the vector control cells. It would thus seem that p204, devoid of functional Rb‐binding motifs, can become oncogenic.
Self‐assembly of ATP synthase subunit c rings
Authors: Arechaga Ignacio , Butler P.Jonathan G. , Walker John E.
Published: October 2002

Subunit c of the H+ transporting ATP synthase is an essential part of its membrane domain that participates in transmembrane proton conduction. The annular architecture of the subunit c from different species has been previously reported. However, little is known about the type of interactions that affect the formation of c‐rings in the ATPase complex. Here we report that subunit c over‐expressed in Escherichia coli and purified in non‐ionic detergent solutions self‐assembles into annular structures in the absence of other subunits of the complex. The results suggest that the ability of subunit c to form rings is determined by its primary structure.
Involvement of NADPH in the cyclization reaction of carotenoid biosynthesis
Authors: Hornero-Méndez D , Britton G
Published: October 2002

Cyclic carotenoids, e.g. β‐carotene, are formed by cyclization of an acyclic precursor, lycopene. The gene, crtY, which encodes lycopene β‐cyclase, has a partial sequence characteristic of a pyridine nucleotide binding domain, and NAD(P)H has been reported to be an absolute requirement for the cyclization reaction in vitro. By complementary incubations with lycopene as substrate and with (4R)‐[4‐2H]NADPH in 1H2O or with unlabelled NADPH in 2H2O in the presence of the purified enzyme, it has now been shown that the hydrogen atom introduced at C(2) in the cyclization comes from water and not from NADPH. The previously proposed mechanism involving the initiation of cyclization by H+ attack at C(2) of the folded acyclic end group of the precursor is thus confirmed. No hydrogen is transferred from NADPH, which is therefore not involved directly in the cyclization reaction, but must play an indirect role, e.g. as an allosteric activator.
NMR measurement of the off rate from the first calcium‐binding site of the synaptotagmin I C2A domain
Authors: Millet Oscar , Bernadó Pau , Garcia Jesús , Rizo Josep , Pons Miquel
Published: October 2002

The off rate from the first calcium‐binding site of the C2A domain of synaptotagmin I, a putative calcium receptor in neurotransmitter release, has been determined by 15N‐nuclear magnetic resonance relaxation dispersion measurements. The exchange rate was obtained by fitting the dependence of the transverse relaxation rates on the interval between 180° pulses in relaxation‐compensated CPMG experiments at 3.2 μM calcium concentration. The measured k
ex is 2.0×103 s−1. The calcium on rate of 3.5±1×107 s−1, determined from the measured off rate and the dissociation constant (5.3×10−5 M), is close to the diffusion limit. These results are consistent with the proposed role of synaptotagmin I as a calcium sensor in release, but suggest that additional factors may help to accelerate the diffusion of Ca2+ to the sensor.
Thiolsubtilisin acts as an acetyltransferase in organic solvents
Authors: Tai Dar-Fu , Liaw Wen-Chen
Published: October 2002

The catalytic mechanism of arylamine N‐acetyltransferase has been proposed to involve Cys‐His‐Asp as its catalytic triad. Thiolsubtilisin, a chemically modified enzyme that has a catalytic triad of Cys‐His‐Asp at the active site, mimics the catalysis of arylamine N‐acetyltransferase, serotonin N‐acetyltransferase, histone N‐acetyltransferase and amino acid N‐acetyltransferase. Thiolsubtilisin not only can catalyze amino acid transacetylation, but is also able to catalyze amine transacetylation. Ethyl acetate was used as the acylating reagent to form N‐acetyl amino acids and amines in organic solvents with moderate yield. Hence, these findings broaden our understanding of the structural features required for N‐acetyltransferases activity as well as provide a structural relationship between cysteine protease and other N‐acyltransferases.
Cathepsin D is specifically inhibited by deoxyribonucleic acids
Authors: Shibata Masahiro , Koike Masato , Waguri Satoshi , Zhang Guiqin , Koga Tomoyuki , Uchiyama Yasuo
Published: October 2002

A cathepsin D (CD) inhibitor was searched using mouse embryonic fibroblasts deficient for CD. Synthetic DNA fragments specifically inhibited CD activity in a dose‐dependent manner, but not the activities of other serine or cysteine proteinases. Cathepsin E activity was also inhibited by DNA fragments when hemoglobin was used as a substrate. CD inhibition by DNA fragments appeared to be electrostatic in nature and dependent on T
m values. Moreover, CD activity was partly inhibited by exogenously ingested DNA fragments, suggesting that DNA fragments with high T
m values are potent inhibitors of CD in vitro and partly in vivo.
Carbohydrate carriers affect adhesion of H. pylori to immobilized Leb‐oligosaccharide
Authors: Kojima Naoya , Seino Keita , Sato Yoshia , Mizuochi Tsuguo
Published: October 2002

The present study involved comparison of adhesion of Helicobacter pylori KH202 to immobilized Leb‐oligosaccharide carried on different carriers, i.e. Leb‐oligosaccharide conjugated with polyacrylamide, bovine serum albumin, and dipalmitoylphosphatidylethanolamine (Leb‐PAA, Leb‐BSA, and Leb‐DPPE). All of the Leb‐oligosaccharide‐carrying neoglycoconjugates served as ligands for H. pylori. However, H. pylori required 10‐fold and 100‐fold quantities of Leb‐antigen to adhere to Leb‐PAA and to Leb‐DPPE in comparison to the quantity of Leb‐antigen needed to adhere to Leb‐BSA, respectively. H. pylori adhesion to Leb‐PAA and Leb‐DPPE was clearly inhibited by Leb‐oligosaccharide, but adhesion to Leb‐BSA was hardly inhibited by the oligosaccharide. Therefore, the carbohydrate carrier affects the affinity of H. pylori KH202 toward Leb‐antigen, although the bacteria recognize Leb‐antigen regardless of the carbohydrate carrier.
Heteronuclear NMR studies of human serum apolipoprotein A‐I
Authors: Okon Mark , Frank Philippe G , Marcel Yves L , Cushley Robert J
Published: October 2002

The apolipoprotein A‐I (apoA‐I) solution structure in the presence of sodium dodecyl sulfate (SDS) was determined by combination of chemical shift index and torsion angle likelihood obtained from shift and sequence similarity methods. ApoA‐I in lipid‐mimetic solution is composed of α‐helices (residues 8–32, 45–64, 67–77, 82–86, 90–97, 100–118, 122–140, 146–162, 167–205, 210–216 and 221–239), with 2–5 residue irregular segments between helical repeats, and the irregular segment 78–81 within helical repeat 2. ApoA‐I is a monomer in the SDS complex and no evidence of interhelical interactions is found. Comparison of the apoA‐I and apoA‐I(1–186) [Okon et al., FEBS Lett. 487 (2001) 390–396] solution structures revealed that apoA‐I undergoes a conformational change around Pro121.
Selective dephosphorylation of the threonine183 residue of ERK2 upon αllbβ3 engagement in platelets
Authors: Pawlowski Marc , Ragab Ashraf , Rosa Jean-Philippe , Bryckaert Marijke
Published: October 2002

Thrombin‐induced extracellular signal‐regulated kinase 2 (ERK2) activation is negatively regulated in conditions of αllbβ3 integrin engagement and platelet aggregation. Here we show by Western blotting with antibodies against mono‐ and biphosphorylated forms of ERK2 that the dephosphorylation of ERK2 by αllbβ3 engagement affects threonine183 and not tyrosine185. Addition of a potent serine/threonine phosphatase inhibitor, okadaic acid (OA), restored thrombin‐induced threonine phosphorylation of ERK2 in conditions of platelet aggregation, whereas OA had no effect in the absence of αllbβ3 engagement. These observations are consistent with αllbβ3 engagement acting via at least one serine/threonine phosphatase, which dephosphorylates the phosphothreonine183 residue of ERK2. Moreover, a small amount (14%) of ERK2 was translocated to the αllbβ3‐dependent cytoskeleton, mostly in a monophosphorylated (i.e. inactive) form, suggesting that cytoskeleton‐associated ERK2 plays only a minor role, if any. Finally, we show that negative regulation (i.e. dephosphorylation) occurs primarily or totally in the cytosol and that the αllbβ3‐dependent ERK2 Thr183‐specific phosphatase is different from phosphatase 1 (PP1) or PP2A. We conclude that αllbβ3 engagement down‐regulates ERK2 through selective dephosphorylation of the phosphothreonine183 residue by a cytosolic serine/threonine phosphatase different from known platelet phosphatases.
Role of C‐terminal cytoplasmic domain of the AT2 receptor in ligand binding and signaling
Authors: Pulakat Lakshmidevi , Gray Amanda , Johnson Janean , Knowle Dieter , Burns Veronica , Gavini Nara
Published: October 2002

A stop codon at position 322 was introduced to generate a truncated, C‐terminal‐deleted AT2 receptor. Expression studies in Xenopus oocytes showed that C‐terminal‐deleted AT2 had reduced affinity to [125I]angiotensin II (K
d=1.7 nM) and enhanced binding of the AT2‐specific peptidic ligand [125I]CGP42112A (K
d=0.097 nM). AT2 activation by angiotensin II resulted in reduction of cGMP levels in oocytes and this reduction was further enhanced by C‐terminal deletion, implying that the C‐terminus may have a negative effect on the AT2‐mediated cGMP reduction. Moreover, interaction of the AT2 with the ATP‐binding domain of the human ErbB3 receptor in yeast two‐hybrid assay was abolished by C‐terminal deletion. In summary, the C‐terminal cytoplasmic tail of AT2 modulates its ligand binding and signaling properties.
Molecular cloning and characterization of a novel member of the UDP‐GalNAc:polypeptide N‐acetylgalactosaminyltransferase family, pp‐GalNAc‐T121
Authors: Guo Jian-Ming , Zhang Yan , Cheng Lamei , Iwasaki Hiroko , Wang Han , Kubota Tomomi , Tachibana Kouichi , Narimatsu Hisashi
Published: October 2002

We cloned in silico a novel human UDP‐GalNAc:polypeptide N‐acetylgalactosaminyltransferase (pp‐GalNAc‐T), pp‐GalNAc‐T12. The deduced amino acid sequence of pp‐GalNAc‐T12 contains all conserved motifs in pp‐GalNAc‐T family proteins. Quantitative real time polymerase chain reaction analysis revealed that the pp‐GalNAc‐T12 transcript was expressed mainly in digestive organs such as stomach, small intestine and colon. The recombinant pp‐GalNAc‐T12 transferred GalNAc to the mucin‐derived peptides such as the Muc1a, Muc5AC, EA2 peptides and the GalNAc‐Muc5AC glycopeptide. Since mucins are glycoproteins mainly produced in the digestive organs, our results suggest that pp‐GalNAc‐T12 plays an important role in the initial step of mucin‐type oligosaccharide biosynthesis in digestive organs.
Estrogen receptor β
Authors: Lewandowski Sebastian , Kalita Katarzyna , Kaczmarek Leszek
Published: October 2002

Recent cloning of estrogen receptor β (ERβ) was followed by the discovery of a variety of its isoforms. This review describes the complexity of ERβ mRNAs in various species for which most data have been gathered so far. The most surprising finding is the great variation in isoform structure among various mammalian species. This may reflect either the fact that only a very limited number of isoforms have been described so far or between‐species specificity, especially as common elements in closely related species could still be noted. Isoform variations, as detected mainly at the mRNA sequence level, should result in profound functional differences at the level of proteins as already shown in selected cases. Thus, it is proposed that the diversity of ERβ isoforms implies a functional role of this phenomenon in cellular physiology and pathology of estrogen response.
The C‐terminus of myogenin, but not MyoD, targets upregulation of MEF2C expression
Authors: Rogerson Parker J , Jamali Mina , Skerjanc Ilona S
Published: October 2002

The myogenic regulatory family of basic helix‐loop‐helix transcription factors, including MyoD and myogenin, functions cooperatively with the myocyte‐specific enhancer binding factor 2 (MEF2) family during skeletal myogenesis. Previously, using aggregated P19 cells, we have shown that myogenin upregulates MEF2C expression while MyoD does not [Ridgeway et al., J. Biol. Chem. 275 (2000) 41–46]. In order to identify the domain of myogenin responsible for activating MEF2C expression, a series of chimeras of MyoD and myogenin were generated. Only chimeras containing the C‐terminal region of myogenin were able to activate MEF2C in aggregated P19 cells, suggesting that the C‐terminus of myogenin is responsible for the regulation of specific target genes.
Potentiation of hematopoietic cell migration with an IGF–interleukin‐3 fusion protein
Authors: DiFalco Marcos R , Congote Luis Fernando
Published: October 2002

A chimera of an N‐terminally modified insulin growth factor (IGF)‐II, NQPQMVHTY‐hIGF‐II(9–67) (BOMIGF), fused to interleukin‐3 (IL‐3) significantly improved the migration of CD34+ human hematopoietic cells with respect to the effects observed during co‐stimulation with BOMIGF and IL‐3. A phosphatidylinositol‐3 (PI‐3) kinase inhibitor specifically inhibited migration in the presence of the chimera, while no significant difference in the inhibition of migration was observed in the presence of a Rho kinase inhibitor. These results suggest a key role of the PI‐3 kinase pathway in the potentiation of migration caused by the linkage of BOMIGF and IL‐3.
NMR analysis of in vitro‐synthesized proteins without purification: a high‐throughput approach
Authors: Guignard Laurent , Ozawa Kiyoshi , Pursglove Sharon E , Otting Gottfried , Dixon Nicholas E
Published: October 2002

A cell‐free protein expression system was established that provides protein samples of adequate concentration and purity for direct NMR analysis. The Escherichia coli peptidyl–prolyl cis–trans isomerase PpiB was expressed in this system with dual amino acid‐selective isotope labeling to identify the NMR signal from the active site‐residue Arg87. Addition of the substrate succinyl‐Ala‐Ala‐Pro‐Phe‐p‐nitroanilide selectively shifted its 15N‐HSQC cross peak, confirming binding to the active site. As cell‐free protein expression provides high yields of protein per unit mass of labeled amino acid and sample handling is minimal, this strategy presents an exceptionally inexpensive and rapid approach to protein analysis.
Mutagenesis identifies a conserved tyrosine residue important for the activity of uroporphyrinogen III synthase from Anacystis nidulans
Authors: Roessner Charles A , Ponnamperuma Krishan , Scott A.I
Published: October 2002

Uroporphyrinogen III synthase from the cyanobacterium Anacystis nidulans was overproduced in Escherichia coli and analyzed by site specific mutagenesis. Of the nine conserved amino acids altered, only a single tyrosine mutant (Y166F) showed any significant decrease in activity suggesting this residue is critical for proper substrate binding and/or catalysis.
Influence of lipids on membrane assembly and stability of the potassium channel KcsA
Authors: van Dalen Annemieke , Hegger Sander , Killian J.Antoinette , de Kruijff Ben
Published: October 2002

Recently we observed in an in vitro system that newly synthesized KcsA assembles efficiently into a tetramer in lipid vesicles [van Dalen et al. (2002) FEBS Lett. 511, 51–58]. Here we used this system to get insight into the importance of the lipid composition for KcsA membrane association and tetramerization and we compared this to the lipid dependency of the thermo‐stability of the KcsA tetramer. It was found that a large amount of phosphatidylethanolamine (>40 mol%) and a lower amount of phosphatidylglycerol (∼20–30 mol%) were optimal for efficient KcsA membrane association and tetramerization. Strikingly, vesicles of the abundant and commonly used membrane lipid phosphatidylcholine did not support assembly, further demonstrating the importance of membrane lipid composition for KcsA assembly. The in vitro assembled KcsA tetramer showed similar thermo‐stability in biological and pure lipid membranes, demonstrating that both tetramers are alike. In addition, we show that solubilization of the membrane with detergent reduces the thermo‐stability of the tetramer. The highest KcsA tetramer stability was observed in intact bilayers in the presence of anionic lipids.
Inhibition of hepatitis C virus NS3 protease by peptides derived from complementarity‐determining regions (CDRs) of the monoclonal antibody 8D4: tolerance of a CDR peptide to conformational changes of a target
Authors: Tsumoto Kouhei , Misawa Satoru , Ohba Yoichi , Ueno Takamasa , Hayashi Hideya , Kasai Nobuhiro , Watanabe Hideki , Asano Ryutaro , Kumagai Izumi
Published: October 2002

We have synthesized and characterized peptides derived from complementarity‐determining regions (CDRs) of 8D4, a mouse monoclonal antibody against NS3 protease domain of hepatitis C virus. 8D4 inhibits enzymatic activity without its cofactor, NS4A peptide. One of the synthetic peptides derived from CDRs, CDR1 of the heavy‐chain (CDR‐H1) peptide strongly inhibited NS3 protease activity competitively in the absence of NS4A and non‐competitively in the presence of NS4A. Moreover, cyclic CDR‐H1 peptides bridged by disulfide inhibited NS3 protease more potently. The chain length of the CDR‐H1 peptide is critical for strong inhibition, even when the peptide is circularized. This finding suggests the importance of peptide conformation. In contrast to a cognate antibody molecule, CDR‐derived peptides may provide good ligands for target molecules by having a tolerance to conformational changes of the targets caused by cofactor binding or mutation.
Magnesium sulfate compared with lytic cocktail for women with eclampsia
Authors: Duley L. , Gülmezoglu A.M.
Published: October 2002

Objective: To compare magnesium sulfate with lytic cocktail for the treatment of eclampsia. Methods: The register of trials kept by the Cochrane Pregnancy and Childbirth Group and the Cochrane Controlled Trials Register were searched for randomized trials comparing magnesium sulfate with lytic cocktail for the treatment of eclampsia. Results: Two studies were included (a total of 199 women). Magnesium sulfate was more effective than lytic cocktail at preventing further convulsions [relative risk (RR) 0.09, 95% confidence interval (C.I.)=0.03–0.24; risk difference=0.43, 95% C.I.=−0.53 to −0.34; number needed to treat=3, 95% C.I.=2–3) and was associated with less respiratory depression (RR=0.12, 95% C.I.=0.02–0.91) and fewer baby deaths (RR=0.26, 95% C.I.=0.26–0.79). There were non‐significantly fewer maternal deaths associated with magnesium sulfate (RR=0.25, 95% C.I.=0.04–1.43). Conclusions: Magnesium sulfate is the anticonvulsant of choice for eclampsia. Lytic cocktail should be abandoned.
HIV and cervical cancer in Kenya
Authors: Gichangi P. , De Vuyst H. , Estambale B. , Rogo K. , Bwayo J. , Temmerman M.
Published: October 2002

Objectives: To determine the effect of the HIV epidemic on invasive cervical cancer in Kenya. Methods: Of the 3902 women who were diagnosed with reproductive tract malignancies at Kenyatta National Hospital (KNH) from 1989 to 1998, 85% had invasive cervical cancer. Age at presentation and severity of cervical cancer were studied for a 9‐year period when national HIV prevalence went from 5% to 5–10%, to 10–15%. Results: There was no significant change in either age at presentation or severity of cervical cancer. Of the 118 (5%) women who were tested for HIV, 36 (31%) were seropositive. These women were 5 years younger at presentation than HIV‐negative women. Conclusions: A two‐ to three‐fold increase in HIV prevalence in Kenya did not seem to have a proportional effect on the incidence of cervical cancer. Yet, HIV‐positive women who presented with cervical cancer were significantly younger than HIV‐negative women.
Expression of peroxisome proliferator‐activated receptors alpha and gamma in differentiating human colon carcinoma Caco‐2 cells
Authors: Huin Cécile , Schohn Hervé , Hatier Renée , Bentejac Marc , Antunes Laurent , Plénat François , Bugaut Maurice , Dauça Michel
Published: October 2002

The expression of peroxisome proliferator‐activated receptors α (PPARα) and γ (PPARγ) was studied in the human adenocarcinoma Caco‐2 cells induced to differentiate by long term culture (15 days). The differentiation of Caco‐2 cells was attested by increases in the activities of sucrase‐isomaltase and alkaline phosphatase (two brush border enzymes), fatty acyl‐CoA oxidase (AOX) and catalase (two peroxisomal enzymes), by an elevation in the protein levels of villin (a brush border molecular marker), AOX, peroxisomal bifunctional enzyme (PBE), catalase and peroxisomal membrane protein of 70 kDa (PMP70), and by the appearance of peroxisomes. The expression of PPARα and PPARγ was investigated by Western blotting, immunocytochemistry, Northern blotting and S1 nuclease protection assay during the differentiation of Caco‐2 cells. The protein levels of PPARα, PPARγ, and PPARγ2 increased gradually during the time‐course of Caco‐2 cell differentiation. Immunocytochemistry revealed that PPARα and γ were localized in cell nuclei. The PPARγ1 protein was encoded by PPARγ3 mRNA because no signal was obtained for PPARγ1 mRNA using a specific probe in S1 nuclease protection assay. The amount of PPARγ3 mRNA increased concomitantly to the resulting PPARγ1 protein. On the other hand, the mRNA of PPARα and PPARγ2 were not significantly changed, suggesting that the increase in their respective protein was due to an elevation of the translational rate. The role played by the PPAR subtypes in Caco‐2 cell differentiation is discussed.
Sources of volume flexibility and their impact on performance
Authors: Jack Eric P. , Raturi Amitabh
Published: October 2002

This research uses three in‐depth case studies to establish the drivers and sources of volume flexibility. We find that in all three firms, there is significant concern among managers for gaining competitiveness through volume flexibility. We also find that there are several avenues for developing a volume flexible response and that deployment of these tactics is dependent on the availability of resources and systems. To verify some of these propositions we report on the outcomes of a field survey that measures the importance firms place on volume flexibility as well as the corresponding actions they take to remain volume flexible. Our critical finding is that short‐ and long‐term sources of volume flexibility have a positive, albeit differential, impact on a firm’s performance.
Subchondral bone thickness, hardness and remodelling are influenced by short‐term exercise in a site‐specific manner
Authors: Murray R. C. , Vedi S. , Birch H. L. , Lakhani K. H. , Goodship A. E.
Published: October 2002

It was hypothesised that subchondral bone thickness, hardness and remodelling are influenced by exercise intensity, and by location within a joint. Dorsal carpal osteochondral injury is a major cause of lameness in horses undergoing high intensity training. This project aimed to determine the subchondral bone thickness, formation, resorption and hardness at sites with high and low incidence of pathology in 2 year‐old horses undergoing 19 weeks high intensity treadmill training or low intensity exercise, and to compare these factors between exercise groups. Dorsal and palmar test sites were identified on radial, intermediate and third carpal articular surfaces after euthanasia. Adjacent osteochondral samples from each test site underwent histomorphometric analysis (for subchondral bone thickness, osteoid perimeter, osteoid seam width, eroded cavity area and eroded cement line surface length) and microhardness testing. Bone from horses undergoing high intensity training was thicker with a greater osteoid perimeter, and at individual sites had a smaller osteoid seam width and eroded cavity. Exercise‐related differences were most marked at dorsal locations. Maximal differences in bone formation indices were observed at dorsal radial and medial third carpal locations. Overall subchondral bone from dorsal sites was thicker with a greater osteoid perimeter. Subchondral bone from dorsal sites was approximately 35% harder than bone from palmar sites. These results show topographical variations in subchondral bone structure, formation, resorption and material properties and a site‐specific response to exercise. The maximal response to exercise was at high load sites with a clinical predisposition to injury. These findings indicate that the combined effect of exercise and local load variations within a joint may lead to maximal adaptive responses or overload of these responses at sites predisposed to injury. © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Gait analysis after total hip arthroplasty with leg‐length equalization in women with unilateral congenital complete dislocation of the hip – comparison with untreated patients
Authors: Lai Kou‐An , Lin Chii‐Jen , Jou I‐Ming , Su Fon‐Chin
Published: October 2002

Patients with Crowe Type‐IV congenital dislocation of the hip (CDH) show significant clinical improvement after total hip arthroplasty (THA) because this surgery greatly reduces pain. Concomitant leg‐length equalization in unilateral patients — a controversial procedure — theoretically should significantly improve these patients' ability to walk efficiently and comfortably. To understand the impact of leg‐length equalization on these patients, we compared their gait parameters with those of untreated patients without pain but with leg‐length discrepancy. Using a motion analysis system, three force platforms and computer calculation, the gait parameters during level walking of 22 women with unilateral Crowe Type‐IV CDH were studied at an average of 58 months (27–98 months) following a successful cementless THA. The socket was placed in the best bone stock, which was close to the level of the true acetabulum. The leg‐length discrepancy was equalized to within 2 cm in all patients. The Harris hip score averaged 94.8 (range, 88–100) at the time of the study. Nine women with untreated unilateral Crowe Type‐IV CDH without major pain but with an average leg‐length discrepancy of 4.7 cm (range, 2.5–6 cm) were also studied for comparison. The treated subjects (Group 1; THA and leg‐length equalization) walked faster and had gait parameters with better bilateral symmetry than the untreated subjects (Group 2). We concluded that leg‐length equalization in addition to THA in patients with unilateral Crowe Type‐IV CDH significantly improves gait symmetry and efficiency. © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Efficacy of ex vivo OPG gene therapy in preventing wear debris induced osteolysis
Authors: Goater J. Jeffrey , O'Keefe Regis J. , Rosier Randy N. , Puzas J. Edward , Schwarz Edward M.
Published: October 2002

Aseptic loosening of prosthetic implants remains a serious orthopaedic problem and the greatest limitation to total joint arthroplasty. Central to the etiology of aseptic loosening is periprosthetic osteolysis at the bone‐implant interface, which is caused by wear debris‐induced inflammation. This inflammation produces the critical osteoclast differentiation factor RANKL, which directly stimulates osteoclastogenesis and osteoclastic bone resorption. A dominant factor known to counteract this process is the natural RANKL receptor antagonist protein OPG. Here we explore the potential of ex vivo OPG gene therapy for aseptic loosening by evaluating the efficacy of stably transfected fibroblast‐like synoviocytes (FLS) expressing OPG in preventing wear debris‐induced osteoclastogenesis, in a mouse calvaria model. Although the stably transfected fibroblasts produced small amounts of OPG (0.3 ng/ml/72 h/106 cells), this protein was very effective in preventing osteoclastic resorption as determined in a bone wafer assay. More importantly, implantation of 107 FLS–OPG, together with 30 mg of Ti wear debris, onto the calvaria of mice, completely inhibited osteoclastogenesis 3 days after surgery. Animals given FLS‐LacZ control cells, which persisted for 3 days as determined by X‐gal staining, together with the Ti particles, had a 6‐fold increase in osteoclastogenesis compared to controls without Ti. This increased osteoclastogenesis was completely inhibited by the FLS‐OPG, as osteoclast numbers in the calvaria of these animals were similar to that seen in the SHAM controls. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Tumor necrosis factor‐α mediates polymethylmethacrylate particle‐induced NF‐κB activation in osteoclast precursor cells
Authors: Clohisy John C. , Teitelbaum Steven , Chen Shaoping , Erdmann Jeanne M. , Abu‐Amer Yousef
Published: October 2002

Tumor necrosis factor‐α (TNF) is a potent osteoclastogenic cytokine that has a fundamental role in the pathogenesis of implant particle‐induced osteolysis. The nuclear transcription factor NF‐κB mediates TNF signaling and this transcription complex is necessary for osteoclastogenesis. Because polymethylmethacrylate (PMMA) particles cause osteolysis, we reasoned the PMMA would induce NF‐κB activation. In fact, we find that exposure of osteoclast precursors, in the form of colony stimulating factor‐1 (CSF‐1) dependent murine bone marrow macrophages, to PMMA particles prompts nuclear translocation and activation of NF‐κB. Supershift assays confirm the presence of the p50 and p65 NF‐κB subunits in the activated transcription factor. Particle‐induced NF‐κB activation is equal in both wild type and LPS‐ hyporesponsive cells indicating that the phenomenon does not represent endotoxin contamination. A soluble, competitive inhibitor of TNF (huTNF:Fc) dampens particle‐directed NF‐κB activation and this response is also abrogated in TNF−/− osteoclast precursors. Thus, PMMA particle activation of NF‐κB is a secondary event resulting from enhanced TNF expression and is independent of LPS contamination. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Geometric analysis of selected press fit prosthetic systems for proximal humeral replacement
Authors: Pearl Michael L. , Kurutz Sam , Robertson Douglas D. , Yamaguchi Ken
Published: October 2002

The ability of a prosthetic system to replicate a wide range of normal anatomy appears dependent in part to its capacity to produce variable prosthetic geometries. Several modern designs have recently been developed in order to provide multiple prosthetic options. The purpose of this study was to compare the geometry of select press fit prosthetic systems in terms of their ability to match normal three‐dimensional geometry of the proximal humerus. The anatomy of 60 humeral specimens from 30 cadavers known from CT data and computer‐aided design (CAD) analysis was compared to a 1996 database of four conventional shoulder prosthetic systems (fixed inclination angle, one medial‐lateral offset position per head) and one so‐called anatomic system (variable inclination angles, multiple head offset positions). The prosthetic system with greatest geometric options allowed for a significantly better replication of the anatomy. Average displacement of the center of rotation was 2.1 mm compared to 9.7 mm for the other systems combined. Reduction in surface arc was 12° compared to 32°. The most problematic feature of conventional prosthetic systems in terms of replicating normal humeral anatomy is the gap created by the prosthetic collar and Morse taper. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.