bluuebunny/crossref_metadata_2025 huggingface dataset:
Stimulation of platelet glycoprotein IIb‐IIIa (αIIbβ3‐integrin) functional activity by a monoclonal antibody to the N‐terminal region of glycoprotein IIIa
Authors: Mazurov Alexey V. , Khaspekova Svetlana G. , Byzova Tatjana V. , Tikhomirov Oleg Yu. , Berndt Michael C. , Steiner Beat , Kouns William C.
Published: April 2003

Platelet glycoprotein (GP) IIb‐IIIa complex (αIIbβ3‐integrin) changes its conformation upon platelet activation that results in binding of RGD‐containing ligands and expression of ligand‐induced binding site (LIBS) neoepitopes. Anti‐GIIb‐IIIa monoclonal antibody (monAB) CRC54 bound to ≤10% of GPIIb‐IIIa on resting platelets but binding was enhanced by the occupation of GPIIb‐IIIa with RGDS peptide and by platelet activation indicating that CRC54 is directed against LIBS epitope. The epitope was located within the first 100 N‐terminal residues of GPIIIa and differed from other LIBS epitopes. CRC54 as well as its Fab fragments were able to induce platelet aggregation. CRC54 also stimulated interaction of GPIIb‐IIIa with its ligands (fibrinogen and fibronectin) and conformation‐dependent antibodies. The results indicated that changes of GPIIb‐IIIa conformation, binding of ligands and platelet aggregation could be stimulated via interaction of anti‐LIBS antibody with the N‐terminal part of GPIIIa.
In vivo activation of recombinant cAPK catalytic subunit active site mutants by coexpression of the wild‐type enzyme, evidence for intermolecular cotranslational phosphorylation
Authors: Girod Andreas , Kinzel Volker , Bossemeyer Dirk
Published: April 2003

The catalytic subunit of cAMP dependent protein kinase (cAPK) carries two stable autophosphorylated residues. One of them, Thr197, residues in the so‐called protein kinase activation segment, and needs to be phosphorylated for full activity and protein kinase inhibitor binding of the enzyme. While wild‐type recombinant mammalian C‐subunit, expressed in E. coli, can fully autoactivate itself by phosphorylation at Thr197, many active site mutants lack this autophosphorylation activity, so that the primary effects of the mutations become obscured. Two active site mutants of bovine C‐subunit, defective in protein kinase inhibitor peptide binding, were activated by wild‐type enzyme in vivo, but could not be activated in vitro, demonstrating intermolecular and presumably cotranslational autophosphorylation. The results may delineate strategies for the expression and mutagenesis of other protein kinases with requirements for activation segment phosphorylation.
Transport of the glutathione conjugate of ethacrynic acid by the human multidrug resistance protein MRP
Authors: Zaman Guido J.R. , Cnubben Nicole H.P. , van Bladeren Peter J. , Evers Raymond , Borst Piet
Published: April 2003

The multidrug resistance protein MRP has been shown to mediate the transport of glutathione S‐conjugates across membranes. In this study we demonstrate that the glutathione S‐conjugate of the diuretic drug ethacrynic acid, which is an efficient inhibitor of glutathione S‐transferases, is a high‐affinity substrate and inhibitor of the glutathione S‐transferase activity, but also pump associated with MRP. This implies that ethacrynic acid may modulate drug resistance of tumor cells not only by inhibiting glutathione S‐transferase activity, but also by inhibiting the export of drug conjugates from the cell by MRP.
Structural requirements for α‐mating factor activity
Authors: Houen G. , Nielsen O. , Flanagan C.
Published: April 2003

The sexual hormone of S. cerevisiae, α‐mating factor (α‐MF, WHWLQLKPGQPMY) has structural homology with mammalian luteinizing hormone releasing hormone (LHRH, pEHWSYGLRPG‐NH2) and has been shown to exhibit LHRH activity [Loumaye et al. (1982) Science 218, 1323–1325]. We have tested whether LHRH has α‐MF activity in yeast and found that it does not. We therefore synthesized a series of hybrid peptides of α‐MF and LHRH to study the structural features which determine α‐MF and LHRH activities. A hybrid peptide consisting of the LHRH sequence with the C‐terminal tetrapeptide (QPMY) of α‐MF did not exhibit α‐MF activity. Thus, the lack of α‐MF activity of LHRH is not due solely to the absence of the C‐terminal residues. Substitution of Lys7 in α‐MF with Arg, as is found in LHRH, did not affect the α‐MF activity, nor did an additional substitution of Trp1 with pGlu. However, the C‐terminal four amino acids of α‐MF were necessary for α‐MF activity. Our results indicate that insertion of a Ser residue in position 4 as found in LHRH abolishes α‐MF activity. These results suggest that, in addition to an intact C‐terminus correct spacing of the N‐terminal His2 and the C‐terminus is required for α‐MF activity. The hybrid peptides all exhibited less LHRH activity than either LHRH or α‐MF. These structure‐function studies indicate that the structural homology between these two reproductive hormones may not reflect an evolutionary relationship between them.
Down‐regulation of the protein kinase A pathway by activators of protein kinase C and intracellular Ca2+ in fibroblast cells
Authors: Döbbeling Udo , Berchtold Martin W.
Published: April 2003

Many genese are regulated by the intracellular calcium protein kinase C (PKC) and protein kinase A (PKA) pathways and it has been shown that these pathways synergize in some cell types, whereas they antagonize in others. Here we show that the calcium and PKC pathways suppress the effects mediated by the PKA pathway in a fibroblast cell line. The suppressing effect of elevated intracellular Ca2+ levels, but not of the PKC pathway, can be abrogated by the addition of cyclosporin A (CsA), indicating that the effect of Ca2+ is mediated by phosphatase‐2B (PP‐2B/calcineurin). Suppression by the PKC pathway is not mediated by the proto‐oncogenes c‐fos, c‐jun and junB, as the co‐transfection of these genes does not block the effects of the PKA stimulator 8‐Br‐cAMP. In addition, cotransfection with the catalytic subunit of PKA shows that the inhibitory effect of PKC occurs upstream of PKA activation.
Molecular imaging of Escherichia coli F0F1‐ATPase in reconstituted membranes using atomic force microscopy
Authors: Takeyasu Kunio , Omote Hiroshi , Nettikadan Saju , Tokumasu Fuyuki , Iwamoto-Kihara Atsuko , Futai Masamitsu
Published: April 2003

The structure of Escherichia coli F0F1‐ATPase (ATP synthase), and its F0 sector reconstituted in lipid membranes was analyzed using atomic force microscopy (AFM) by tapping‐mode operation. The majority of F0F1‐ATPases were visualized as spheres with a calculated diameter of , and a height of from the membrane surface. F0 sectors were visualized as two different ring‐like structures (one with a central mass and the other with a central hollow of depth) with a calculated outer diameter of . The two different images possibly represent the opposite orientations of the complex in the membranes. The ring‐like projections of both images suggest inherently asymmetric assemblies of the subunits in the F0 sector. Considering the stoichiometry of F0 subunits, the area of the image observed is large enough to accommodate all three F0 subunits in an asymmetric manner.
Activation of NF‐κB by ER stress requires both Ca2+ and reactive oxygen intermediates as messengers
Authors: Pahl Heike L. , Baeuerle Patrick A.
Published: April 2003

The eukaryotic transcription factor NF‐κB is activated by a large variety of stimuli. We have recently shown that ER stress, caused by an aberrant accumulation of membrane proteins within this organelle, also activates NF‐κB. Here, we show that activation of NF‐κB by ER stress requires an increase in the intracellular levels of both reactive oxygen intermediates (ROIs) and ca2+. Two distinct intracellular Ca2+ chelators and a panel of structurally unrelated antioxidants prevented NF‐κB activation by various ER stress‐eliciting agents, whereas only antioxidants but not the Ca2+ chelators prevented NF‐κB activation by the inflammatory cytokine TNF‐α. Consistent with an involvement of calcium, the ER‐resident Ca2+‐ATPase inhibitors thapsigargin and cyclopiazonic acid (CPA), which trigger a rapid efflux of Ca2+ from the ER, also potently activated NF‐κB. Pretreatment with a Ca2+ chelator abrogated this induction. The Ca2+ chelator BAPTA‐AM inhibited ROI formation in response to thapsigargin and CPA treatment, suggesting that the Ca2+ increase preceded ROI formation during NF‐κB activation. The selective inhibitory effect of the drug tepoxalin suggests that the peroxidase activity of cyclooxygenases or lipoxygenases was responsible for the increased ROI production in response to Ca2+ release by thapsigargin.
IL4 and IL13 receptors share the γc chain and activate STAT6, STAT3 and STAT5 proteins in normal human B cells
Authors: Rolling Christine , Treton Dominique , Pellegrini Sandra , Galanaud Pierre , Richard Yolande
Published: April 2003

IL13 induces the same biological effects as IL4 in normal human B cells. We show that as in the IL4R complex, both IL4Rα and IL2Rγc are components of the IL13R and that both cytokines induced STAT6, STAT3 and STAT5 activation in B cells. In spite of this similar downstream signalling, IL4 and IL13 used a different set of Janus kinases: IL13 is unable to activate JAK1 and JAK3.
Correlation of intrinsic DNA curvature with DNA property periodicity
Authors: Gabrielian Andrei , Pongor Sándor
Published: April 2003

Twelve di‐ and trinucleotide parameter sets representing various structural, thermodynamic or bendability‐related properties of DNA were tested in the prediction of DNA curvature applying Fourier analysis on curved and straight, A/T‐type or G/C‐type DNA sequence motifs. The best predictions were obtained with a new consensus bendability scale created by combining a nucleosome‐based and a deoxyribonuclease I‐based parameter set. Geometry calculations on the same sequences showed that the helical parameters derived from NMR structures can correctly predict curvature, as distinct from the parameters derived from X‐ray crystallographic analysis.
Soluble Nef antigen of HIV‐1 is cytotoxic for human CD4+ T cells
Authors: Fujii Yoichi , Otake Kaori , Tashiro Masato , Adachi Akio
Published: April 2003

We have previously shown that Nef‐gene 10 fusion protein induces marked growth arrest of human primary CD4+ T cells. Here, in vitro cytostatic and cytotoxic activities of human immunodeficiency virus type 1 (HIV‐1) Nef against CD4+ T cells were extensively investigated. Growth of human CD4+ cells was inhibited significantly just by the addition of purified full‐length Nef to cultures. When Nef was cross‐linked by anti‐Nef antibodies, it became very cytocidal for CD4+ T cells. A high percentage of sera from HIV‐1‐infected individuals contained soluble Nef. Thus, soluble Nef in vivo may play an important role in immunodysfunction of CD4+ T lymphocytes in HIV‐1 infection.
Human immunodeficiency virus type 1 Nef protein on the cell surface is cytocidal for human CD4+ T cells
Authors: Fujii Yoichi , Otake Kaori , Tashiro Masato , Adachi Akio
Published: April 2003

We have previously shown that the carboxyl‐terminal region of human immunodeficiency virus type 1 (HIV‐1) Nef antigen present on the outer surface of virus‐infected cells has affinity for uninfected T cells. Here, the in vitro cytotoxic potential of HIV‐1 Nef on the T cell surface against CD4+ T cells was investigated in detail. Human T cells expressing Nef on the cell surface by transfection with non‐infectious mutant HIV‐1 proviruses were demonstrated to kill CD4+ T cells effeciently. Furthermore, it was shown that the carboxyl‐terminal portion of Nef was cytotoxic for CD4+ T cells and that monoclonal antibody against the carboxyl‐terminal region of Nef inhibited Nef induced‐cytolysis. Thus, we concluded that Nef protein on CD4+ T cells may play an important role in the specific loss of CD4+ T lymphocytes during HIV‐1 infection.
Only sphingolipid activator protein B (SAP‐B or saposin B) stimulates the degradation of globotriaosylceramide by recombinant human lysosomal α‐galactosidase in a detergent‐free liposomal system
Authors: Kase Ryoichi , Bierfreund Uwe , Klein Andreas , Kolter Thomas , Itoh Kohji , Suzuki Minoru , Hashimoto Yasuhiro , Sandhoff Konrad , Sakuraba Hitoshi
Published: April 2003

The degradation of globotriaosylceramide (GbOse3Cer) by insect‐cell derived recombinant human α‐galactosidase (EC 3.2.1.22) was carried out in a detergent‐free liposomal system in order to mimic intralysosomal conditions. GbOse3Cer incorporated into unilamellar liposomes was used as the substrate, and naturally occurring sphingolipid activator proteins, rather than detergents, were used to stimulate the enzyme reaction. The degradation of GbOse3Cer was dependent on the presence of both α‐galactosidase and sphingolipid activator protein B (SAP‐B or saposin B). It proceeded optimally at pH 4.6, and was enhanced by increasing amounts of both α‐galactosidase (0.24–24 mU/50 μl assay) and SAP‐B (0–5 μg/50 μl assay). The enzyme reaction was not affected by SAP‐A, SAP‐C, or SAP‐D. Therefore, our results indicate that only SAP‐B is essential for the degradation of GbOse3Cer by α‐galactosidase.
Relative importance of inositol (1,4,5)trisphosphate and inositol (1,3,4,5)tetrakisphosphate in Entamoeba histolytica
Authors: Giri Banabihari , Raha Sanghamitra , Bhattacharyya Bhudeb , Biswas Susweta , Biswas Birendra B.
Published: April 2003

[3H]Inositol tetrakisphosphate (Ins(1,3,4,5)P4) binding sites which were poorly displaced by unlabelled inositol (1,4,5)‐trisphosphate (Ins(1,4,5)P3) were detected in membrane fractions of Entamoeba histolytica. Similarly, unlabelled Ins(1,3,4,5)P4 was 30‐fold less efficient in displacing [3H]Ins(1,4,5)P3 binding. pH sensitivities of binding of the two isomers were markedly different. Scatchard analysis of the data revealed single binding sites and similar receptor densities for each of the two isomers. Formation of both Ins(1,4,5)P3 and Ins(1,3,4,5)P4 in E. histolytica was also demonstrated. Calcium release studies showed that after treatment with a saturating dose of either Ins(1,4,5)P3 or Ins(1,3,4,5)P4 the other inositol polyphosphate could partially revive the response to a subsequent addition of the first inducer. Our data clearly demonstrate that Ins(1,4,5)P3 and Ins(1,3,4,5)P4 are two equally important but independent second messengers in E. histolytica.
The conformational stability of α‐crystallin is rather low: Calorimetric results
Authors: Gesierich Ulrike , Pfeil Wolfgang
Published: April 2003

The eye lens protein and chaperonin, α‐crystallin, was studied by differential scanning microcalorimetry, spectroscopy and size exclusion chromatography. The thermal transition of α‐crystallin proceeds at T

trs
= 59.8 ± 0.6°C with an enthalpy change of ΔH = 336 ± 9 kJ per mol subunit. Disagreement between previous ΔH values could be attributed to a side reaction that leads, depending on the scan rate, to the formation of a non‐productive folding form. The conformational stability of α‐crystallin is rather low (ΔG = 24 ± 5 kJ/mol of subunit). The minimal cooperative unit of α‐crystallin is the monomeric subunit.
Similar effects of α‐ and β‐SNAP on Ca 2+‐regulated exocytosis
Authors: Sudlow Allan W. , McFerran Brian W. , Bodill Helena , Barnard Richard J.O. , Morgan Alan , Burgoyne Robert D.
Published: April 2003

Soluble N‐ethylmaleimide‐sensitive fusion protein attachment proteins (SNAP) proteins function in Ca2+‐regulated exocytosis. Recent work (Schiavo et al. (1996) Nature 378, 733–736) based on in vitro protein interactions has raised the possibility that α‐ and β‐SNAPs have distinct roles in exocytosis. We have examined this possibility by comparing the activities of recombinant α‐ and β‐SNAPs. Both these proteins were able to similarly bind NSF and activate its ATPase activity but to a lesser extent than γ‐SNAP. When introduced into digitoninpermeabilised chromaffin cells, both α‐ and β‐SNAP stimulated Ca2+‐regulated exocytosis in a MgATP‐dependent manner. The dose‐response relationships for these proteins were essentially the same and addition of both proteins did not lead to any further increase in exocytosis above that due to each protein alone. We conclude that α‐ and β‐SNAPs are interchangeable isoforms with similar functions in regulated exocytosis.
High‐level expression of Pinus sylvestris glutamine synthetase in Escherichia coli
Authors: Canton Francisco R. , Garcia-Gutierrez Angel , Crespillo Remedios , Cánovas Francisco M.
Published: April 2003

In a previous work we reported the molecular characterization of a glutamine synthetase (GS; EC 6.3.1.2.) complementary DNA from a woody plant (Cantón et al. (1993) Plant Mol. Biol. 22, 819–828). The isolated cDNA (pGSP114) encoding a Scots pine (Pinus sylvestris) cytosolic subunit, has been subcloned into the expression vector pET3c to overproduce the GS polypeptide in Escherichia coli cells. The recombinant GS protein showed the same molecular size as a native Scots pine GS subunit. Antibodies against the pET3c‐GSP114 encoded protein were raised in rabbits by injecting purified preparations and specificity was determined by immunoprecipitation of GS activity present in pine crude extracts. In spite of the antibodies were able to recognize both cytosolic and chloroplastic GS in tomato plants, they were unable to immunodetect chloroplastic GS in green cotyledons of pine seedlings and cytosolic GS was the unique recognized polypeptide. Unlike to that found in other plant species, cytosolic GS was strongly expressed in green tissues as determined by protein and Northern analysis. Our results suggest a key role for cytosolic GS in photosynthetic tissues of conifers.
Rhizobium fredii synthesizes an array of lipooligosaccharides, including a novel compound with glucose inserted into the backbone of the molecule
Authors: Bec-Ferté Marie-Pierre , Krishnan Hari B. , Savagnac Arlette , Pueppke Steven G. , Promé J.-C.
Published: April 2003

Flavonoid cues from the plant host cause symbiotic, nitrogen‐fixing rhizobia to synthesize lipochitooligosaccharides (LCOs), which act as return signals to initiate the nodulation process. Rhizobium fredii strain USDA257 is known to produce four LCOs, all substituted with vaccenic acid (C18:1). We show here that a mutant strain can replace vaccenic acid with a C16:0 side chain, and that strain USDA191 synthesizes additional LCOs that differ from one another in the length and unsaturation of their fatty acyl substituents. USDA191 and 257DH4 both produce a novel LCO with glucose substituted for in the backbone of the molecule.
Mouse cathepsin K: cDNA cloning and predominant expression of the gene in osteoclasts, and in some hypertrophying chondrocytes during mouse development
Authors: Rantakokko Juho , Aro Hannu T. , Savontaus Mikko , Vuorio Eero
Published: April 2003

We have constructed cDNA clones covering the entire coding region of mouse, human and rabbit preprocathepsin K mRNA for studies on bone turnover. The clone pMCatK‐1 for mouse cathepsin K shares 87% nucleotide homology with the corresponding human and rabbit sequences. Analysis of a panel of mouse tissues for tissue distribution of cathepsin K mRNA revealed the highest levels in musculoskeletal tissues: bone, cartilage and skeletal muscle. In situ hybridization of developing mouse embryos was performed to identify the cellular source of cathepsin K mRNA. The strongest mRNA signal was detected in osteoclasts of bone, identified in serial sections by positive TRAP staining. Cathepsin K mRNA was also observed in some hypertrophic chondrocytes of growth cartilages. Association of cathepsin K production with degradation of bone and cartilage matrix suggests that this enzyme and its mRNA levels could serve as markers for matrix degradation in diseases affecting these tissues.
NADH‐mediated DNA damage induced by a new coenzyme, pyrroloquinoline quinone, in the presence of copper(II) ion
Authors: Hiraku Yusuke , Kawanishi Shosuke
Published: April 2003

Pyrroloquinoline quinone (PQQ) plays a role as a vitamin or growth factor. Low concentrations of PQQ induced DNA cleavage sites frequently at thymine and cytosine residues in the presence of NADH and Cu(II). Catalase and bathocuproine inhibited DNA damage, whereas free hydroxyl radical scavengers did not. Electron spin resonance and UV‐visible spectrometries showed generation of semiquinone radical and superoxide during the reaction of PQQ with NADH. These results suggest that NADH‐dependent PQQ redox cycle generated superoxide and hydrogen peroxide to mediate copper‐dependent DNA damage. The present study has proposed a requirement to investigate the potentiality of PQQ carcinogenicity.
Characterization of the newly constructed domains that replace P5abc within the Tetrahymena ribozyme
Authors: Ikawa Yoshiya , Shiraishi Hideaki , Inoue Tan
Published: April 2003

The P5abc domain of the Tetrahymena ribozyme has been shown to function as an activator that enhances core catalytic activity of the ribozyme. We reported previously that several new domains in that their primary sequences are different from that of P5abc are also capable of activating the ribozyme. It was unclear whether the mechanism of activation by the new domains is identical to that by P5abc. We have investigated structural and functional properties of the new domains and obtained evidence that strongly indicates that a particular domain activates the ribozyme in a different manner from that by P5abc.
α‐Lipoic acid blocks HIV‐1 LTR‐dependent expression of hygromycin resistance in THP‐1 stable transformants
Authors: Merin Jocelyn P. , Matsuyama Mototaka , Kira Toshihiko , Baba Masanori , Okamoto Takashi
Published: April 2003

Gene expression of human immunodeficiency virus (HIV) depends on a host cellular transcription factors including nuclear factor‐κB (NF‐κB). The involvement of reactive oxygen intermediates (ROI) has been implicated as intracellular messengers in the inducible activation of NF‐κB. In this study, we compared the efficacy of two antioxidants, α‐lipoic acid (LA) and N‐acetylcysteine (NAC), which are widely recognized NF‐κB inhibitors. Here, we demonstrate that LA has a more potent activity in inhibiting NF‐κB‐mediated gene expression in THP‐1 cells that have been stably transfected with a plasmid bearing a hygromycin B resistance gene under the control of HIV‐1 long terminal repeat (LTR) promoter. The spontaneous activation of NF‐κB in this cell culture system leads to expression of the hygromycin phosphotransferase gene hence rendering the cells resistance to hygromycin B. In this study, the effect of the test compounds against transcriptional activity of HIV‐1 LTR was evaluated based on the degree of cellular toxicity due to the inhibitory activity on the expression of hygromycin B resistance gene in the presence of hygromycin B. We also found that 0.2 mM LA could cause 40% reduction in the HIV‐1 expression from the TNF‐α‐stimulated OM 10.1, a cell line latently infected with HIV‐1. On the other hand, 10 mM NAC was required to elicit the same effect. Furthermore, the initiation of HIV‐1 induction by TNF‐α was completely abolished by 1 mM LA. These findings confirm the involvement of ROI in NF‐κB‐mediated HIV gene expression as well as the efficacy of LA as a therapeutic regimen for HIV infection and acquired immunodeficiency syndrome (AIDS). Moreover, this study validates the applicability of our present assay system which we primarily designed for the screening of candidate drugs against HIV‐1 gene expression.
Purification and characterization of the Escherichia coli thermoresistant gluconokinase encoded by the gntK gene
Authors: Izu Hanae , Adachi Osao , Yamada Mamoru
Published: April 2003

A thermoresistant gluconokinase encoded by the gntK gene of Escherichia coli K‐12 was purified and characterized. The K

m
values of the purified enzyme for gluconate and ATP are 42 μM and 123 μM, respectively, and the activity was not altered by the presence of pyruvate. The enzyme was shown to function as a dimer with two identical subunits of 18.4 kDa. These characteristics appear to be distinct from those of the gluconokinase reported by E.I. Vivas, A. Liendo, K. Dawidowicz, and T. Istúriz (1994) J. Basic. Microbiol. 16, 117–122.
SNAP‐25 is present on chromaffin granules and acts as a SNAP receptor
Authors: Tagaya Mitsuo , Genma Tomohiro , Yamamoto Akitsugu , Kozaki Shunji , Mizushima Shoji
Published: April 2003

SNAP‐25 is located on the plasma membrane and essential for exocytosis of neurotransmitters. It was suggested that SNAP‐25 and syntaxin 1 via the interaction with VAMP‐2 located on synaptic vesicles mediate the docking of the vesicles with the plasma membrane. In the present study, by means of biochemical and morphological analyses, we showed that SNAP‐25 is present on chromaffin granules as well as on the plasma membrane. Reconstitution and immunoprecipitation analyses revealed that SNAP‐25 on chromaffin granules has essentially the same properties as does SNAP‐25 on the plasma membrane.
Amino acid sequence and crystal structure of buffalo α‐lactalbumin
Authors: Calderone V. , Giuffrida M.G. , Viterbo D. , Napolitano L. , Fortunato D. , Conti A. , Acharya K.R.
Published: April 2003

Isolation, purification, amino acid sequence determination and X‐ray crystal structure of buffalo α‐lactalbumin were performed in order to gain further knowledge of the molecular basis of α‐lactalbumin in the lactose synthase complex. The deduced amino acid sequence differs at one position from the bovine α‐lactalbumin sequence (at position 17). The refined crystal structure at 2.3 Å is very similar to those previously reported for human and baboon α‐lactalbumins. However, a portion of the molecule (residues 105–109) exhibits different conformation. It forms a ‘flexible loop’, and appears to be a functionally important region in forming the lactose synthase complex.
Functional conservation of a natural cysteine peptidase inhibitor in protozoan and bacterial pathogens1
Authors: Sanderson S.J. , Westrop G.D. , Scharfstein J. , Mottram J.C. , Coombs G.H.
Published: April 2003

Cysteine peptidase inhibitor genes (ICP) of the chagasin family have been identified in protozoan (Leishmania mexicana and Trypanosoma brucei) and bacterial (Pseudomonas aeruginosa) pathogens. The encoded proteins have low sequence identities with each other and no significant identity with cystatins or other known cysteine peptidase inhibitors. Recombinant forms of each ICP inhibit protozoan and mammalian clan CA, family C1 cysteine peptidases but do not inhibit the clan CD cysteine peptidase caspase 3, the serine peptidase trypsin or the aspartic peptidases pepsin and thrombin. The functional homology between ICPs implies a common evolutionary origin for these bacterial and protozoal proteins.
IQGAP1 as signal integrator: Ca2+, calmodulin, Cdc42 and the cytoskeleton
Authors: Briggs Michael W , Sacks David B
Published: April 2003

A family of proteins known as IQGAPs have been identified in yeast, amebas and mammals. IQGAPs are multidomain molecules that contain several protein‐interacting motifs which mediate binding to target proteins. Mammalian IQGAP1 is a component of signaling networks that are integral to maintaining cytoskeletal architecture and cell–cell adhesion. Published data suggest that IQGAP1 is a scaffolding protein that modulates cross‐talk among diverse pathways in complex regulatory circuits. These pathways include modulating the actin cytoskeleton, mediating signaling by Rho family GTPases and calmodulin, regulating E‐cadherin and β‐catenin function and organizing microtubules.
Isolation and characterization of a flowering plant male gametic cell‐specific promoter1
Authors: Singh Manjit , Bhalla Prem L , Xu Huiling , Singh Mohan B
Published: April 2003

Flowering plant male gametic cell‐specific gene expression has been reported recently but the regulatory elements controlling specificity of such genes expressed in generative cell and sperm cells have not been identified and studied. Here, we report the 0.8 kb promoter sequence upstream of the start of the transcription site of the generative cell‐specific gene, LGC1, sufficient to regulate the expression of reporter genes in a cell‐specific manner. In addition, the diphtheria toxin A‐chain‐ (DT‐A)‐coding region under the control of the LGC1 promoter sequence confirmed unequivocally the lack of LGC1 expression in vegetative tissues. Transgenic tobacco plants carrying the LGC1‐DT/A construct showed normal phenotype except for anthers of these plants that contained sterile and aborted pollen. Truncation and internal deletion analysis of the LGC1 promoter identified −242 bp as the minimal sequence necessary for male gametic cell‐specific expression. In addition, a regulatory sequence required for determining generative cell‐specific expression of LGC1 was identified. Deletion of this regulatory sequence led to loss of the generative cell specificity resulting in activation of this promoter in other tissues where it is normally repressed. Therefore, male gametic cell specificity of the LGC1 gene seems to be regulated by factors that suppress its activation in other plant cells. This is the first report of a male gametic cell‐specific promoter, hence can be used as a novel tool in molecular analyses and experimental manipulation of flowering plant spermatogenesis and fertilization.
Redox stress regulates cell proliferation and apoptosis of human hepatoma through Akt protein phosphorylation
Authors: Dong-Yun Shi , Yu-Ru Deng , Shan-Lin Liu , Ya-Dong Zhang , Lian Wei
Published: April 2003

Employing a spin trapping agent combined with electron spin resonance spectroscopy, we were able to capture reactive oxygen species (ROS) in living hepatoma cells and first found that the trapped ROS was superoxide anion (O2

−). O2

− suppressed by treatment with diphenylene iodonium, a flavoprotein inhibitor, was generated by the flavoprotein‐containing NADPH‐oxidase complex. Applying endogenous/exogenous pro‐oxidant or antioxidant causes different redox states in hepatoma cells. Akt activity and cell growth were significantly stimulated by treating hepatoma cells with low concentration of ROS, which could be abolished by adding antioxidants. The phosphatidylinositol 3‐kinase (PI3K) inhibitor wortmannin (0.15 μM) inhibited Akt phosphorylation induced by ROS. Our results indicate that hepatoma cell growth is ROS‐dependent, and fluctuation of the intracellular redox state may regulate hepatoma cell growth through Akt phosphorylation and the PI3K/Akt pathway, resulting in a broad array of responses from cellular proliferation to apoptosis.
Abnormal junctional membrane structures in cardiac myocytes expressing ectopic junctophilin type 1
Authors: Komazaki Shinji , Nishi Miyuki , Takeshima Hiroshi
Published: April 2003

Recent studies indicate that junctophilin (JP) subtypes contribute to the formation of the junctional membrane complexes between the plasma membrane and the endoplasmic/sarcoplasmic reticulum (ER/SR) in excitable cells. Cardiac muscle contains the diad, in which the transverse (T) tubule of the invaginated cell membrane is closely associated with the SR membrane, and skeletal muscle bears the triad, in which the T‐tubule is associated with two SR membranes on the both sides. Among defined JP subtypes, JP‐2 is specifically expressed in cardiac muscle, while skeletal muscle cells contain both JP‐1 and JP‐2. These observations, together with other findings, suggest that the triad might be constructed in a JP‐1‐dependent manner after the achievement of JP‐2‐mediated diad formation during skeletal muscle maturation. In this study using transgenic mice, we examined whether the triad can be formed when JP‐1 is additionally expressed in cardiac muscle. Immunochemical analysis demonstrated co‐expression of JP‐1 and JP‐2 in cardiac myocytes from the transgenic mice. In cardiac muscle expressing JP‐1, abnormal junctional membranes were frequently observed under the electron microscope, in which the T‐tubules were rolled up with the SR membranes at several turns, but authentic triad formation could not be detected. Therefore, ectopic JP‐1 expression cannot convert the diad to the triad in cardiac myocytes. The present results suggest that triad formation requires an as yet unknown skeletal muscle‐specific mechanism, in addition to the JP subtypes.
Rho kinase is required for CCR7‐mediated polarization and chemotaxis of T lymphocytes
Authors: Bardi Giuseppe , Niggli Verena , Loetscher Pius
Published: April 2003

We studied the role of Rho kinase and extracellular signal‐regulated kinase (ERK)‐2 in the polarization and migration of T lymphocytes in response to the CCR7 ligands EBI1 ligand chemokine (ELC; CCL19) and secondary lymphoid‐tissue chemokine (SLC; CCL21). Both Rho kinase protein isoforms are expressed in T lymphocytes. Inhibition of the Rho kinases with Y‐27632 strongly inhibited SLC‐ and ELC‐induced polarized morphology and chemotaxis of T lymphocytes. Although the chemokines induced ERK‐2 activation, the blockade of this signaling pathway showed no effect on polarization and migration. This study indicates an important role of Rho kinase in CCR7‐mediated polarization and migration of T lymphocytes, whereas ERK‐2 is not involved in these processes.
Modulation of glucose transporters in rat diaphragm by sodium tungstate
Authors: Girón M.D , Caballero J.J , Vargas A.M , Suárez M.D , Guinovart J.J , Salto R
Published: April 2003

Oral administration of sodium tungstate is an effective treatment for diabetes in animal models. We examined the effects of 6 weeks of oral administration of tungstate on glucose transporters (GLUT) in streptozotocin‐induced diabetic rat diaphragm. Diabetes decreased GLUT4 expression while tungstate treatment normalized not only GLUT4 protein but also GLUT4 mRNA in the diabetic rats. Furthermore, treatment increased GLUT4 protein in plasma and internal membranes, suggesting a stimulation of its translocation to the plasma membrane. Tungstate had no effect on healthy animals. There were no differences in the total amount of GLUT1 transporter in any group. We conclude that the normoglycemic effect of tungstate may be partly due to a normalization of the levels and subcellular localization of GLUT4, which should result in an increase in muscle glucose uptake.
Proton pumping by NADH:ubiquinone oxidoreductase. A redox driven conformational change mechanism?
Authors: Brandt Ulrich , Kerscher Stefan , Dröse Stefan , Zwicker Klaus , Zickermann Volker
Published: April 2003

The modular evolutionary origin of NADH:ubiquinone oxidoreductase (complex I) provides useful insights into its functional organization. Iron–sulfur cluster N2 and the PSST and 49 kDa subunits were identified as key players in ubiquinone reduction and proton pumping. Structural studies indicate that this ‘catalytic core’ region of complex I is clearly separated from the membrane. Complex I from Escherichia coli and Klebsiella pneumoniae was shown to pump sodium ions rather than protons. These new insights into structure and function of complex I strongly suggest that proton or sodium pumping in complex I is achieved by conformational energy transfer rather than by a directly linked redox pump.
The role of electrostatics in proton‐conducting membrane protein complexes
Authors: Lancaster C.Roy D.
Published: April 2003

Electrostatic interactions play a key role in the coupling of electron and proton transfer in membrane protein complexes during the conversion of the energy stored in sunlight or reduced substrates into biochemical energy via a transmembrane electrochemical proton potential. Principles of charge stabilization within membrane proteins are reviewed and discussed for photosynthetic reaction centers, cytochrome c oxidases, and diheme‐containing quinol:fumarate reductases. The impact of X‐ray structure‐based electrostatic calculations on the functional interpretation of these structural coordinates, on providing new explanations for experimental observations, and for the design of more focused additional experiments is illustrated by a number of key examples.
A comparison of two adjunctive treatments for intrauterine adhesions following lysis
Authors: Orhue A.A.E. , Aziken M.E. , Igbefoh J.O.
Published: April 2003

Objectives: To assess which treatment modality has a better outcome: the use of an intrauterine contraceptive device or the Foley catheter balloon, for the adjunctive treatment of intrauterine adhesion (IUA) in patients presenting with infertility. Methods: In a 4‐year initial period, patients with intrauterine adhesion were treated with the insertion of an intrauterine contraceptive device (IUCD) after adhesiolysis. In the next 4 years, a pediatric Foley catheter balloon was used after adhesiolysis instead of the IUCD. The postoperative treatment was the same throughout the 8 years. While the IUCD was removed after three consecutive withdrawal vaginal bleedings, the Foley catheter was removed after 10 days. Hysterosalpingography was repeated in all patients after the third withdrawal vaginal bleeding, and the procedure was repeated if the intrauterine adhesion still persisted. The χ2‐test was used for analysis. Results: There were 51 cases of IUA treated with the IUCD and 59 cases treated with the Foley catheter balloon. In the Foley catheter group, 81.4% of the patients had restoration of normal menstruation compared with 62.7% in the IUCD group (P<0.05). Persistent posttreatment amenorrhea and hypomenorrhea occurred less frequently in the Foley catheter group (18.6%) than in the IUCD group (37.3%) (P<0.03), and the conception rate in the catheter group was 33.9% compared with 22.5% in the IUCD group. The need for repeated treatment was also significantly less in the Foley catheter group. Conclusion: The Foley Catheter is a safer and more effective adjunctive method of treatment of IUA compared with the IUCD.
Pethidine versus tramadol for pain relief during labor
Authors: Keskin H.L. , Aktepe Keskin E. , Avsar A.F. , Tabuk M. , Caglar G.S.
Published: April 2003

Objective: To evaluate and compare the analgesic efficacy and adverse effects of tramadol and pethidine in labor. Method: Fifty‐nine full term parturients were randomly assigned to one of two groups in active labor. Group 1 received 100 mg pethidine; group 2, 100 mg tramadol, intramuscularly. Analgesic efficacy, maternal side effects, changes in the blood pressure, heart rate, and duration of labor were assessed. Result: At 30 and 60 min after drug administration, pain relief was greater in the pethidine group than in tramadol group. The incidence of nausea and fatigue was higher in the tramadol group. Following drug administration the decrease in systolic and diastolic blood pressure and the increase in heart rate were statistically significant in both groups. No significant difference was found between the groups when compared for duration of labor and Apgar scores. None of the neonates developed respiratory depression. Conclusion: Pethidine seems to be a better alternative than tramadol in obstetric analgesia because of its superiority in analgesic efficacy and low incidence of maternal side effects.
Histological classification of endometriosis as a predictor of response to treatment
Authors: Abrao M.S. , Neme R.M. , Carvalho F.M. , Aldrighi J.M. , Pinotti J.A.
Published: April 2003

Objectives: To evaluate the usefulness of the histological classification of endometriosis in predicting responses to treatment. Methods: We evaluated 412 biopsy specimens from 241 patients with pelvic endometriosis. Pain and infertility were evaluated before surgery. Disease location and stage of development were analyzed according to the 1985 American Society of Reproductive Medicine (ASRM) classification. Histological findings were classified as stromal, well‐differentiated, undifferentiated, and mixed endometriosis. Clinical response to pain or infertility was evaluated. Results: Histological findings, disease location and stage of development, and response to treatment were compared. Undifferentiated endometriosis was more frequently associated with stages III/IV than the well‐differentiated and stromal histological types. Pure or mixed undifferentiated patterns were more frequently associated with rectovaginal endometriosis. When considering pain symptoms, patients presenting well‐differentiated or stromal histological patterns responded better to therapeutic treatment than those who presented undifferentiated histological patterns. There were no significant differences in cases related to sterility. Conclusions: The histological categorization of endometriosis can help predict the behavioral patterns of the disease.
The influence of an integration strategy on competitive capabilities and business performance: An exploratory study of consumer products manufacturers
Authors: Rosenzweig Eve D , Roth Aleda V , Dean James W
Published: April 2003

In this paper, we expand upon recent research by Frohlich and Westbrook [J. Operations Manage. 19 (2) (2001) 185] that characterizes the influence of supply chain integration on performance. Introducing supply chain integration intensity as a proxy variable for Frohlich and Westbrook’s [J. Operations Manage. 19 (2) (2001) 185] ‘outward‐facing supply chain strategy’, we investigate the ways that manufacturing‐based competitive capabilities mediate the relationship between supply chain integration and business performance. While previous research suggests that supply chain integration is directly related to superior business performance, the mediating role of manufacturing capabilities has not been explored. Using hierarchical regression analysis, we develop and test a theory‐based model using a sample of consumer products manufacturers. Contrary to Frohlich and Westbrook’s [J. Operations Manage. 19 (2) (2001) 185] assertions regarding the applicability of the ‘outward‐facing strategy’ to the consumer goods sector, our results provide empirical evidence that supply chain integration intensity leads directly to improved business performance, thus corroborating the conventional wisdom concerning the increasing importance of supply chain integration in the consumer products sector. In addition, this study uncovers empirical evidence for the mediating role of manufacturing‐based competitive capabilities in supply chain management. These results support the growing call for a broader, more generalized view of manufacturing strategy.
Inhibition of MMP‐1 expression by antisense RNA decreases invasiveness of human chondrosarcoma
Authors: Jiang Xiaoling , Dutton Charyl M. , Qi Wen‐Ning , Block Joel A. , Brodt Pnina , Durko Margaret , Scully Sean P.
Published: April 2003

We previously reported that an elevated level of matrix metalloproteinase‐1 (MMP‐1) gene expression in patients with chondrosarcoma has a strong statistical correlation with recurrence and in vitro invasion. In the present study, we used an antisense RNA strategy for MMP‐1 inhibition to determine if this would affect the invasive characteristics of the cells. We transfected a human chondrosarcoma cell line with a retroviral plasmid expressing a 770 bp genomic fragment of the human MMP‐1 gene in the sense or antisense orientation. The results show that cells transfected with the MMP‐1 antisense fragment had a significant decrease in both MMP‐1 protein and enzyme activity (p < 0.05) as compared to cells transfected with an empty plasmid or the parental cells. Cells transfected with the MMP‐1 antisense fragment demonstrated a significant decrease in their ability to invade the collagen I barrier (p < 0.05). The gene expression for MMP‐8 and MMP‐13 were unaffected in cells transfected with the MMP‐1 antisense fragment, MMP‐1 sense fragment, or empty plasmid. These results support the hypothesis that MMP‐1 facilitates tumor cell egress from chondrosarcoma tissue and demonstrate the potential of MMP‐1 as a promising target for a novel biologic therapy in chondrosarcoma. © 2003 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Patient reporting of adverse drug reactions: useful information for pain management?
Authors: Jarernsiripornkul Narumol , Krska Janet , Richards R. Michael E. , Capps Phillip A.G.
Published: April 2003

Background. patients' perceptions of adverse effects caused by the medicines they are prescribed may influence how they use these medicines. Little is known about patients' perceptions of the adverse effects of specific drugs in everyday use and whether these differ from those identified by clinical trials and standard post‐marketing surveillance methods.

Aim. To compare reports of perceived adverse drug reactions (ADRs) obtained directly from patients taking tramadol to those found in clinical trials and two methods of post‐marketing surveillance.

Method. Postal questionnaire distributed to 1048 patients who had a prescription for tramadol dispensed over a 3‐month period.

Results. Most (84%) of the 344 respondents reported at least one symptom perceived as an ADR to tramadol. Dry mouth, light‐headedness and constipation were most commonly reported. Almost half (48%) rated their most bothersome symptom as at least moderate and 43% claimed to have reported symptoms to their doctor. Perceived problems had led 38 respondents to stop taking tramadol. The 10 most frequently reported symptoms were all previously reported ADRs to tramadol. Although relatively minor, all 10 also appeared in reports to the UK Committee on the Safety of Medicines (CSM) and in prescription event monitoring. For many symptoms, the estimated range of frequency was in line with published reports, but considerably higher than that of post‐marketing surveillance methods.

Conclusions. Symptoms were reported by the majority of respondents and for many symptoms the frequency was high. Many patients did not report symptoms they perceived to be adverse effects to their doctor. The results indicate that patient perceptions of potential ADRs are relevant and should be an integral part of a pain management strategy.
Pain as presenting symptom in Lyme neuroborreliosis
Authors: Dotevall Leif , Eliasson Tore , Hagberg Lars , Mannheimer Clas
Published: April 2003

Neurogenic pain with radiculitis is often the starting symptom in adult patients with tick‐borne Lyme neuroborreliosis and in some cases the only clinical manifestation. Cranial paresis and other neurologic signs usually occur after the onset of pain. The present paper describes four patients who had severe pain as the main presenting symptom of Lyme neuroborreliosis. Opioids had good short‐term effect in two of the cases. Oral doxycycline treatment was used successfully to eliminate the infection.
Non-linear normalization and background correction in
one-channel cDNA microarray studies
Authors: Edwards David
Published: April 2003

Motivation: Data from one-channel cDNA microarray studies may exhibit poor reproducibility due to spatial heterogeneity, non-linear array-to-array variation and problems in correcting for background. Uncorrected, these phenomena can give rise to misleading conclusions.

Results: Spatial heterogeneity may be corrected using two-dimensional loess smoothing (Colantuoni et al., 2002). Non-linear between-array variation may be corrected using an iterative application of one-dimensional loess smoothing. A method for background correction using a smoothing function rather than simple subtraction is described. These techniques promote within-array spatial uniformity and between-array reproducibility. Their application is illustrated using data from a study of the effects of an insulin sensitizer, rosiglitazone, on gene expression in white adipose tissue in diabetic db/db mice. They may also be useful with data from two-channel cDNA microarrays and from oligonucleotide arrays.

Availability: R functions for the methods described are available on request from the author.

Contact: DEd@novonordisk.com
Do current sequence analysis algorithms
disclose multifunctional (moonlighting) proteins?
Authors: Gómez Antonio , Domedel Nuria , Cedano Juan , Piñol Jaume , Querol Enrique
Published: April 2003

Sumary: A number of multifunctional, ‘moonlighting’, proteins have been analyzed by different current programs to test wheter they identify both functions. PSI-BLAST and PRODOM perform best in predicting the alternative function.

Contact: enric.querol@uab.es

Supplementary information: Full tables and additional information available at http://ibb.uab.es/moonlighting

* The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.

† To whom correspondence should be addressed.