bluuebunny/crossref_metadata_2025 huggingface dataset:
Large‐scale purification of functional recombinant human aquaporin‐2
Authors: Werten Paul J.L. , Hasler Lorenz , Koenderink Jan B. , Klaassen Corné H.W. , de Grip Willem J. , Engel Andreas , Deen Peter M.T.
Published: October 2002

The homotetrameric aquaporin‐2 (AQP2) water channel is essential for the concentration of urine and of critical importance in diseases with water dysregulation, such as nephrogenic diabetes insipidus, congestive heart failure, liver cirrhosis and pre‐eclampsia. The structure of human AQP2 is a prerequisite for understanding its function and for designing specific blockers. To obtain sufficient amounts of AQP2 for structural analyses, we have expressed recombinant his‐tagged human AQP2 (HT‐AQP2) in the baculovirus/insect cell system. Using the protocols outlined in this study, 0.5 mg of pure HT‐AQP2 could be obtained per liter of bioreactor culture. HT‐AQP2 had retained its homotetrameric structure and exhibited a single channel water permeability of 0.93±0.03×10−13 cm3/s, similar to that of other AQPs. Thus, the baculovirus/insect cell system allows large‐scale expression of functional recombinant human AQP2 that is suitable for structural studies.
Requirement of ceramide for adhesion of Helicobacter pylori to glycosphingolipids
Authors: Tang Wei , Seino Keita , Ito Mieko , Konishi Toshiro , Senda Hisato , Makuuchi Masatoshi , Kojima Naoya , Mizuochi Tsuguo
Published: October 2002

Direct adhesion of Helicobacter pylori to immobilized glycosphingolipids (GSLs) was compared to that of their corresponding oligosaccharide‐conjugated neoglycoconjugates in order to clarify the roles of the carbohydrate and lipid portions of GSLs in H. pylori adhesion. These bacteria were found to adhere to sulfatide, GM3, GalCer and LacCer, but not to ceramide, sphingomyelin, or polyacrylamides conjugated with β‐galactose, lactose, 3′‐sialyllactose or 3′‐sulfo‐β‐galactose. Furthermore, neoglycolipids or bovine serum albumin derivatives with corresponding oligosaccharides were unable to serve as the ligands. H. pylori adhesion to GalCer with α‐hydroxyl fatty acid was much stronger than GalCer with the non‐hydroxyl fatty acid. These results suggest that H. pylori recognize the conformation of GSL with α‐hydroxyl fatty acid on solid phase.
Pregnancy‐associated plasma protein‐A (PAPP‐A) cleaves insulin‐like growth factor binding protein (IGFBP)‐5 independent of IGF: implications for the mechanism of IGFBP‐4 proteolysis by PAPP‐A
Authors: Laursen Lisbeth S. , Overgaard Michael T. , Søe Rikke , Boldt Henning B. , Sottrup-Jensen Lars , Giudice Linda C. , Conover Cheryl A. , Oxvig Claus
Published: October 2002

Pregnancy‐associated plasma protein‐A (PAPP‐A) has recently been identified as the proteinase responsible for cleavage of insulin‐like growth factor binding protein (IGFBP)‐4, an inhibitor of IGF action, in several biological fluids. Cleavage of IGFBP‐4 by PAPP‐A is believed to occur only in the presence of IGF. We here report that in addition to IGFBP‐4, PAPP‐A also cleaves IGFBP‐5. Cleavage occurs at one site, between Ser‐143 and Lys‐144 of IGFBP‐5. In the presence of IGF, IGFBP‐4 and ‐5 are cleaved with similar rates by PAPP‐A. Interestingly, cleavage of IGFBP‐5 by PAPP‐A does not require the presence of IGF, but is slightly inhibited by IGF. These findings have implications for the mechanism of proteolysis of IGFBP‐4 by PAPP‐A, suggesting that IGFBP‐4 binds IGF, which then becomes a PAPP‐A substrate. Using highly purified, recombinant proteins, we establish that (1) PAPP‐A cleavage of IGFBP‐4 can occur in the absence of IGF, although the rate of hydrolysis is very slow, and (2) IGF is unable to bind to PAPP‐A. We thus conclude that IGF enhances proteolysis by binding to IGFBP‐4, not by interaction with PAPP‐A, which could not previously be ruled out.
The Pla surface protease/adhesin of Yersinia pestis mediates bacterial invasion into human endothelial cells
Authors: Lähteenmäki Kaarina , Kukkonen Maini , Korhonen Timo K
Published: October 2002

The plasminogen activator Pla of Yersinia pestis belongs to the omptin family of enterobacterial surface proteases and is responsible for the highly efficient invasion of the plague bacterium from the subcutaneous infection site into the circulation. Y. pestis has been reported to invade human epithelial cells. Here, we investigated the role of Pla in bacterial invasion into human endothelial cells. Expression of Pla in recombinant Escherichia coli XL1(pMRK1) enhanced bacterial invasion into ECV304 cells. The invasiveness was not affected by substitution mutation at the residues S99 or D206 that are needed for the proteolytic activity of Pla. Pla‐expressing bacteria adhered to the extracellular matrix of ECV304 cells. Only weak adhesion and poor invasion were seen with the recombinant E. coli XL1(pMRK2), which expresses the omptin homolog from E. coli. The results identify Pla as an invasion protein of Y. pestis and show that the invasive function does not involve the proteolytic activity of Pla.
The carboxy‐terminal domain of Grp94 binds to protein kinase CK2α but not to CK2 holoenzyme
Authors: Roher Nerea , Sarno Stefania , Miró Francesc , Ruzzene Maria , Llorens Franc , Meggio Flavio , Itarte Emilio , Pinna Lorenzo A , Plana Maria
Published: October 2002

Surface plasmon resonance analysis shows that the carboxy‐terminal domain of Grp94 (Grp94‐CT, residues 518–803) physically interacts with the catalytic subunit of protein kinase CK2 (CK2α) under non‐stressed conditions. A K
D of 4×10−7 was determined for this binding. Heparin competed with Grp94‐CT for binding to CK2α. CK2β also inhibited the binding of Grp94‐CT to CK2α, and CK2 holoenzyme reconstituted in vitro was unable to bind Grp94‐CT. The use of CK2α mutants made it possible to map the Grp94‐CT binding site to the four lysine stretch (residues 74–77) present in helix C of CK2α. Grp94‐CT stimulated the activity of CK2α wild‐type but was ineffective on the CK2α K74–77A mutant.
FKBP12 associates tightly with the skeletal muscle type 1 ryanodine receptor, but not with other intracellular calcium release channels
Authors: Carmody Mark , Mackrill John J. , Sorrentino Vincenzo , O'Neill Cora
Published: October 2002

This study compared the relative levels of ryanodine receptor (RyR) isoforms, inositol 1,4,5‐trisphosphate receptor (IP3R) isoforms, and calcineurin, plus their association with FKBP12 in brain, skeletal and cardiac tissue. FKBP12 demonstrated a very tight, high affinity association with skeletal muscle microsomes, which was displaced by FK506. In contrast, FKBP12 was not tightly associated with brain or cardiac microsomes and did not require FK506 for removal from these organelles. Furthermore, of the proteins solubilised from skeletal muscle, cardiac muscle and brain microsomes, only skeletal muscle RyR1 bound to an FKBP12–glutathione‐S‐transferase fusion protein, in a high affinity FK506 displaceable manner. These results suggest that RyR1 has distinctive FKBP12 binding properties when compared to RyR2, RyR3, all IP3R isoforms and calcineurin.
Two‐dimensional gel electrophoresis and FTIR spectroscopy reveal both forms of yeast plasma membrane H+‐ATPase in activated and basal‐level enzyme preparations
Authors: Lapathitis Georgios , Tanfani Fabio , Kotyk Arnost , Bertoli Enrico
Published: October 2002

Plasma membrane H+‐ATPase of the yeast Saccharomyces cerevisiae was isolated and purified in its two forms, the activated A‐ATPase from glucose‐metabolizing cells, and the basal‐level B‐ATPase from cells with endogenous metabolism only. Using two‐dimensional gel electrophoretic analysis, we showed that both enzyme preparations are actually mixtures of the non‐active, i.e. non‐phosphorylated, and the active, i.e. phosphorylated, forms of the enzyme. Previous deliberations suggesting that the B‐ATPase displays some activity which is lower than that of A‐ATPase were apparently wrong. It seems that, molecularly speaking, the B‐form is actually not active at all, and what activity we measure in our preparation is due to an admixture of the true active form (A‐form). Fourier transform infrared spectroscopic study of the secondary structure and particularly thermal denaturation data suggest the possibility that the two enzyme forms interact to form complexes less stable than the single forms. On the whole then, there apparently is a different ratio of the active and inactive forms and/or complexes between the two forms present in all enzyme preparations.
D1′ centers are less efficient than normal photosystem II centers
Authors: Funk Christiane , Wiklund Ronney , Schröder Wolfgang P. , Jansson Christer
Published: October 2002

One prominent difference between the photosystem II (PSII) reaction center protein D1′ in Synechocystis 6803 and normal D1 is the replacement of Phe‐186 in D1 with leucine in D1′. Mutants of Synechocystis 6803 producing only D1′, or containing engineered D1 proteins with Phe‐186 substitutions, were analyzed by 77 K fluorescence emission spectra, chlorophyll a fluorescence induction yield and decay kinetics, and flash‐induced oxygen evolution. Compared to D1‐containing PSII centers, D1′ centers exhibited a 50% reduction in variable chlorophyll a fluorescence yield, while the flash‐induced O2 evolution pattern was unaffected. In the F186 mutants, both the P680+/QA
− recombination and O2 oscillation pattern were noticeably perturbed.
High‐pressure NMR study of the complex of a GTPase Rap1A with its effector RalGDS
Authors: Inoue Kyoko , Maurer Till , Yamada Hiroaki , Herrmann Christian , Horn Gudrun , Kalbitzer Hans Robert , Akasaka Kazuyuki
Published: October 2002

Unusually large non‐linear 1H and 15N nuclear magnetic resonance chemical shifts against pressure have been detected for individual amide groups of the Ras‐binding domain of Ral guanine dissociation stimulator (GDS). The non‐linear response is largest in the region of the protein remote from the Rap1A‐binding site, which increases by about two‐fold by the complex formation with its effector protein Rap1A. The unusual non‐linearity is explained by the increasing population of another conformer (N′), lying energetically above the basic native conformer (N), at higher pressure. It is considered likely that the conformational change from N to N′ in the Ras‐binding domain of RalGDS works as a switch to transmit the effector signal further to molecules of different RalGDS‐dependent signaling pathways.
Role of the sequence of the rne‐dependent site in 3′ processing of M1 RNA, the catalytic component of Escherichia coli RNase P
Authors: Sim Soyeong , Kim Semi , Lee Younghoon
Published: October 2002

The 3′ processing of M1 RNA, the catalytic component of Escherichia coli RNase P, occurs by two pathways involving multiple steps. The precursor of M1 RNA has an rne‐dependent site downstream of the processing site, whose sequence variation affects the processing efficiency. In this study, we showed that the sequence itself of the rne‐dependent site possessed the ability to determine the processing pathways and that it also affected the cleavage specificity with the generation of the processing products at one nucleotide upstream or downstream of the normal cleavage sites.
The P2Y12 receptor induces platelet aggregation through weak activation of the αIIbβ3 integrin – a phosphoinositide 3‐kinase‐dependent mechanism
Authors: Kauffenstein G. , Bergmeier W. , Eckly A. , Ohlmann P. , Léon C. , Cazenave J.P. , Nieswandt B. , Gachet C.
Published: October 2002

High concentrations of adenosine‐5′‐diphosphate ADP are able to induce partial aggregation without shape change of P2Y1 receptor‐deficient mouse platelets through activation of the P2Y12 receptor. In the present work we studied the transduction pathways selectively involved in this phenomenon. Flow cytometric analyses using R‐phycoerythrin‐conjugated JON/A antibody (JON/A‐PE), an antibody which recognizes activated mouse αIIbβ3 integrin, revealed a low level activation of αIIbβ3 in P2Y1 receptor‐deficient platelets in response to 100 μM ADP or 1 μM 2MeS‐ADP. Adrenaline induced no such activation but strongly potentiated the effect of ADP in a dose‐dependent manner. Global phosphorylation of 32P‐labeled platelets showed that P2Y12‐mediated aggregation was not accompanied by an increase in the phosphorylation of myosin light chain (P20) or pleckstrin (P47) and was not affected by the protein kinase C (PKC) inhibitor staurosporine. On the other hand, two unrelated phosphoinositide 3‐kinase inhibitors, wortmannin and LY294002, inhibited this aggregation. Our results indicate that (i) the P2Y12 receptor is able to trigger a P2Y1 receptor‐independent inside‐out signal leading to αIIbβ3 integrin activation and platelet aggregation, (ii) ADP and adrenaline use different signaling pathways which synergize to activate the αIIbβ3 integrin, and (iii) the transduction pathway triggered by the P2Y12 receptor is independent of PKC but dependent on phosphoinositide 3‐kinase.
ATRA‐regulated Asb‐2 gene induced in differentiation of HL‐60 leukemia cells
Authors: Kohroki Junya , Fujita Sayaka , Itoh Norio , Yamada Yukiko , Imai Harue , Yumoto Noboru , Nakanishi Tsuyoshi , Tanaka Keiichi
Published: October 2002

Suppressors of cytokine signaling (SOCS) proteins possess common structures, a SOCS box at the C‐terminus and a SH2 domain at their center. These suppressors are inducible in response to cytokines and act as negative regulators of cytokine signaling. The ASB proteins also contain the SOCS box and the ankyrin repeat sequence at the N‐terminus, but do not have the SH2 domain. Although Socs genes are directly induced by several cytokines, no Asb gene inducers have been identified. In this study, we screened the specific genes expressed in the course of differentiation of HL‐60 cells, and demonstrated that ASB‐2, one of the ASB proteins, was rapidly induced by all‐trans retinoic acid (ATRA). Typical retinoid receptors (RARs) or retinoid X receptors (RXRs) binding element (RARE/RXRE) were presented in the promoter of the Asb‐2 gene. We showed that RARα, one of the RARs, binds to the RARE/RXRE in the Asb‐2 promoter. In addition, we demonstrated by luciferase reporter assay that this element was a functional RARE/RXRE. These findings indicate that ASB‐2 is directly induced by ATRA and may act as a significant regulator, underlying such physiological processes as cell differentiation.
A non‐hypoxic, ROS‐sensitive pathway mediates TNF‐α‐dependent regulation of HIF‐1α
Authors: Haddad John J , Land Stephen C
Published: October 2002

A non‐hypoxic, reactive oxygen species (ROS)‐sensitive pathway mediating tumor necrosis factor‐α (TNF‐α)‐dependent regulation of hypoxia‐inducible factor‐1α (HIF‐α) was investigated in vitro. TNF‐α mediated the translocation of HIF‐1α, associated with up‐regulating its activity under normoxia. Analysis of the mode of action of TNF‐α revealed the accumulation of hydrogen peroxide (H2O2), superoxide anion (O2
−
) and hydroxyl radical (OH). Antioxidants purported as prototypical scavengers of H2O2 and OH, attenuated TNF‐α‐induced HIF‐1α activation, and blockading NADPH‐oxidase by scavenging O2
−
reduced the activity of HIF‐1α. Inhibition of the mitochondrion complex I abrogated TNF‐α‐dependent activation of HIF‐1α. Interrupting the respiratory chain reversed the excitatory effect of TNF‐α on HIF‐1α. These results indicate a non‐hypoxic pathway mediating cytokine‐dependent regulation of HIF‐1α in a ROS‐sensitive mechanism.
Ci‐opsin1, a vertebrate‐type opsin gene, expressed in the larval ocellus of the ascidian Ciona intestinalis
Authors: Kusakabe Takehiro , Kusakabe Rie , Kawakami Isao , Satou Yutaka , Satoh Nori , Tsuda Motoyuki
Published: October 2002

A novel gene encoding visual pigment, Ci‐opsin1, was identified in a primitive chordate, the ascidian, Ciona intestinalis. Molecular phylogenetic analysis and the exon–intron organization suggest that Ci‐opsin1 is closely related to the retinal and pineal opsins of vertebrates. During embryogenesis, Ci‐opsin1 transcripts were first detected in part of the brain of mid tailbud embryos; its expression was confined to photoreceptor cells of the ocellus (eye spot) in the larval brain as development proceeded. These results suggest a common descent of the ascidian ocellus and the vertebrate eyes. The ocellus of ascidian larvae may represent an ancestral state of the vertebrate eye.
How carcinogens (or telomere dysfunction) induce genetic instability: associated‐selection model
Authors: Blagosklonny Mikhail V
Published: October 2002

Carcinogens induce carcinogen‐specific genetic instability (defects in DNA repair). According to the ‘direct‐selection’ model, defects in DNA repair per se provide an immediate growth advantage. According to the ‘associated‐selection’ model, carcinogens merely select for cells with adaptive mutations. Like any mutations, adaptive mutations occur predominantly in genetically unstable cells. The ‘associated‐selection’ model predicts that carcinogen‐driven selection minimizes cytotoxic but maximizes mutagenic effects of carcinogens. A purely mutagenic (neither cytotoxic, nor cytostatic) environment will favor effective DNA repair, whereas any growth‐limiting conditions (telomerase deficiency, anticancer drugs) will select for genetically unstable cells. Genetic instability is a postmark of selective pressure rather than a hallmark of cancer per se. Once selected, genetic instability facilitates the development of resistance to any other growth‐limiting conditions. As an example, a putative link between prior exposure to carcinogens and the ability to develop a telomerase‐independent growth is discussed.
Induction of CHOP and apoptosis by nitric oxide in p53‐deficient microglial cells
Authors: Kawahara Kohichi , Oyadomari Seiichi , Gotoh Tomomi , Kohsaka Shinichi , Nakayama Hitoshi , Mori Masataka
Published: October 2002

Excessive nitric oxide (NO) has been implicated in neurotoxicity after stresses such as ischemia. NO toxicity is generally thought to be mediated by the DNA damage–p53 pathway or mitochondrial dysfunction. We investigated the mechanism of NO toxicity by using murine microglial MG5 cells established from p53‐deficient mice. When MG5 cells were exposed to bacterial lipopolysaccharide plus interferon‐γ, mRNA and protein for inducible NO synthase (iNOS) were markedly induced, and apoptosis occurred. Under these conditions, we found that mRNA and protein for CHOP/GADD153, a C/EBP family transcription factor which is involved in endoplasmic reticulum (ER) stress‐induced apoptosis, are induced. iNOS mRNA was induced 2 h after treatment, whereas CHOP mRNA began to increase at 6 h with a time lag. CHOP mRNA was also induced by NO donors S‐nitroso‐N‐acetyl‐DL‐penicillamine (SNAP) or NOC18, or a peroxynitrite generator 3‐(4‐morpholinyl)‐sydnonimine hydrochloride (SIN‐1). Bip/GRP78, an ER chaperone which is known to be induced by ER stress, was also induced by SNAP or SIN‐1, indicating that NO causes ER stress. These results suggest that NO‐induced apoptosis in MG5 cells occurs through the ER stress pathway involving CHOP, but is independent of p53.
A vesicle transport system inside chloroplasts
Authors: Westphal Sabine , Soll Jürgen , Vothknecht Ute C
Published: October 2002

Intracellular transport via membrane vesicle traffic is a well known feature of eukaryotic cells. Yet, no vesicle transport system has been described for prokaryotes or organelles of prokaryotic origin, such as chloroplasts and mitochondria. Here we show that chloroplasts possess a vesicle transport system with features similar to vesicle traffic in homotypic membrane fusion. Vesicle formation and fusion is affected by specific inhibitors, e.g. nucleotide analogues, protein phosphatase inhibitors and Ca2+ antagonists. This vesicle transfer is an ongoing process in mature chloroplasts indicating that it represents an important new pathway in the formation and maintenance of the thylakoid membranes.
Adiponectin gene expression is inhibited by β‐adrenergic stimulation via protein kinase A in 3T3‐L1 adipocytes
Authors: Fasshauer Mathias , Klein Johannes , Neumann Susanne , Eszlinger Markus , Paschke Ralf
Published: October 2002

Recently, it has been shown that the fat‐derived factor adiponectin is downregulated in insulin resistance and obesity and that replenishment of this adipocytokine reverses insulin resistance in mice. Growing evidence, on the other hand, suggests that raised levels of catecholamines due to increased activity of the sympathetic nervous system are an integral part in the development of insulin resistance. To clarify whether catecholamines might exert their insulin resistance‐inducing effects at least partly via downregulation of adiponectin gene expression, 3T3‐L1 adipocytes were treated with isoproterenol, and adiponectin mRNA was measured by quantitative real‐time reverse transcription‐polymerase chain reaction. In fact, isoproterenol treatment reduced the level of adiponectin mRNA by about 75% in a dose‐dependent fashion with significant inhibition detectable at concentrations as low as 10 nM isoproterenol. Furthermore, the inhibitory effect of isoproterenol was almost completely reversed by pretreatment of 3T3‐L1 cells with the β‐adrenergic antagonist propranolol and the protein kinase A (PKA) inhibitor H‐89. Moreover, the effects of isoproterenol could be mimicked by stimulation of stimulatory guanine nucleotide‐binding (GS)‐proteins with cholera toxin and adenylyl cyclase with forskolin. Thus, our results suggest that adiponectin gene expression is severely suppressed by β‐adrenergic agents via activation of a GS‐protein–PKA‐dependent pathway. The data support a possible role of adiponectin in catecholamine‐induced insulin resistance.
Arabidopsis thaliana expresses a second functional phytochelatin synthase
Authors: Cazalé Anne-Claire , Clemens Stephan
Published: October 2002

Phytochelatins represent a major detoxifying pathway for heavy metals in plants and many other organisms. The Arabidopsis thaliana CAD1 (=AtPCS1) gene encodes a phytochelatin synthase and cad1 mutants are phytochelatin deficient and cadmium hypersensitive. The Arabidopsis genome contains a highly homologous gene, AtPCS2, of which expression and function were studied in order to understand the apparent non‐redundancy of the two genes. Low constitutive AtPCS2 expression is detected in all plant organs analyzed. The AtPCS2 gene encodes a functional phytochelatin synthase as shown by expression in Saccharomyces cerevisiae and the complementation of a Schizosaccharomyces pombe phytochelatin synthase knockout strain.
Green fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichia coli
Authors: Drew David E , von Heijne Gunnar , Nordlund Pär , de Gier Jan-Willem L
Published: October 2002

Escherichia coli is one of the most widely used vehicles to overexpress membrane proteins (MPs). Currently, it is not possible to predict if an overexpressed MP will end up in the cytoplasmic membrane or in inclusion bodies. Overexpression of MPs in the cytoplasmic membrane is strongly favoured to overexpression in inclusion bodies, since it is relatively easy to isolate MPs from membranes and usually impossible to isolate them from inclusion bodies. Here we show that green fluorescent protein (GFP), when fused to an overexpressed MP, can be used as an indicator to monitor membrane insertion versus inclusion body formation of overexpressed MPs in E. coli. Furthermore, we show that an overexpressed MP can be recovered from a MP–GFP fusion using a site specific protease. This makes GFP an excellent tool for large‐scale MP target selection in structural genomics projects.
Induction of H2O2 synthesis by β‐glucan elicitors in soybean is independent of cytosolic calcium transients
Authors: Mithöfer Axel , Fliegmann Judith , Daxberger Andrea , Ebel Chantal , Neuhaus-Url Gabriele , Bhagwat Arvind A , Keister Donald L , Ebel Jürgen
Published: October 2002

Soybean cell suspension cultures have been used to investigate the role of the elevation of the cytosolic Ca2+ concentration in β‐glucan elicitors‐induced defence responses, such as H2O2 and phytoalexin production. The intracellular Ca2+ concentration was monitored in transgenic cells expressing the Ca2+‐sensing aequorin. Two lines of evidence showed that a transient increase of the cytosolic Ca2+ concentration is not necessarily involved in the induction of H2O2 generation: (i) a Bradyrhizobium japonicum cyclic β‐glucan induced the H2O2 burst without increasing the cytosolic Ca2+ concentration; (ii) two ion channel blockers (anthracene‐9‐carboxylate, A9C; 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoate, NPPB) could not prevent a Phytophthora soja β‐glucan elicitor‐induced H2O2 synthesis but did prevent a cytosolic Ca2+ concentration increase. Moreover, A9C and NPPB inhibited P. sojae β‐glucan‐elicited defence‐related gene inductions as well as the inducible accumulation of phytoalexins, suggesting that the P. sojae β‐glucan‐induced transient cytosolic Ca2+ increase is not necessary for the elicitation of H2O2 production but is very likely required for phytoalexin synthesis
Reanalysis of the involvement of γ‐glutamyl transpeptidase in the cell activation process
Authors: Antczak Christophe , Karp David R , London Robert E , Bauvois Brigitte
Published: October 2002

The inhibitor of γ‐glutamyl transpeptidase (γ‐GT) acivicin modulates cellular responses including growth, myeloid maturation and apoptosis. Whether these effects result from the inhibition of γ‐GT enzyme activity remains unclear. We compared the cellular effects of acivicin against a more potent and specific inhibitor of γ‐GT (L‐2‐amino‐4‐boronobutanoic acid (L‐ABBA)) in γ‐GT‐negative (B lymphoblastoid Ramos) and γ‐GT‐positive (myelomonocytic HL‐60, γ‐GT‐transfected Ramos) cell lines. Under non‐oxidative stress conditions, acivicin‐induced cell growth arrest, apoptosis and macrophage maturation occurred independent of γ‐GT while L‐ABBA did not influence any of these processes. Acivicin triggered tyrosine phosphorylation and increased nuclear factor κB activity. Further insight into the role of γ‐GT in cellular processes is needed.
Nuclear translocation of PDCD5 (TFAR19): an early signal for apoptosis?
Authors: Chen Yingyu , Sun Ronghua , Han Wenling , Zhang Yingmei , Song Quansheng , Di Chunhui , Ma Dalong
Published: October 2002

The programmed cell death 5 (PDCD5) protein is a novel protein related to regulation of cell apoptosis. In this report, we demonstrate that the level of PDCD5 protein expressed in cells undergoing apoptosis is significantly increased compared with normal cells, then the protein translocates rapidly from the cytoplasm to the nucleus of cells. The appearance of PDCD5 in the nuclei of apoptotic cells precedes the externalization of phosphatidylserine and fragmentation of chromosome DNA. This phenomenon is parallel to the loss of mitochondrial membrane potential, independent of the feature of apoptosis‐inducing stimuli and also independent of the cell types and the apoptosis modality. In conclusion, the nuclear translocation of PDCD5 is a universal earlier event of the apoptotic process, and may be a novel early marker for apoptosis.
Identification of a new human lens UV filter compound
Authors: Taylor Lisa M. , Aquilina J.Andrew , Willis Richard H. , Jamie Joanne F. , Truscott Roger J.W.
Published: October 2002

A new UV filter compound, 4‐(2‐amino‐3‐hydroxyphenyl)‐4‐oxobutanoic acid O‐diglucoside, has been identified in human lenses. The structure suggests that it is a further metabolic product of the second most abundant UV filter compound, 4‐(2‐amino‐3‐hydroxyphenyl)‐4‐oxobutanoic acid O‐glucoside. Quantification studies on the new compound show that it decreases towards zero in both the nucleus and cortex as a function of age. The discovery of this novel disaccharide completes the identification of the major UV filter compounds present in the human lens.
A lipid transfer protein binds to a receptor involved in the control of plant defence responses
Authors: Buhot N. , Douliez J.-P. , Jacquemard A. , Marion D. , Tran V. , Maume B.F. , Milat M.-L. , Ponchet M. , Mikès V. , Kader J.-C. , Blein J.-P.
Published: October 2002

Lipid transfer proteins (LTPs) and elicitins are both able to load and transfer lipidic molecules and share some structural and functional properties. While elicitins are known as elicitors of plant defence mechanisms, the biological function of LTP is still an enigma. We show that a wheat LTP1 binds with high affinity sites. Binding and in vivo competition experiments point out that these binding sites are common to LTP1 and elicitins and confirm that they are the biological receptors of elicitins. A mathematical analysis suggests that these receptors could be represented by an allosteric model corresponding to an oligomeric structure with four identical subunits.
Identification of mouse trp homologs and lipid rafts from spermatogenic cells and sperm
Authors: Treviño Claudia L. , Serrano Carmen J. , Beltrán Carmen , Felix Ricardo , Darszon Alberto
Published: October 2002

Intracellular Ca2+ has an important regulatory role in the control of sperm motility, capacitation, and the acrosome reaction (AR). However, little is known about the molecular identity of the membrane systems that regulate Ca2+ in sperm. In this report, we provide evidence for the expression of seven Drosophila transient receptor potential homolog genes (trp1–7) and three of their protein products (Trp1, Trp3 and Trp6) in mouse sperm. Allegedly some trps encode capacitative Ca2+ channels. Immunoconfocal images showed that while Trp6 was present in the postacrosomal region and could be involved in sperm AR, expression of Trp1 and Trp3 was confined to the flagellum, suggesting that they may serve sperm to regulate important Ca2+‐dependent events in addition to the AR. Likewise, one of these proteins (Trp1) co‐immunolocalized with caveolin‐1, a major component of caveolae, a subset of lipid rafts potentially important for signaling events and Ca2+ flux. Furthermore, by using fluorescein‐coupled cholera toxin B subunit, which specifically binds to the raft component ganglioside GM1, we identified caveolin‐ and Trp‐independent lipid rafts residing in the plasma membrane of mature sperm. Notably, the distribution of GM1 changes drastically upon completion of the AR.
Molecular cloning and characterization of six novel isoforms of human Bim, a member of the proapoptotic Bcl‐2 family1
Authors: U Mami , Miyashita Toshiyuki , Shikama Yoshiaki , Tadokoro Keiko , Yamada Masao
Published: October 2002

Bim protein is one of the BH3‐only proteins, members of the Bcl‐2 family that have only one of the Bcl‐2 homology regions, BH3. BH3‐only proteins are essential initiators of apoptotic cell death. Thus far, three isoforms of Bim have been reported, i.e. BimEL, BimL and BimS. Here we report the cloning and characterization of six novel isoforms of human Bim, designated as Bimα1, α2, and β1–β4, which are generated by alternative splicing. Unlike the three known isoforms, none of these novel isoforms contained a C‐terminal hydrophobic region. Among the novel isoforms, only Bimα1 and α2 contained a BH3 domain and were proapoptotic, although less potent than the classical isoforms. These two isoforms localized, at least in part, in mitochondria when transiently expressed in HeLa cells as a green fluorescent protein‐fused form. These results suggest that the BH3 domain is necessary for induction of apoptosis and mitochondrial localization but not sufficient for the full proapoptotic activity. While the classical isoforms were always predominantly expressed in transformed cells, expression profiles of bim isoforms were highly variable among normal tissues at least in humans, suggesting a tissue‐specific transcriptional regulation of bim.
Adenovirus‐mediated preproinsulin gene transfer into adipose tissues ameliorates hyperglycemia in obese diabetic KKAy mice
Authors: Nagamatsu Shinya , Nakamichi Yoko , Ohara-Imaizumi Mica , Ozawa Sachihiko , Katahira Hitoshi , Watanabe Takashi , Ishida Hitoshi
Published: October 2002

We investigated whether adenovirus‐mediated preproinsulin gene transfer into insulin target tissues (adipocytes) ameliorates hyperglycemia in diabetic mice. KKAy mice, a genetically obese type 2 diabetic animal model, were treated with a single subcutaneous injection of recombinant adenovirus, Adex1CA‐human preproinsulin (Adex1CA‐pchi), into the epididymal fat pads. pchi mRNA was expressed only in adipose tissue in which mature insulin was produced. Three days after virus injection these mice showed a marked decrease of blood glucose levels (from about 400 to 200 mg/dl), and an intraperitoneal glucose tolerance test revealed the markedly improved glucose tolerance. There was no significant difference in serum insulin levels between control and recombinant adenovirus‐treated KKAy mice. The normalized glucose levels in diabetic mice were maintained for at least 2 weeks after the virus injection. This strategy could provide a novel and, most importantly, a simple and convenient gene therapy for obese type 2 diabetes patients.
Inter‐ and intramolecular domain interactions of the catalase‐peroxidase KatG from M. tuberculosis
Authors: Wilming Martin , Johnsson Kai
Published: October 2002

The inter‐ and intramolecular interactions between the different domains of the catalase‐peroxidase KatG from Mycobacterium tuberculosis were analyzed using the two‐hybrid assay. It was shown that the dimerization of the enzyme is due to a strong interaction of the first 99 amino acids of the N‐terminal domain whereas the C‐terminal domain does not play a role in the dimerization. In addition, an intramolecular interaction between the N‐ and C‐terminal domains was detected which might play a functional role in the mechanism of the enzyme.
PPARγ‐dependent and PPARγ‐independent effects on the development of adipose cells from embryonic stem cells
Authors: Vernochet C. , Milstone D.S. , Iehlé C. , Belmonte N. , Phillips B. , Wdziekonski B. , Villageois P. , Amri E.Z. , O'Donnell P.E. , Mortensen R.M. , Ailhaud G. , Dani C.
Published: October 2002

Peroxisome proliferator‐activated receptor (PPAR) γ was shown to be required for adipocyte formation both in vivo and in vitro. However, the role of PPARγ in the initial steps of adipose cell development was not distinguished from its role in the terminal steps. We now show that PPARγ is expressed early in embryoid bodies (EBs) derived from embryonic stem cells and in E.8.5 mouse embryos. Addition of a specific ligand for PPARγ in developing EBs over‐expressing PPARγ did not commit stem cells towards the adipose lineage. In differentiated PPARγ−/− EBs, only markers characteristic of preadipocytes were found to be expressed. PPARδ is present in EBs but did not compensate for the lack of PPARγ in terminal differentiation. Taken together, these results favor a critical PPARγ‐independent phase culminating in preadipocyte formation that precedes a PPARγ‐dependent phase in the development of adipose cells from pluripotent stem cells.
Pleckstrin homology domains and the cytoskeleton
Authors: Lemmon Mark A. , Ferguson Kathryn M. , Abrams Charles S.
Published: October 2002

Pleckstrin homology (PH) domains are 100–120 amino acid protein modules best known for their ability to bind phosphoinositides. All possess an identical core β‐sandwich fold and display marked electrostatic sidedness. The binding site for phosphoinositides lies in the center of the positively charged face. In some cases this binding site is well defined, allowing highly specific and strong ligand binding. In several of these cases the PH domains specifically recognize 3‐phosphorylated phosphoinositides, allowing them to drive membrane recruitment in response to phosphatidylinositol 3‐kinase activation. Examples of these PH domain‐containing proteins include certain Dbl family guanine nucleotide exchange factors, protein kinase B, PhdA, and pleckstrin‐2. PH domain‐mediated membrane recruitment of these proteins contributes to regulated actin assembly and cell polarization. Many other PH domain‐containing cytoskeletal proteins, such as spectrin, have PH domains that bind weakly, and to all phosphoinositides. In these cases, the individual phosphoinositide interactions may not be sufficient for membrane association, but appear to require self‐assembly of their host protein and/or cooperation with other anchoring motifs within the same molecule to drive membrane attachment.
A novel bifunctional molybdo‐enzyme catalyzing both decarboxylation of indolepyruvate and oxidation of indoleacetaldehyde from a thermoacidophilic archaeon, Sulfolobus sp. strain 7
Authors: Wakagi Takayoshi , Fukuda Eriko , Ogawa Yoko , Kino Hiroyasu , Matsuzawa Hiroshi
Published: October 2002

An enzyme, which catalyzes both decarboxylation of indolepyruvate and subsequent oxidation of indoleacetaldehyde into indoleacetate, was purified from a thermoacidophilic archaeon, Sulfolobus sp. strain 7. The enzyme showed a M
r of 280 kDa on gel filtration and was composed of three subunits (a, 89; b, 30; and c, 19 kDa), possibly in a stoichiometry of 2:2:2. Mo and Fe were detected. Thiamine pyrophosphate was absent. Biotin was suggested to bind to the b‐subunit. The first step, the decarboxylation reaction, was specific for 2‐oxoacids with an aromatic group, while in the second reaction, various aldehydes including glyceraldehyde, which is a glycolytic intermediate in the organism, were oxidized.
KCO1 is a component of the slow‐vacuolar (SV) ion channel
Authors: Schönknecht Gerald , Spoormaker Petra , Steinmeyer Ralf , Brüggeman Lioubov , Ache Peter , Dutta Rajiv , Reintanz Birgit , Godde Matthias , Hedrich Rainer , Palme Klaus
Published: October 2002

The Arabidopsis double pore K+ channel KCO1 was fused to green fluorescent protein and expressed in tobacco protoplasts. Microscopic analysis revealed a bright green fluorescence at the vacuolar membrane. RT‐PCR experiments showed that KCO1 is expressed in the mesophyll. Vacuoles from Arabidopsis wild‐type and kco1 knockout plants were isolated for patch‐clamp analyses. Currents mediated by slow‐activating vacuolar (SV) channels of mesophyll cell vacuoles were significantly smaller in kco1 plants compared to the wild‐type. This shows that KCO1 is involved in the formation of SV channels.
Molecular characterization of frog chromogranin B reveals conservation of selective sequences encoding potential novel regulatory peptides1
Authors: Aït-Ali Djida , Turquier Valérie , Alexandre David , Grumolato Luca , Jégou Sylvie , Vaudry Hubert , Anouar Youssef
Published: October 2002

Chromogranin B (CgB) is a member of the granin family of neuroendocrine secretory proteins, which has been proposed to play a role in secretory granule biogenesis and as a precursor to bioactive peptides. The cloning of CgB in a phylogenetically distant vertebrate, the frog Rana ridibunda, reveals a modest overall homology (35–40%) with mammalian CgB. However, the sequences of the N‐ and C‐terminal regions are more highly conserved (57–65% amino acid identity) and may give rise to novel regulatory peptides. In frog, intense expression of CgB mRNA was observed in particular structures of the brain and in the distal lobe of the pituitary.
The spectrin repeat: a structural platform for cytoskeletal protein assemblies
Authors: Djinovic-Carugo Kristina , Gautel Mathias , Ylänne Jari , Young Paul
Published: October 2002

Spectrin repeats are three‐helix bundle structures which occur in a large number of diverse proteins, either as single copies or in tandem arrangements of multiple repeats. They can serve structural purposes, by coordination of cytoskeletal interactions with high spatial precision, as well as a ‘switchboard’ for interactions with multiple proteins with a more regulatory role. We describe the structure of the α‐actinin spectrin repeats as a prototypical example, their assembly in a defined antiparallel dimer, and the interactions of spectrin repeats with multiple other proteins. The α‐actinin rod domain shares several features common to other spectrin repeats. (1) The rod domain forms a rigid connection between two actin‐binding domains positioned at the two ends of the α‐actinin dimer. The exact distance and rigidity are important, for example, for organizing the muscle Z‐line and maintaining its architecture during muscle contraction. (2) The spectrin repeats of α‐actinin have evolved to make tight antiparallel homodimer contacts. (3) The spectrin repeats are important interaction sites for multiple structural and signalling proteins. The interactions of spectrin repeats are, however, diverse and defy any simple classification of their preferred interaction sites, which is possible for other domains (e.g. src‐homology domains 3 or 2). Nevertheless, the binding properties of the repeats perform important roles in the biology of the proteins where they are found, and lead to the assembly of complex, multiprotein structures involved both in cytoskeletal architecture as well as in forming large signal transduction complexes.
Bromodomain: an acetyl‐lysine binding domain
Authors: Zeng Lei , Zhou Ming-Ming
Published: October 2002

Bromodomains, an extensive family of evolutionarily conserved protein modules originally found in proteins associated with chromatin and in nearly all nuclear histone acetyltransferases, have been recently discovered to function as acetyl‐lysine binding domains. More recent structural studies of bromodomain/peptide ligand complexes have enriched our understanding of differences in ligand selectivity of bromodomains. These new findings demonstrate that bromodomain/acetyl‐lysine recognition can serve as a pivotal mechanism for regulating protein–protein interactions in numerous cellular processes including chromatin remodeling and transcriptional activation, and reinforce the concept that functional diversity of a conserved protein modular structure is achieved by evolutionary changes of amino acid sequences in the ligand binding site.
Evidence for cardiac sodium–calcium exchanger association with caveolin‐3
Authors: Bossuyt Julie , Taylor Bonnie E. , James-Kracke Marilyn , Hale Calvin C.
Published: October 2002

The interaction of cardiac Na+–Ca2+ exchange (NCX1) with caveolin proteins was investigated in sarcolemmal vesicles. Western blots of sarcolemmal vesicles revealed the presence of caveolin‐1, ‐2, and ‐3. NCX1 co‐fractionated more closely with caveolin‐3 than caveolin‐1 on sucrose density gradients. NCX1 has five possible caveolin‐binding motifs and NCX1 co‐precipitated specifically with caveolin‐3. Molecular sieve column chromatography indicated that this co‐precipitation was not due to incomplete solubilization of lipid raft microdomains. Cholesterol chelation in vesicles decreased NCX1 transport activity and caveolin‐3 co‐precipitation. NCX1 may play a role in caveolar transmembrane signaling in addition to its role in excitation–contraction coupling.
The proton‐pumping NADH:ubiquinone oxidoreductase (complex I) of Aquifex aeolicus
Authors: Scheide Dierk , Huber Robert , Friedrich Thorsten
Published: October 2002

The proton‐pumping NADH:ubiquinone oxidoreductase, also called complex I, is the first energy‐transducing complex of many respiratory chains. Homologues of complex I are present in the three domains of life. Here, we report the properties of complex I in membranes of the hyperthermophilic bacterium Aquifex aeolicus. The complex reacted with NADH but not with NADPH and F420H2 as electron donors. Short‐chain analogues of ubiquinone like decyl‐ubiquinone and ubiquinone‐2 were suitable electron acceptors. The affinities towards NADH and ubiquinone‐2 were comparable to the ones obtained with the Escherichia coli complex I. The reaction was inhibited by piericidin A at the same concentration as in E. coli. The complex showed an unusual pH optimum at pH 9 and a maximal rate at 80°C. We found no evidence for the presence of an alternative, single subunit NADH dehydrogenase in A. aeolicus membranes. The NADH:ferricyanide reductase activity of detergent extracts of A. aeolicus membranes sedimented as a protein with a molecular mass of approximately 550 kDa. From the data we concluded that A. aeolicus contains a NADH:ubiquinone oxidoreductase resembling complex I of mesophilic bacteria.
Identification of functional VEGF receptors on human platelets
Authors: Selheim Frode , Holmsen Holm , Vassbotn Flemming S
Published: October 2002

Platelets secrete platelet‐derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) upon stimulation. We have demonstrated that platelets have functionally active PDGF α‐receptors, a transmembrane tyrosine kinase involved in negative feedback regulation. Here we demonstrate the presence of the related VEGF receptors fms‐like tyrosine kinase‐1 and kinase‐insert domain region on human platelets. VEGF itself did not cause platelet aggregation. However, addition of exogenous VEGF to SFRLLN or thrombin‐stimulated platelets potentiated platelet aggregation. Moreover, thrombin‐induced phosphoinositide 3‐kinase and mitogen‐activated protein kinase activity were enhanced in the presence of VEGF.
Reversible inhibition of photophosphorylation in chloroplasts by nitric oxide
Authors: Takahashi Shunichi , Yamasaki Hideo
Published: October 2002

Nitric oxide (NO) is a bioactive molecule involved in diverse physiological functions in plants. Here we demonstrate that NO is capable of regulating the activity of photophosphorylation in chloroplasts. The electron transport activity in photosystem II determined from chlorophyll a fluorescence was inhibited by NO. NO also inhibited light‐induced ΔpH formation across the thylakoid membrane. High concentrations of nitrite and nitrate did not show such inhibitory effects, suggesting that the inhibition is not due to uncoupling effects of the oxidized products of NO. ATP synthesis activity upon illumination was severely inhibited by NO (IC50=0.7 μM). The inhibition was found to be temporary and the activity was completely recovered by removing NO. Bovine hemoglobin and bicarbonate were effective in preventing NO‐dependent inhibition of photophosphorylation. These results indicate that NO is a reversible inhibitor of photosynthetic ATP synthesis.
Structure and functional properties of the ubiquitin binding protein p62
Authors: Geetha Thangiah , Wooten Marie W.
Published: October 2002

Several highly conserved p62 homologs have recently been isolated, e.g. the rat atypical protein kinase C‐interacting protein (ZIP), the murine A170/signal transduction and adapter protein, and the human p62, a protein that binds the Src homology 2 domain of p56lck. These proteins share striking similarity in amino acid sequence and structural motifs, thereby suggesting conserved functional properties. ZIP/p62 has been shown to play an important role as a scaffold leading to the activation of the transcription factor nuclear factor κB. In addition, a nuclear form of p62 has been characterized that can serve as a transcriptional co‐activator. Moreover, p62 is capable of binding ubiquitin (Ub) non‐covalently through its Ub‐associated domain. In this review, we will focus on the structure and function of ZIP/p62.
Novel approach reveals localisation and assembly pathway of the PsbS and PsbW proteins into the photosystem II dimer
Authors: Thidholm Ellinor , Lindström Viktoria , Tissier Christophe , Robinson Colin , P. Schröder Wolfgang , Funk Christiane
Published: October 2002

A blue‐native gel electrophoresis system was combined with an in organello import assay to specifically analyse the location and assembly of two nuclear‐encoded photosystem II (PSII) subunits. With this method we were able to show that initially the low molecular mass PsbW protein is not associated with the monomeric form of PSII. Instead a proportion of newly imported PsbW is directly assembled in dimeric PSII supercomplexes with very fast kinetics; its negatively charged N‐terminal domain is essential for this process. The chlorophyll‐binding PsbS protein, which is involved in energy dissipation, is first detected in the monomeric PSII subcomplexes, and only at later time points in the dimeric form of PSII. It seems to be bound tighter to the PSII core complex than to light harvesting complex II. These data point to radically different assembly pathways for different PSII subunits.