bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural characterization of a Lymnaea putative endoprotease related to human furin
Authors: Smit August B. , Spijker Sabine , Nagle Gregg T. , Knock Susan L. , Kurosky Alexander , Geraerts Wijnand P.M.
Published: July 2002

A number of peptides have been identified in the central nervous system of the freshwater snail, Lymnaea stagnalis, that function as hormones and neurotransmitters/neuromodulators. These peptides are typically proteolytically processed from larger prohormones mostly at sites composed of single or multiple basic amino acid residues. Previously we demonstrated a diversity of putative prohormone convertases that may be involved in prohormone processing in the Lymnaea brain. In the present report, we have characterized a cDNA clone encoding a putative endoprotease of 837 amino acids. The primary structure of the endoprotease (Lfur2) was comparable to that of human furin and contained a putative catalytic domain, a Cys‐rich domain, and a transmembrane region. The catalytic domain of Lfur2 demonstrated about 70% residue identity when compared with human furin, PACE4 and Drosophila Dfur1 and dKLIP‐1. The Lfur2 gene was expressed in the central nervous system as well as various peripheral tissues of Lymnaea.
Limited proteolysis of cytochrome c in trifluoroethanol
Authors: Fontana Angelo , Zambonin Marcello , De Filippis Vincenzo , Bosco Manuela , de Laureto Patrizia Polverino
Published: July 2002

Horse heart cytochrome c is cleaved by thermolysin in 50% aqueous TFE (v/v) at neutral pH (25°C, 24 h) at the Gly56‐Ile57 peptide bond of the 104‐residue chain of the protein. Additional, but anyway minor, fragmentation at the Gly45‐Phe46 and Met80‐Ile81 peptide bonds is also observed. On the other hand, in buffer only and in the absence of TFE, cytochrome c is digested by thermolysin to numerous small peptides. Considering the broad substrate specificity of the TFE‐resistant thermolysin, clearly the conformational state of the protein substrate dictates the observed selective proteolysis. It is proposed that the highly helical secondary structure acquired by cytochrome c when dissolved in aqueous TFE hampers binding and adaptation of the protein substrate at the active site of the protease and that peptide bond fission occurs at flexible chain segments characterized by a low α‐helix propensity.
Thrombopoietin (TPO) induces tyrosine phosphorylation and activation of STAT5 and STAT3
Authors: Bacon Chris M. , Justin Tortolani P. , Shimosaka Akihiro , Rees Robert C. , Longo Dan L. , O'Shea John J.
Published: July 2002

The growth and differentiation of megakaryocytes are regulated by thrombopoietin (TPO), a recently characterized cytokine which exerts its effects via a member of the hematopoietin receptor superfamily, c‐Mpl. Since many cytokines which bind hematopoietin receptors activate the STAT family of transcription factors, we investigated whether STAT proteins were activated by TPO. TPO induced the formation of a DNA‐binding complex recognizing a known STAT binding sequence. STAT5 was a major component of this DNA‐binding complex, and STAT5 was tyrosine phosphorylated in response to TPO. Additionally, TPO‐induced the tyrosine phosphorylation and DNA‐binding activity of STAT3. Together with the recent demonstration of JAK2 activation in response to TPO, the data presented here define a rapid signaling pathway likely to be important in TPO‐induced gene regulation.
Amino‐terminal deletions in the decorin core protein leads to the biosynthesis of proteoglycans with shorter glycosaminoglycan chains
Authors: Oldberg Åke , Antonsson Per , Moses Jonatan , Fransson Lars-Åke
Published: July 2002

Analysis of the N‐terminal sequence of decorin purified from connective tissues and comparison with the sequence deduced from the cDNA indicate that the nascent proteoglycan has a 14 amino acid residue N‐terminal propeptide. Mammalian expression vectors encoding wild‐type decorin and decorin with deletions in the propeptide were used to transform COS and CHO cells. Cells transformed with vectors encoding deletion variants of decorin synthesize proteoglycans with shorter galactosaminoglycan chains than cells transformed with wild‐type decorin. This effect on the polysaccharide chain length may be due to a lower affinity between the core protein and the glycosyltransferases synthesizing the linkage region. Alternatively, the deletions may affect the intracellular transport of decorin. An antiserum prepared against the N‐terminal propeptide immunoprecipitated decorin secreted by cultured cells, showing that decorin is exported with the N‐terminal region intact.
Oligomerization of VIP21‐caveolin in vitro is stabilized by long chain fatty acylation or cholesterol
Authors: Monier Solange , Dietzen Dennis J. , Hastings W.Randall , Lublin Douglas M. , Kurzchalia Teymuras V.
Published: July 2002

VIP21‐caveolin is one of the components which form the cytoplasmic surface of caveolae. In vivo, this integral membrane protein is found in homo‐oligomers with molecular masses of approximately 200, 400 and 600 kDa. These oligomers are also formed by the addition of cytosol to the in vitro synthesized and membrane inserted VIP21‐caveolin. Here we show that long chain fatty acyl coenzyme A esters can completely substitute for cytosol in inducing 200 kDa and 400 kDa complexes, whereas 25‐hydroxy‐cholesterol can produce the 200 kDa oligomer. In order to understand whether acylation of VIP21‐caveolin itself is a prerequisite for oligomerization, we studied a mutant protein lacking all three cysteines. When analyzed by velocity sucrose gradient centrifugation in the presence of the non‐ionic detergent octylglucoside, both palmitoylated and non‐palmitoylated VIP21‐caveolin formed oligomers that were indistinguishable. However, only the oligomers of the non‐palmitoylated protein are disrupted when analyzed by SDS‐PAGE without boiling. These data suggest that the protein domains of VIP21‐caveolin are the primary determinants of oligomerization, but that palmitoylation of cysteine residues can increase the stability of the oligomers.
Basic fibroblast growth factor (bFGF) increases the survival of embryonic and postnatal basal forebrain cholinergic neurons in primary culture
Authors: Perkins Linda A. , Cain Lisa D.
Published: July 2002

Basic fibroblast growth factor (bFGF) is found in high concentrations in the mammalian central nervous system. It is a mitogen for glia and it influences the development and survival of specific populations of neurons. In this study, we investigated the effect of various concentrations of bFGF on the survival of embryonic and postnatal cholinergic basal forebrain neurons plated at low and high density in the presence and absence of glia. We observed that 50 and 100 ng/ml of bFGF increased the survival of embryonic cholinergic neurons plated at high density. This effect was observed only in the presence of glia. Lower concentrations of 10 and 20 ng/ml had no effect on cholinergic neuronal survival. The number of GFAP (glial fibrillary acidic protein)‐positive cells in high‐density embryonic cultures was increased by all concentrations of bFGF. In low‐density embryonic cultures, an increase in cholinergic neuron survival was observed at concentrations ranging from 20 to 100 ng/ml. The number of GFAP‐positive cells in low‐density cultures was also increased by all concentrations of bFGF. Similar to low‐density embryonic cultures, the survival of cholinergic neurons from postnatal day 2 cultures was significantly increased in the presence of glia at concentrations of 20, 50 and 100 ng/ml of bFGF. Postnatal glia was affected by all concentrations of bFGF, as was observed in embryonic cultures. This study indicates that high concentrations of bFGF can influence cholinergic neuronal survival by stimulating and increasing glia, which may produce factor(s) that are necessary for cholinergic neuron survival.
EXPRESSION OF THE AVIAN α7‐INTEGRIN IN DEVELOPING NERVOUS SYSTEM AND MYOTOME
Authors: Kil Sung H. , Bronner‐Fraser Marianne
Published: July 2002

Integrins are cell surface receptors for a variety of extracellular matrix molecules including fibronectin, laminin and collagens. Although their role in development is not completely understood, they are likely to have important functions in cell migration and axon guidance. To characterize the types of integrins expressed in the developing nervous system, we have used monoclonal antibodies against α7‐ and αv‐integrin subunits to examine the distribution of these subunits in the early chick embryo. Low levels of α7 immunoreactivity were first observed in the neural tube and developing myotome of stage 17 embryos (E2.5). Although low levels of α7 expression were associated with most neuroepithelial cells, distinct α7 immunoreactivity was first detected in the ventrolateral portions of the neural tube at a stage corresponding to the time when the first neurons differentiate. Its distribution pattern overlapped with that of commissural neurons in the developing spinal cord. α7 was also prominently localized to the motor neurons and their axons emanating from the neural tube. In addition, α7 immunoreactivity was observed on a subpopulation of trunk neural crest cells migrating through the somitic sclerotome. At later stages, α7 expression was observed in other nervous system structures such as the pigmented retinal epithelial cells. In addition to its distribution in the developing nervous system, α7 immunoreactivity was associated with early myotomal cells shortly after myotome formation and its expression persisted throughout myotome development. In contrast to α7, αv‐integrin had a limited distribution in the nervous system, being expressed only at low levels in the neural tube. However, αv displayed prominent immunoreactivity in the myotome and in endothelial cells of the dorsal aorta. The results suggest that α7‐integrin is one of the prevalent integrin subunits on neurons and axons in the developing spinal cord.
Psychiatric rehabilitation in Switzerland
Authors: Uchtenhagen A
Published: July 2002

Psychiatric rehabilitation at present has a focus on the prevention of chronic disability and on active rehabilitation of socalled new long-stay patients. This is a major public health problem, concerning about one-third of all disabled persons in need of rehabilitation. Close co-operation between the various agencies concerned, with funding organizations, patients organizations etc, is of high priority in assuring effective rehabilitation. Various structures and models have been realized in order to facilitate such cooperation. The main axes of rehabilitation are lodging, occupation, financial independence/support, need for out-patient psychiatric care and quality of life. Rehabilitation results are measured along those axes, based on an assessment of patients' needs and satisfaction. Specific programmes have been implemented for various diagnostic groups, especially schizophrenic psychosis, mental retardation, substance dependence and organic brain damage. Specifications of these diversified programmes are described. Evaluation of rehabilitation efforts uses different designs, methods and instruments; a harmonization on the level of instruments is intended. A few examples regarding evaluation results (concerning schizophrenic and drug dependent patients) are presented. Finally, external factors limiting the efficacy of rehabilitation efforts are described.
Analysis of the interaction between single‐chain variable fragments and their antigen in a reducing intracellular environment using the two‐hybrid system
Authors: De Jaeger Geert , Fiers Esbjörn , Eeckhout Dominique , Depicker Ann
Published: July 2002

The coding sequences of three single‐chain variable (scFv) fragments (A4, G4 and H3), which bind to dihydroflavonol‐4‐reductase (DFR) of Petunia hybrida, and the DFR‐encoding sequence were cloned in two‐hybrid vectors. The vectors were transformed in the yeast strain HF7c (his3‐200, trp1‐901, leu2‐3) and the scFv‐DFR interaction was analyzed by measuring yeast growth on medium without histidine. ScFv‐G4 and, to a lesser extent, scFv‐A4 could interact with DFR in the yeast nucleus. On the contrary, scFv‐H3 showed no interaction with its antigen in yeast. The results of a previous expression analysis of the same scFv fragments in the plant cytosol correlate with those of the two‐hybrid test. This suggests that it is possible to evaluate the antigen‐scFv interaction in a reducing subcellular environment with the two‐hybrid test. Therefore, the yeast two‐hybrid system can be useful to identify candidate scFv fragments for intracellular antibody applications.
The first peak of the UVB irradiation‐dependent biphasic induction of vascular endothelial growth factor (VEGF) is due to phosphorylation of the epidermal growth factor receptor and independent of autocrine transforming growth factor α
Authors: Blaudschun Ralf , Brenneisen Peter , Wlaschek Meinhard , Meewes Christian , Scharffetter-Kochanek Karin
Published: July 2002

Ultraviolet B (UVB) irradiation, the major damaging component of sunlight, has earlier been reported to enhance cutaneous angiogenesis in chronically sun‐exposed skin. We herein provide first evidence for a biphasic induction of the vascular endothelial growth factor (VEGF) following UVB irradiation of the human epidermal cell line HaCaT. The first VEGF peak occurred on mRNA level at 1 h and on protein level at 4 h postirradiation and is fully mediated by the UVB‐dependent phosphorylation of the epidermal growth factor receptor, which subsequent to its phosphorylation also initiates at least in part the synthesis of transforming growth factor α that confers as shown previously the second late VEGF peak at 8 h on mRNA and at 24 h on protein level.
MSSP, a protein binding to an origin of replication in the c‐myc gene, interacts with a catalytic subunit of DNA polymerase α and stimulates its polymerase activity
Authors: Niki Takeshi , Galli Ivo , Ariga Hiroyoshi , Iguchi-Ariga Sanae M.M.
Published: July 2002

MSSP has been identified as a protein that binds to both single‐ and double‐stranded sequences of a putative DNA replication origin sequence in the human c‐myc gene. MSSP possesses versatile functions, including stimulation of DNA replication, transcriptional regulation, apoptosis induction, and cell transformation coordinated by c‐Myc. MSSP contains two RNP domains, RNP1‐A and RNP1‐B, both of which are necessary for all of the functions of MSSP. In this study, we found that MSSP binds to the N‐terminal region of a catalytic subunit of a human DNA polymerase α via its RNP domains both in vitro and in human cells. Furthermore, MSSP was released from the putative DNA replication origin of the c‐myc gene after it complexed with DNA polymerase α, and MSSP stimulated DNA polymerase activity in vitro.
Identification of the histidine and aspartic acid residues essential for enzymatic activity of a family I.3 lipase by site‐directed mutagenesis
Authors: Hyun-Ju Kwon , Amada Kei , Haruki Mitsuru , Morikawa Masaaki , Kanaya Shigenori
Published: July 2002

A lipase from Pseudomonas sp. MIS38 (PML) is a member of the lipase family I.3. We analyzed the roles of the five histidine residues (His30, His274, His291, His313, and His365) and five acidic amino acid residues (Glu253, Asp255, Asp262, Asp275, and Asp290), which are fully conserved in the amino acid sequences of family I.3 lipases, by site‐directed mutagenesis. We showed that the mutation of His313 or Asp255 to Ala almost fully inactivated the enzyme, whereas the mutations of other residues to Ala did not seriously affect the enzymatic activity. Measurement of the far‐ and near‐UV circular dichroism spectra suggests that inactivation by the mutation of His313 or Asp255 is not due to marked changes in the tertiary structure. We propose that His313 and Asp255, together with Ser207, form a catalytic triad in PML.
Studies on the interaction between TWEAK and the death receptor WSL‐1/TRAMP (DR3)
Authors: Kaptein Allard , Jansen Mieke , Dilaver Gonul , Kitson Jeremy , Dash Laura , Wang Edward , Owen Michael J , Bodmer Jean-Luc , Tschopp Jurg , Farrow Stuart N
Published: July 2002

WSL‐1/TRAMP (DR3) is a member of the tumour necrosis factor (TNF) receptor superfamily which exhibits effects on NF‐κB activation and apoptosis. TWEAK, a novel TNF‐related molecule, has been proposed as the ligand for this receptor. Utilising both human and murine TWEAK ligand, it is shown that TWEAK and WSL‐1/TRAMP do not interact in an in vitro binding assay and that TWEAK binds strongly to cells that do not express WSL‐1/TRAMP on the cell surface. Biological activity of TWEAK is also observed in these cells. Finally, cells isolated from WSL‐1/TRAMP knockout mice are shown to retain their ability to interact with TWEAK. These results suggest that WSL‐1/TRAMP is not the major receptor for TWEAK
Hypericin photo‐induced apoptosis involves the tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) and activation of caspase‐8
Authors: Schempp Christoph M , Simon-Haarhaus Birgit , Termeer Christian C , Simon Jan C
Published: July 2002

Hypericin (HYP) is a photosensitizing pigment from Hypericum perforatum that displays cytotoxic effects in neoplastic cell lines. Therefore, HYP is presently under consideration as a new anticancer drug in photodynamic therapy. Here, we investigated the mechanism of action of HYP photo‐induced apoptosis of Jurkat cells compared to the cytostatic drug paclitaxel (PXL). Both photoactivated HYP and PXL similarly increased the activity of caspase‐8 and caspase‐3, and drug‐induced apoptosis of Jurkat cells was completely blocked by inhibitors of caspase‐8 (Z‐IETD‐FMK) and caspase‐3 (Z‐DEVD‐FMK). The involvement of death receptors was analyzed using neutralizing monoclonal antibodies against Fas (SM1/23), FasL (NOK‐2) and TNF‐R1 (MAB225), and a polyclonal rabbit anti‐human TNF‐related apoptosis‐inducing ligand (TRAIL) antiserum. TRAIL antibody blocked TRAIL‐induced and HYP photo‐induced, but not PXL‐induced apoptosis of Jurkat cells. In contrast, PXL‐induced, but not HYP‐induced apoptosis was blocked by the SM1/23 and NOK‐2 antibodies. Anti‐TNF‐R1 antibody had no effect. These findings suggest that HYP photo‐induced apoptosis of Jurkat cells is mediated in part by the TRAIL/TRAIL‐receptor system and subsequent activation of upstream caspases.
Synergistic effects of transforming growth factor‐β on the expression of c‐fms, macrophage colony‐stimulating factor receptor gene, in vascular smooth muscle cells
Authors: Inaba Toshimori , Ishibashi Shun , Harada Kenji , Osuga Jun-ichi , Yagyu Hiroaki , Ohashi Ken , Yazaki Yoshio , Yamada Nobuhiro
Published: July 2002

Vascular smooth muscle cells (SMC) transform to foam cells in the process of atherosclerosis. We have reported that SMC derived from the intima of atherosclerotic lesions express c‐fms, macrophage colony‐stimulating factor receptor gene, which is not normally expressed in medial SMC. In the present study, we demonstrated that transforming growth factor‐β (TGF‐β) synergistically induced expression of c‐fms in the presence of platelet‐derived growth factor‐BB in human medial SMC, a level comparable to that observed in the intima. The induction of c‐fms was not inhibited by protein kinase C (PKC) inhibitor, suggesting that TGF‐β induces c‐fms via a PKC‐independent pathway. These results suggest that TGF‐β plays an important role in the phenotypic change of smooth muscle cells to macrophage‐like cells in the process of atherosclerosis.
Role of Csk in neural differentiation of the embryonic carcinoma cell line P19
Authors: Takayama Yoshiharu , Nada Shigeyuki , Nagai Katsuya , Okada Masato
Published: July 2002

To examine the neural function of Csk (C‐terminal Src kinase), a membrane‐targeted form of Csk (Src/Csk) and its kinase‐defective variant (DK‐Src/Csk) were expressed in the embryonic carcinoma cell line P19. Expression of Src/Csk, but not DK‐Src/Csk, caused reduction of the specific activities of Src and Fyn in the differentiated P19 cells. During neural differentiation, the specific activity of Src was elevated in the control P19 cells, whereas the activation was completely eliminated in the Src/Csk transfectant. In normally differentiated P19 cells, cross‐linking of a cell adhesion molecule, L1, induced a short‐term activation of Src and Fyn. In the Src/Csk transfectant, L1 stimulation induced delayed activation of Src and Fyn peaking at much lower levels than in the control cells. Src/Csk transfectants developed normally in the initial stages of neural differentiation, but exhibited an apparent defect in cell‐to‐cell interaction, i.e. neurite fasciculation and aggregation of cell bodies, in the latter stages. These findings imply that Csk is involved in the regulation of Src family kinases that play roles in cell‐to‐cell interaction mediated by cell adhesion molecules.
Susceptibility of different subsets of immature thymocytes to apoptosis
Authors: Chow Sek C , Snowden Roger , Orrenius Sten , Cohen Gerald M
Published: July 2002

In the present study the susceptibility of different subsets of immature rat thymocytes to undergo apoptosis was examined. Unfractionated rat thymocytes were negatively enriched into immature double positive (CD4+CD8+), immature single positive (CD4−CD8+CD3−) and triple negative (CD4−CD8−CD3−) thymocytes. These enriched subsets of immature thymocytes were then exposed to various apoptotic stimuli such as dexamethasone, etoposide and thapsigargin which readily induced apoptosis in unfractionated rat thymocytes. We found that the double positive thymocytes and their precursor cells, i.e. the single positive immature thymocytes, were equally sensitive to apoptosis after treatment with the apoptotic stimuli. In sharp contrast, the early migrants or precursor‐containing thymocytes which are triple negative have a lower spontaneous apoptosis rate and were relatively resistant to all the apoptotic stimuli. These findings showed a breakpoint in thymocyte sensitivity to apoptosis which occurs after the onset of CD8 expression, suggesting that susceptibility of thymocytes to apoptosis is developmentally regulated.
A new transglutaminase‐like from the ascidian Ciona intestinalis1
Authors: Cariello Lucio , Ristoratore Filomena , Zanetti Laura
Published: July 2002

A cDNA clone encoding a transglutaminase (TGase) was isolated from a cDNA library prepared from the larval stage of Ciona intestinalis. The cDNA sequence has an open reading frame encoding a protein of 696 amino acids and is about 36% identical to 11 other TGase sequences. In addition, the critical residues thought to form the catalytic center are conserved. The Ciona TGase (CiTGase) has an extension of 39 amino acids in the NH2‐terminal region similar to that reported for keratinocyte TGases. A phylogenetic analysis among other types of TGases demonstrated that CiTGase represents a new type of the enzyme.
Glucose activation of the yeast plasma membrane H+‐ATPase requires the ubiquitin–proteasome proteolytic pathway
Authors: de la Fuente Natalia , Maldonado Ana M , Portillo Francisco
Published: July 2002

Glucose triggers transcriptional and post‐transcriptional mechanisms that increase the level and activity of Saccharomyces cerevisiae plasma membrane H+‐ATPase. We have studied the post‐transcriptional activation of the enzyme by glucose and have found that Rsp5, a ubiquitin–protein ligase enzyme, Ubc4, a ubiquitin‐conjugating enzyme, and the 26S proteasome complex are implicated in this activation. These results suggest that ATPase activation by glucose requires the ubiquitin–proteasome proteolytic pathway. This is supported by the fact that over‐expression of the ubiquitin‐specific protease Ubp2, which cleaves ubiquitin from its branched conjugates, inhibits this activation. We propose that glucose triggers degradation of an inhibitory protein resulting in enzyme activation.
Modulation by (iso)flavonoids of the ATPase activity of the multidrug resistance protein
Authors: Hooijberg J.H , Broxterman H.J , Heijn M , Fles D.L.A , Lankelma J , Pinedo H.M
Published: July 2002

The multidrug resistance protein (MRP) is an ATP‐dependent transport protein for organic anions, as well as neutral or positively charged anticancer agents. In this study we report that dinitrophenyl‐S‐glutathione increases ATPase activity in plasma membrane vesicles prepared from the MRP‐overexpressing cell line GLC4/ADR. This ATPase stimulation parallels the uptake of DNP‐SG in these vesicles. We also show that the (iso)flavonoids genistein, kaempferol and flavopiridol stimulate the ATPase activity of GLC4/ADR membranes, whereas genistin has no effect. The present data are consistent with the hypothesis that certain (iso)flavonoids affect MRP‐mediated transport of anticancer drugs by a direct interaction with MRP.
Cytochrome b6/f complex from the cyanobacterium Synechocystis 6803: evidence of dimeric organization and identification of chlorophyll‐binding subunit
Authors: Poggese Chiara , Polverino de Laureto Patrizia , Giacometti Giorgio M , Rigoni Fernanda , Barbato Roberto
Published: July 2002

Fractionation of photosynthetic membranes from the cyanobacterium Synechocystis 6803 by polyacrylamide gel electrophoresis in the presence of Deriphat‐160 allowed the isolation of a number of pigmented bands. Two of them, with molecular masses of 240±20 and 110±15 kDa respectively, showed peroxidase activity and, by means of polypeptide composition, immunoblotting and N‐terminal sequencing, were identified as dimeric and monomeric cytochrome b
6/f complexes, containing 1.3±0.35 chlorophyll molecules per cytochrome f. Further fractionation of monomeric complexes by mild gel electrophoresis in the presence of sodium dodecyl sulfate indicated that it is the cytochrome b
6 polypeptide which provides the actual binding site for the chlorophyll molecule observed in the complex.
The βγ subunits of heterotrimeric G proteins acquire detergent insolubility directly at the plasma membrane
Authors: Rehm Armin , Ploegh Hidde L.
Published: July 2002

The subunits of heterotrimeric G proteins, Gα and Gβγ, are found in association with detergent‐resistant domains in most mammalian cell types, implicating such domains in G protein‐coupled signaling. The pathway by which the βγ complexes are targeted to these detergent‐resistant domains was unaffected by the brefeldin A‐imposed block on endoplasmic reticulum‐to‐Golgi transport. We have used subcellular fractionation and β subunit‐specific immunoprecipitation to localize the acquisition of detergent insolubility of newly synthesized βγ complexes. The β subunits cofractionate with plasma membranes, and acquire detergent insolubility coincident with arrival in the plasma membrane fractions. This association was not affected by phorbol 12‐myristate 13‐acetate‐induced activation of Proetin kinase C.
Functional lysophosphatidic acid receptor in bovine luteal cells1
Authors: Budnik L.T , Mukhopadhyay A.K
Published: July 2002

The aim of this study was to determine whether luteal cells possess functional receptors for lysophosphatidic acid (LPA). We present evidence that [3H]LPA binds to a 38–40 kDa protein in a membrane fraction prepared from luteal cells and that this phospholipid is able to induce tyrosine phosphorylation of several proteins (65–125 kDa). Furthermore, LPA upregulates forskolin‐ and LH/GTP‐stimulated adenylyl cyclase activity by changing its Vmax. Although a pertussis toxin‐sensitive G‐protein has been reported to transmit the inhibitory signals between the LPA receptor and adenylyl cyclase, the observed upregulation of the enzyme activity in luteal cells is not abolished after pre‐treating the cells with the toxin, suggesting that a different mechanism is operative in these cells. According to the pharmacological regulatory pattern it is suggested that the modulated adenylyl cyclase isoform is the enzyme subtype V expressed in luteal cells.
Role of two conserved glycine residues in the β‐propeller domain of the integrin α4 subunit in VLA‐4 conformation and function1
Authors: Guerrero-Esteo Mercedes , Ruiz-Velasco Natividad , Muñoz Marisa , Teixidó Joaquı́n
Published: July 2002

The N‐terminal region of the α integrin subunits is predicted to fold into a β‐propeller domain. Using K562 α4 transfectants we show that mutations at α4 subunit residues Gly130 and Gly190 affect the conformation of this domain causing a reduction in the recognition of α4 by anti‐α4 antibodies which map to the β‐propeller. The improper α4 conformation also led to an altered association with the β1 subunit, and to a lack of α4β1 adhesion to VCAM‐1 and CS‐1/fibronectin, as well as an abolishment of anti‐α4‐ and anti‐β1‐dependent homotypic aggregation. The total conservation of Gly130 and Gly190 among integrin α subunits suggests their importance in the correct folding of their respective β‐propeller domains, and thus, in the adhesive activity of the integrins.
Shared motifs of the capsid proteins of hepadnaviruses and retroviruses suggest a common evolutionary origin
Authors: Zlotnick Adam , Stahl Steven J , Wingfield Paul T , Conway James F , Cheng Naiqian , Steven Alasdair C
Published: July 2002

The structure of the dimeric C‐terminal domain of the HIV‐1 capsid protein (CA), recently determined by X‐ray crystallography (Gamble et al. (1997)), has a notable resemblance to the structure of the hepatitis B virus (HBV) capsid protein (Cp) dimer, previously determined by cryo‐electron microscopy (Conway et al. (1997), Böttcher et al. (1997)). In both proteins, dimerization is effected by formation of a four‐helix bundle, whereby each subunit contributes a helix‐loop‐helix and most of the interaction between subunits is mediated by one pair of helices. These are the first two observations of a motif that is common to the capsid proteins of two enveloped viruses and quite distinct from the eight‐stranded anti‐parallel β‐barrel found in most other virus capsid proteins solved to date (Harrison et al. (1996)). Motivated by the structural resemblance, we have examined retroviral and HBV capsid protein sequences and found weak but significant similarities between them. These similarities further support an evolutionary relationship between these two virus families of great medical importance – the hepadnaviruses (e.g. HBV) and retroviruses (e.g. HIV).
The mouse gene encoding the peroxisomal membrane protein 1‐like protein (PXMP1‐L): cDNA cloning, genomic organization and comparative expression studies1
Authors: Holzinger Andreas , Muntau Ania , Mayerhofer Peter , Kammerer Stefan , Albet Sabrina , Bugaut Maurice , Roscher Adelbert A
Published: July 2002

PXMP1‐L (synonyms: PMP69, P70R) is a peroxisomal protein that belongs to the ABC‐transporter superfamily. Its closest homolog is the peroxisomal membrane protein 1 (PMP70). We have cloned the mouse PXMP1‐L gene. It encodes a 606 amino acid protein. In contrast to the human and the rat, mouse PXMP1‐L is predominantly expressed in the liver. The mouse PXMP1‐L gene consists of 19 exons and spans 21 kb of genomic sequence. No obvious peroxisome proliferator response element has been found in 1.1 kb of the putative promoter region. No coordination of constitutive or fenofibrate‐induced expression of PXMP1‐L with other peroxisomal ABC transporters was observed so that an obligate exclusive heterodimer formation is not likely to occur. The data presented will be particularly useful for the generation of a mouse model defective in PXMP1‐L in order to elucidate the yet unknown function of this protein.
Evidence for the archaebacterial‐type conformation about the bond between the β‐ionone ring and the polyene chain of the chromophore retinal in chlamyrhodopsin
Authors: Sakamoto Masaru , Wada Akimori , Akai Akiko , Ito Masayoshi , Goshima Takehiko , Takahashi Tetsuo
Published: July 2002

Previous studies using retinal analogs (Wada, A., Sakai, M., Kinumi, T., Tsujimoto, K., Yamauchi, M. and Ito, M. (1994) J. Org. Chem. 59, 6992–6997; Wada, A., Sakai, M., Imamoto, Y., Shichida., Y., Yoshizawa, T. and Ito, M. (1993) Chem. Pharm. Bull. 41, 793–795) revealed that both retinochrome and visual pigments share a common chromophoric conformation in which the ring region of retinal is twisted ca. 50° with respect to the plane of the polyene chain, suggesting a highly conserved 6‐s‐cis conformation throughout rhodopsin‐like pigments in animals. By contrast, 6‐s‐trans conformation was commonly observed or suggested in archaebacterial rhodopsins examined thus far. Here we have reconstituted in vivo both the photoreceptor for photobehavioral responses of the unicellular alga Chlamydomonas reinhardtii and the second archaeal sensory photoreceptor phoborhodopsin from a series of retinal analogs. Results exclusively point to the conclusion that in both photoreceptors retinal has the coplanar 6‐s‐trans conformation, though recent molecular cloning studies revealed no homology between Chlamydomonas photoreceptor (chlamyrhodopsin) and archaeal rhodopsins.
Membrane potential generation coupled to oxidation of external NADH in liver mitochondria
Authors: Bodrova M.E. , Dedukhova V.I. , Mokhova E.N. , Skulachev V.P.
Published: July 2002

Oxidation of added NADH by rat liver mitochondria has been studied. It is found that exogenous NADH, when oxidized by rat liver mitochondria in sucrose hypotonic medium supplemented with Mg2+ and EGTA, generates a membrane potential (ΔΨ) even in the absence of added cytochrome c. ADP and phosphate decrease ΔΨ, the effect being reversed by oligomycin. Rotenone and myxothiazol do not inhibit ΔΨ generated by oxidation of exogenous NADH. Added cytochrome c increases the rate of the exogenous NADH oxidation and coupled ΔΨ formation. In sucrose isotonic medium, or in hypotonic medium without Mg2+, exogenous NADH fails to stimulate respiration and to form a membrane potential. In the presence of Mg2+, exogenous NADH appears to be effective in ΔΨ generation in isotonic sucrose medium if mitochondria were treated with digitonin. In isotonic KCl without Mg2+, oxidation of exogenous NADH is coupled to the ΔΨ formation and MgCl2 addition before mitochondria prevents this effect. In hypotonic (but not in isotonic) sucrose medium, Mg2+ makes a portion of the cytochrome c pool reducible by exogenous NADH or ascorbate. It is assumed that (i) hypotonic treatment or digitonin causes disruption of the outer mitochondrial membrane, and, as a consequence, desorption of the membrane‐bound cytochrome c in a Mg2+‐dependent fashion; (ii) incubation in isotonic KCl without Mg2+ results in swelling of mitochondrial matrix, disruption of the outer membrane and cytochrome c desorption whereas Mg2+ lowers the K+ permeability of the inner membrane and, hence, prevents swelling; (iii) desorbed cytochrome c is reduced by added NADH via NADH‐cytochrome b
5 reductase and cytochrome b
5 or by ascorbate and is oxidized by cytochrome oxidase. The role of desorbed cytochrome c in oxidation of superoxide and cytoplasmic NADH as well as possible relations of these events to apoptosis are discussed.
Bacillus subtilis DnaG primase stabilises the bacteriophage SPP1 G40P helicase‐ssDNA complex
Authors: Ayora Silvia , Langer Uwe , Alonso Juan C
Published: July 2002

Purified Bacillus subtilis DnaG primase (predicted molecular mass 68.8 kDa) behaves as a monomer in solution. We demonstrate that DnaG physically interacts with bacteriophage SPP1 hexameric helicase G40P (G40P6) in the absence of ATP. G40P6‐ATP forms an unstable complex with ssDNA, and by itself carries out ATP‐driven translocation along a ssDNA template with low processivity. The presence of DnaG in the reaction mixture increased the helicase activity of G40P6 about 3‐fold, but not the ATPase activity. The results presented here suggest that the DnaG protein stabilises the G40P6‐ssDNA complexes.
Hyperthermophilic redox chemistry: a re‐evaluation
Authors: Hagedoorn Peter L , Driessen Martijn C.P.F , van den Bosch Marieke , Landa Ilse , Hagen Wilfred R
Published: July 2002

The redox chemistry of Pyrococcus furiosus rubredoxin and ferredoxin has been studied as a function of temperature in direct voltammetry and in EPR monitored bulk titrations. The E
ms of both proteins, measured with direct voltammetry, have a normal (linear) temperature dependence and show no pH dependence. EPR monitoring is not a reliable method to determine the temperature dependence of the E
m: upon rapid freezing the proteins take their conformation corresponding to the freezing point of the solution.
R73A and H144Q mutants of the yeast mitochondrial cyclophilin Cpr3 exhibit a low prolyl isomerase activity in both peptide and protein‐folding assays
Authors: Scholz Christian , Maier Peter , Dolinski Kara , Heitman Joseph , Schmid Franz X
Published: July 2002

Previously we reported that the R73A and H144Q variants of the yeast cyclophilin Cpr3 were virtually inactive in a protease‐coupled peptide assay, but retained activity as catalysts of a proline‐limited protein folding reaction [Scholz, C. et al. (1997) FEBS Lett. 414, 69–73]. A reinvestigation revealed that in fact these two mutations strongly decrease the prolyl isomerase activity of Cpr3 in both the peptide and the protein‐folding assay. The high folding activities found previously originated from a contamination of the recombinant Cpr3 proteins with the Escherichia coli protein SlyD, a prolyl isomerase that co‐purifies with His‐tagged proteins. SlyD is inactive in the peptide assay, but highly active in the protein‐folding assay.
High polymorphism level of genomic sequences flanking insertion sites of human endogenous retroviral long terminal repeats
Authors: Lavrentieva Irina , Broude Natalia E , Lebedev Yuri , Gottesman Irving I , Lukyanov Sergei A , Smith Cassandra L , Sverdlov Eugene D
Published: July 2002

The polymorphism at the multitude of loci adjacent to human endogenous retrovirus long terminal repeats (LTRs) was analyzed by a technique for whole genome differential display based on the PCR suppression effect that provides selective amplification and display of genomic sequences flanking interspersed repeated elements. This strategy is simple, target‐specific, requires a small amount of DNA and provides reproducible and highly informative data. The average frequency of polymorphism observed in the vicinity of the LTR insertion sites was found to be about 12%. The high incidence of polymorphism within the LTR flanks together with the frequent location of LTRs near genes makes the LTR loci a useful source of polymorphic markers for gene mapping.
Submitochondrial and subcellular distributions of the carnitine‐acylcarnitine carrier
Authors: Fraser Fiona , Zammit Victor A
Published: July 2002

The submitochondrial and subcellular distributions of the carnitine‐acylcarnitine translocase (CAC) have been studied. CAC is enriched to a much lesser extent than the carnitine palmitoyltransferases within the contact sites of mitochondria. A high‐abundance protein of identical molecular size as the mitochondrial CAC that is immunoreactive with an anti‐peptide antibody raised against a linear epitope of mitochondrial CAC is present in peroxisomes but not in microsomes. This suggests that CAC is targeted to at least two different locations within the liver cell and that acylcarnitine transport into peroxisomes is CAC mediated.
Key role of barstar Cys‐40 residue in the mechanism of heat denaturation of bacterial ribonuclease complexes with barstar
Authors: Protasevich I.I , Schulga A.A , Vasilieva L.I , Polyakov K.M , Lobachov V.M , Hartley R.W , Kirpichnikov M.P , Makarov A.A
Published: July 2002

The mechanism by which barnase and binase are stabilized in their complexes with barstar and the role of the Cys‐40 residue of barstar in that stabilization have been investigated by scanning microcalorimetry. Melting of ribonuclease complexes with barstar and its Cys‐82‐Ala mutant is described by two 2‐state transitions. The lower‐temperature one corresponds to barstar denaturation and the higher‐temperature transition to ribonuclease melting. The barstar mutation Cys‐40‐Ala, which is within the principal barnase‐binding region of barstar, simplifies the melting to a single 2‐state transition. The presence of residue Cys‐40 in barstar results in additional stabilization of ribonuclease in the complex.
Identification of the sequence responsible for the nuclear localization of human Cdc6
Authors: Takei Yoshinori , Yamamoto Koshi , Tsujimoto Gozoh
Published: July 2002

The Cdc6 is the essential protein for the initiation of DNA replication. Cdc6 is localized in the G1 nucleus, and abnormal nuclear localization of this protein induces irregular initiation of DNA replication. We identified here that amino acids K57 and R58 in the human Cdc6 protein play an important role in the nuclear localization of the protein. The fundamental features of the mechanism regulating the localization of Cdc6 seem to be maintained in yeast, Xenopus, and human, since the amino acid sequence surrounding K57 and R58, (S/T)PXKR(L/I), is conserved in these species. Substitution of amino acid residue S54 with E and not Q blocked partially the nuclear localization of the protein, implying that the phosphorylation at S54 is involved in the regulating mechanism of the cell cycle‐dependent localization of Cdc6.
Casein kinase II is responsible for phosphorylation of NF‐L at Ser‐473
Authors: Nakamura Yu , Hashimoto Ryota , Kashiwagi Yujiro , Wada Yoshinao , Sakoda Saburo , Miyamae Yasuyuki , Kudo Takashi , Takeda Masatoshi
Published: July 2002

Ser‐473 is solely phosphorylated in vivo in the tail region of neurofilament L (NF‐L). With peptides including the native phosphorylation site, it was not possible to locate responsible kinases. We therefore adopted full‐length dephosphorylated NF‐L as the substrate, and employed MALDI/TOF (matrix‐assisted laser desorption and ionization/time of flight) mass spectrometry and a site‐specific phosphorylation‐dependent antibody recognizing Ser‐473 phosphorylation. The antibody showed that casein kinase I (CK I) as well as casein kinase II (CK II) phosphorylated Ser‐473 in vitro, while neither GSK‐3β nor calcium/calmodulin‐dependent protein kinase II did so. However, the mass spectra of the tail fragments of the phosphorylated NF‐L indicated that CK II was the kinase mediating Ser‐473 phosphorylation in vitro as opposed to CK I, because CK I phosphorylated another site as well as Ser‐473 in vitro. The antibody also demonstrated that NF‐L phosphorylated at Ser‐473 was abundant in the neuronal perikarya of the rat cortex, indicating that phosphorylation of Ser‐473 may take place there. This result may support the suggestion that CK II is the kinase responsible for Ser‐473 phosphorylation. Despite many reports showing that CK I mediates phosphorylation of neurofilaments, CK II may phosphorylate NF‐L in vivo.
Light‐induced degradation of cytochrome b559 during photoinhibition of the photosystem II reaction center
Authors: Ortega José M , Roncel Mercedes , Losada Manuel
Published: July 2002

The behaviour of cytochrome (cyt) b559 during acceptor‐ and donor‐side photoinhibition has been investigated in oxygen‐evolving and non‐evolving photosystem II (PSII) membranes. Strong illumination at 20°C under aerobiosis induced a strong decrease in the absorbance of the cyt b559 α‐band in the two preparations. This absorbance decline was observed only in non‐oxygen‐evolving PSII samples when illumination was performed under aerobiosis but at 4°C, or under anaerobiosis at 20°C. These results suggest that acceptor‐side photoinhibition induces the degradation of cyt b559 by a mechanism related to an enzymatic reaction mediated by singlet oxygen. Donor‐side photoinhibition may induce, however, a non‐enzymatic photocleavage of the protein.
Hepatitis C virus NS3 serine protease interacts with the serpin C1 inhibitor
Authors: Drouet Christian , Bouillet Laurence , Csopaki Françoise , Colomb Maurice G
Published: July 2002

Both NS3 protein (1007–1657) and its protease moiety (NS3p, 1027–1207) were able to interact in vitro with C1 Inhibitor (C1Inh) to give a 95‐kDa M
r C1Inh cleavage product similar to that obtained upon proteolysis by complement protease C1s. High‐M
r reaction products were also detected after incubation of C1Inh with NS3 but not with NS3p; they correspond to ester‐bonded complexes from their hydroxylamine lability. Similar reactivity of NS3 was observed upon incubation with α2‐antiplasmin. Serpin cleavage was prevented by treatment of NS3 with synthetic serine protease inhibitors. This interaction between viral NS3 and host serpins suggests that NS3 is likely to be controlled by infected cell protease inhibitors.
Ascorbate‐mediated electron transfer in protein thiol oxidation in the endoplasmic reticulum
Authors: Csala Miklós , Braun László , Mile Valéria , Kardon Tamás , Szarka András , Kupcsulik Péter , Mandl József , Bánhegyi Gábor
Published: July 2002

Addition of, or gulonolactone oxidase‐dependent in situ generation of, ascorbate provoked the oxidation of protein thiols, which was accompanied by ascorbate consumption in liver microsomal vesicles. The maximal rate of protein thiol oxidation was similar upon gulonolactone, ascorbate or dehydroascorbate addition. Cytochrome P450 inhibitors (econazole, proadifen, quercetin) decreased ascorbate consumption and the gulonolactone or ascorbate‐stimulated thiol oxidation. The results demonstrate that the ascorbate/dehydroascorbate redox couple plays an important role in electron transfer from protein thiols to oxygen in the hepatic endoplasmic reticulum, even in gulonolactone oxidase deficient species.
Thyrotropin‐releasing hormone‐induced depletion of Gqα/G11α proteins from detergent‐insensitive membrane domains
Authors: Pešanová Zuzana , Novotný Jiřı́ , Černý Jan , Milligan Graeme , Svoboda Petr
Published: July 2002

The role of detergent‐insensitive membrane domains (DIMs) in desensitisation of the G protein‐coupled receptor‐mediated hormone response was studied in clone E2M11 of HEK293 cells which stably express high levels of both thyrotropin‐releasing hormone (TRH) receptors and G11α G protein. DIMs were prepared by flotation in equilibrium sucrose density gradients and characterised by a panel of membrane markers representing peripheral, glycosylphosphatidylinositol‐bound as well as integral membrane proteins (caveolin, CD29, CD55, CD59, CD147, the α subunit of Na,K‐ATPase) and enzyme activities (alkaline phosphatase, adenylyl cyclase). Caveolin‐containing DIMs represented only a small fraction of the overall pool of Gqα/G11α‐rich domains. Prolonged stimulation of E2M11 cells with TRH resulted in dramatic depletion of Gqα/G11α from all DIMs, which was paralleled by a concomitant Gqα/G11α increase in the high‐density gradient fractions containing the bulk‐phase membrane constituents soluble in 1% Triton X‐100. Distribution of membrane markers was unchanged under these conditions. Membrane domains thus represent a substantial structural determinant of the G protein pool relevant to desensitisation of hormone action.