bluuebunny/crossref_metadata_2025 huggingface dataset:
VIP21‐Caveolin, a protein of the trans‐Golgi network and caveolae
Authors: Kurzchalia Teymuras V. , Dupree Paul , Monier Solange
Published: July 2002

VIP21‐Caveolin is a component of the filamentous coat surrounding the invaginations of the plasma membrane called caveolae. Unlike the vesicular coat proteins identified so far, VIP21‐Caveolin can be classified as an integral membrane protein. Furthermore, it is found in high molecular mass oligomers. Based on its localisation in specialised membrane subdomains, a role for VIP21‐Caveolin in membrane protein sorting has been proposed.
The ‘Hinge’ protein of cytochrome c reductase from potato lacks the acidic domain and has no cleavable presequence
Authors: Braun Hans-Peter , Jänsch Lothar , Kruft Volker , Schmitza Udo K.
Published: July 2002

The ‘Hinge’ protein of cytochromecreductase from fungi and mammals is thought to support electron transport from cytochromec1to cytochromecand was reported to be one of the most acidic proteins known. Isolation and analysis of cDNA clones of the first ‘Hinge’ protein from a plant source reveals that it has a surplus of basic residues in potato. While the overall identity between the deduced amino acid sequence of the potato ‘Hinge’ protein and the proteins from yeast and bovine is in the range of 40%, the characteristic acidic domain is lacking. Therefore the numerous theories on the function of the mitochondrial ‘Hinge’ protein seem not to apply for the protein from potato. Also the atypical acidic presequence of the ‘Hinge’ protein from fungi and mammals is absent as revealed by N‐terminal sequencing of the isolated potato ‘Hinge’ protein. Functional implications of these results for the ‘Hinge’ proteins from other organisms are discussed.
Structural diversity of triadin in skeletal muscle and evidence of its existence in heart
Authors: Peng Mei , Fan Hongran , Kirley Terence L. , Caswell Anthony H. , Schwartz Arnold
Published: July 2002

Triadin has been characterized as an abundant protein co‐localized with the calcium release channel on the terminal cisternae of the sarcoplasmic reticulum of the skeletal muscle. Its localization to terminal cisternae of the sarcoplasmic reticulum and functional studies suggest that it has an important role in excitation‐contraction coupling. In this study we identify three triadin isoforms in rabbit skeletal muscle and by Northern blot analysis demonstrate that triadin also exists in the heart.
Ca2+‐dependent cosecretion of adrenomedullin and catecholamines mediated by nicotinic receptors in bovine cultured adrenal medullary cells
Authors: Katoha Fumi , Niina Hiromi , Kitamura Kazuo , Ichiki Yoshinari , Yamamoto Yoshitaka , Kangawa Kenji , Eto Tanenao , Wada Akihiko
Published: July 2002

Bovine cultured adrenal medullary cells (4 × 106) contained 4266.5 ± 370.0 fmol of immunoreactive adrenomedullin and 373.4 ± 32.6 mnol of catecholamines. Nicotinic (but not muscarinic) receptors mediated the Ca2+‐dependent co‐secretion of adrenomedullin and catecholamines, with the molar ratio of adrenomedullin/catecholamines secreted into the medium being equal to the ratio stored in the cells. The concentration‐response curve of carbachol for adrenomedullin secretion (EC50 42 μM) was similar to that for catecholamine secretion (EC50 63 μM). Reverse phase HPLC analysis showed that immunoreactive adrenomedullins in the cells and secreted into the medium were both eluted exclusively at the position almost identical to synthetic human adrenomedullin[1–52]NH2.
Participation of Mn(II) in the catalysis of laccase, manganese peroxidase and lignin peroxidase from Phlebia radiata
Authors: Lundell Taina , Hatakka Annele
Published: July 2002

Oxidation capacities of laccase, manganese peroxidase (MnP) and lignin peroxidase (LiP) from Phlebia radiata were compared using non‐phenolic (veratryl alcohol and ABTS) and phenolic (syringaldazine, vanillalacetone and Phenol red) compounds as reducing substrates. The effect of Mn(II) on enzyme reactions was also studied. Highest specific activities were recorded with laccase in the oxidation of phenolic compounds or ABTS and irrespective of MN(II) concentration. LiP and MnP oxidized all these substrates but only the catalysis of MnP was dependent upon Mn(II). Only LiP clearly oxidized veratryl alcohol. However, Mn(II) interfered with this reaction by repressing veratraldehyde formation. These results point to multiple participation of manganese ions, either as a reducing (Mn(II)) or oxidizing (Mn(III)) agent in the enzymatic reactions.
Influence of the conformation of a macromolecule on the generation of T‐cell proliferative response A study with model polypeptides
Authors: Lacassie E. , Delmas A. , Trudelle Y.
Published: July 2002

To study the influence of the conformation of polypeptidic macromolecules on the generation of T‐cell epitopes, sequential polypeptides with an octamer repeat unit were designed and synthesized. They adopt mainly unordered and α‐helical conformations. Among these polypeptides, those containing proline are fully or partly unordered, and are more effective at inducing T‐cell proliferation than a proline‐free very stable α‐helical polypeptide. This extremely stable α‐helical conformation, probably stabilized by aggregation, would enhance its stability against proteolytic processing.
Cloning and characterization of the Drosophila melanogaster CDK5 homolog
Authors: Hellmich Mark R. , Kennison James A. , Hampton Lori L. , Battey James F.
Published: July 2002

The D. melanogaster homolog of mammalian CDK5 has been cloned and its chromosomal location determined. The gene for Cdk5 consists of 4 exons separated by 3 short introns ranging in size from 61–160 bp. Northern blot analysis revealed a single mRNA of approximately 1.6 kb that is expressed at highest levels in the adult fly. The putative amino acid sequence for Drosophila Cdk5 predicts a protein with a mass of approximately 32 kDa that is 77% identical to its mammalian counter‐parts. Drosophila Cdk5 gene is located in polytene chromosomal region 52BC of the right arm of chromosome 2. This study provides the framework for a molecular genetic analysis of CDK5 function.
Src homology 2 domains of protein tyrosine phosphatase are associated in vitro with both the insulin receptor and insulin receptor substrate‐1 via different phosphotyrosine motifs
Authors: Ugi Satoshi , Maegawa Hiroshi , Olefsky Jerrold M. , Shigeta Yukio , Kashiwagi Atsunori
Published: July 2002

To clarify the role of protein tyrosine phosphatase containing Src homology 2 (SH2) regions on insulin signaling, we investigated the interactions among the insulin receptor, a pair of SH2 domains of SH‐PTP2 coupled to glutathione‐S‐transferase (GST) and insulin receptor substrate‐1 (IRS‐1)‐GST fusion proteins (amino‐portion, IRS‐1N; carboxyl portion, IRS‐1C). GST‐SH2 protein of SH‐PTP2 bound to the wild type insulin receptor, but not to that with a carboxyl‐terminal mutation (Y/F2). Furthermore, even though Y/F2 receptors were used, the SH2 protein was also co‐immunoprecipitated with IRS‐1C, but not with IRS‐1N. These results indicate that SH2 domains of SH‐PTP2 can directly associate with the Y1322TXM motif on the carboxyl terminus of insulin receptors and also may bind to the carboxyl portion of IRS‐1, possibly via the V1172IDL motif in vitro.
Deletion of highly conserved C‐terminal sequences in the Kv1 K+ channel sub‐family does not prevent expression of currents with wild‐type characteristics
Authors: Uebele V.N. , Yeola S.W. , Snyders D.J. , Tamkun M.M.
Published: July 2002

The C‐terminal regions of Kv1 K+ channels show little conservation between isoforms except for the last four C‐terminal residues, (E/L)TDV, which are well conserved from Drosophila to man. Deletions of the 4,16, and 57 C‐terminal residues of the human Kv1.5 channel did not affect whole cell current amplitude, midpoint of activation, degree of inactivation, or activation kinetics following expression in mouse L‐cells. Similar results were obtained with the rat Kv1.1 channel. Therefore, the conserved (E/L)TDV motif, and most of the C‐terminal amino acids, are not required for Kv1 channel expression.
Radioimmunoassay for circulating human guanylin
Authors: Kuhn Michaela , Kulaksiz Hasan , Adermann Knut , Rechkemmer Gerhurd , Forssmann Wolf-Georg
Published: July 2002

A highly specific and sensitive radioimmunoassay for circulating human guanylin (guanylin‐22–115) has been developed. Antibodies were raised against the amino‐terminus (positions 4–16) of the peptide. Western blot analysis confirmed that the antibody selected for radioimmunoassay recognizes circulating high molecular weight (10.3 kDa) guanylin. Extraction and purification of guanylin from blood hemofiltrate and from blood plasma showed that circulating guanylin is detectable in corresponding amounts by the radioimmunoassay and by a specific bioassay. In 30 healthy subjects, the mean plasma concentration of immunoreactive (IR) guanylin was 42 ± 3 . In 22 patients with chronic renal insufficiency, the concentrations of IR‐guanylin were significantly enhanced (1,074 ± 24 ), indicating that kidneys metabolize and/or eliminate the circulating hormone.
Two intestinal specific nuclear factors binding to the lactase‐phlorizin hydrolase and sucrase‐isomaltase promoters are functionally related oligomeric molecules
Authors: Troelsen J.T. , Olsen J. , Mitchelmore C. , Hansen G.H. , Sjöström H. , Norén O.
Published: July 2002

Lactase‐phlorizin hydrolase (LPH) and sucrase‐isomaltase (SI) are enterocyte‐specific gene products. The identification of regulatory cis‐elements in the promoter of these two genes has enabled us to carry out comparative studies of the corresponding intestinal‐specific nuclear factors (NF‐LPH1 and SIF1‐BP). Electrophoretic mobility shift assays demonstrated that the two nuclear factors compete for binding on the same cis‐elements. The molecular size of the DNA binding polypeptide is estimated to be approximately 50 kDa for both factors. In the native form the factors are found as 250 kDa oligomeric complexes. Based on these results NF‐LPH1 and SIF1‐BP are suggested to be either identical or closely related molecules.
Protein expression of the α, γ, δ and ε subspecies of protein kinase C changes as C6 glioma cells become contact inhibited and quiescent in the presence of serum
Authors: Moreton Kevin , Turner Rebecca , Blake Nicola , Paton Alex , Groome Nigel , Rumsby Martin
Published: July 2002

Total protein kinase C (PKC) activity and protein expression of the α and δ subspecies of PKC increases markedly as C6 glioma cells grow from low cell density to the contact‐inhibited quiescent state (also known as G0) in the presence of serum. At the same time protein expression of PKC subspecies γ and ϵ decreases while the β
I, β
II, ι and ζ subspecies did not change. Serum deprivation of growing C6 glioma cells does not induce the same changes in PKC subspecies protein expression. The findings support the growing view that there are significant differences between the G0 states brought about by contact inhibition or serum deprivation.
Urinary output of hydroxylysine glycosides and pyridinium cross‐links in detecting rat bone collagen turnover rate
Authors: Grazioli Vittorio , Alfano Massimo , Stenico Alberta , Casari Erminia
Published: July 2002

Glucosylgalactosylhydroxylysine (GGHYL), galactosylhydroxylysine (GHYL), pyridinoline (PYD) and deoxypyridinoline (DPD) were measured in the urine (6 h serial specimens over 96 and 24 h urine specimens for 4 days) collected from four adult Sprague Dawley rats and in the femoral and tibial bone as well as in the dorsal skin of the same rats. No significant daily variations were found in the urine excretion of GGHYL, GHYL, PYD and DPD but significant diurnal variations. The GGHYL/GHYL ratio in rat urine (0.46±0.1) reflected neither the bone collagen ratio (1.9 to 2.4) nor the skin collagen ratio (1.22±1.07), a finding that may reflect GGHYL conversion into GHYL. The content of both pyridinolines was very low in the skin and high in the bone collagen and the urinary PYD/DPD ratio (1.46±0.15) reflected essentially the bone collagen ratio (0.8‐3.0). These results suggest the usefulness of measuring GGHYL, GHYL, PYD and DPD in 24 h urine specimen and, based on the inter‐animal variations, the necessity to consider each animal as its own control when bone turnover needs to be monitored.
Results of treatment in 182 consecutive patients with genital fistulas
Authors: Ayhan A. , Tuncer Z.S. , Doǧan L. , Pekin S. , Kişnişçi H.A.
Published: July 2002

Objectives: To evaluate genital fistulas with the aim of improving strategies to prevent them. Method: An institutional, retrospective, clinical analysis. One hundred eighty‐two patients with genital fistulas managed consecutively between 1970 and 1993 were analyzed. Results: About 60% of all fistulas were attributable to obstetric traumas whereas gynecologic surgery constituted only 24.7% of cases. The contribution of gynecologic surgery increased from 11.1% in 1970–1975 to 60.6% in 1988–1993. Approximately half of the patients had vesico‐vaginal fistulas. Of the 45 patients with fistulas occurring following gynecologic surgery, 19 had undergone simple hysterectomy and eight had undergone radical hysterectomy. All cesarean sections had been performed after failed labor. Hysterectomy was performed due to myoma in nine patients, endometrial hyperplasia in two patients and pelvic relaxation in eight patients. As regards management of fistulas, the transvaginal approach was chosen in the vast majority of patients. The overall success rate after the primary repair of fistulas was 91.5% (162/177). Fifteen patients with failure of the primary repair were subjected to repeat repairs. The second repair was successful in 11 patients but the remaining four needed further repairs. The maximum was seven repairs to a recto‐vaginal fistula before a satisfactory result could be achieved, which had initially occurred after a cesarean section. Conclusion: Although the results of reparative surgery are promising, it would be more useful to encourage national measures to prevent in particular obstetric trauma.
Mirror image mutations reveal the significance of an intersubunit ion cluster in the stability of 3‐isopropylmalate dehydrogenase
Authors: Németh Attila , Svingor Ádám , Pócsik Márta , Dobó József , Magyar Csaba , Szilágyi András , Gál Péter , Závodszky Péter
Published: July 2002

The comparison of the three‐dimensional structures of thermophilic (Thermus thermophilus) and mesophilic (Escherichia coli) 3‐isopropylmalate dehydrogenases (IPMDH, EC 1.1.1.85) suggested that the existence of extra ion pairs in the thermophilic enzyme found in the intersubunit region may be an important factor for thermostability. As a test of our assumption, glutamine 200 in the E. coli enzyme was turned into glutamate (Q200E mutant) to mimic the thermophilic enzyme at this site by creating an intersubunit ion pair which can join existing ion clusters. At the same site in the thermophilic enzyme we changed glutamate 190 into glutamine (E190Q), hereby removing the corresponding ion pair. These single amino acid replacements resulted in increased thermostability of the mesophilic and decreased thermostability of the thermophilic enzyme, as measured by spectropolarimetry and differential scanning microcalorimetry.
Rab37 is a novel mast cell specific GTPase localized to secretory granules
Authors: Masuda Esteban S. , Luo Ying , Young Chi , Shen Mary , Rossi Alex B. , Huang Betty C.B. , Yu Sandra , Bennett Mark K. , Payan Don G. , Scheller Richard H.
Published: July 2002

GTPases regulate a myriad of cellular functions including signal transduction, cytoskeletal organization and membrane trafficking. Rab GTPases act to coordinate the membrane dynamics of cells by organizing and regulating the activity of effector proteins important in vesicle trafficking. Rab37 is a novel Rab GTPase specifically expressed in the MC‐9 mast cell line and bone marrow mast cells. Rab37 is 74% identical to Rab26 and 47% identical to Rab8, a GTPase important in Golgi to plasma membrane vesicle trafficking in mammalian cells. When green fluorescent protein tagged Rab37 is expressed in bone marrow mast cells, the secretory granules are labeled. These data suggest that Rab37 may play an important role in mast cell degranulation making this protein a potentially important target for therapeutic intervention in the treatment of allergy.
Neuronal nitric oxide synthase localizes through multiple structural motifs to the sarcolemma in mouse myotubes
Authors: Abdelmoity Ahmed , Padre Roanna C , Burzynski Kimberley E , Stull James T , Lau Kim S
Published: July 2002

In skeletal muscle, neuronal nitric oxide synthase is localized at the sarcolemma in association with the dystrophin glycoprotein complex (DGC). The nNOS N‐terminal 231 amino acids comprise a PDZ domain (residues 1–100) and a β‐hairpin finger loop (residues 101–130) which binds α‐syntrophin located in the DGC. Endogenous nNOS and GFP‐tagged nNOS localize to the sarcolemma in mouse C2C12 myotubes. Expression of GFP‐tagged nNOS domains in C2C12 myotubes reveals that the PDZ domain and the β‐hairpin finger loop of nNOS are independently capable of localizing to the sarcolemma of C2C12 myotubes. Binding studies indicate that α‐syntrophin binds only to the β‐hairpin finger loop and not the PDZ domain of nNOS. nNOS may bind to proteins in addition to α‐syntrophin at muscle sarcolemma.
Learning from hydrogenases: location of a proton pump and of a second FMN in bovine NADH–ubiquinone oxidoreductase (Complex I)
Authors: Albracht Simon P.J. , Hedderich Reiner
Published: July 2002

Hydrogenases have clear evolutionary links to the much more complex NADH–ubiquinone oxidoreductases (Complex I). Certain membrane‐bound [NiFe]‐hydrogenases presumably pump protons. From a detailed comparison of hydrogenases and Complex I, it is concluded here that the TYKY subunit in these enzymes is a special 2[4Fe–4S] ferredoxin, which functions as the electrical driving unit for a proton pump. The comparison further revealed that the flavodoxin fold from [NiFe]‐hydrogenases is presumably conserved in the PSST subunit of Complex I. It is proposed that bovine Complex I and the soluble NAD+‐reducing hydrogenase from Ralstonia eutropha each contain a second FMN group.
Studies of isopenicillin N synthase enzymatic properties using a continuous spectrophotometric assay
Authors: Dubus Alain , Sami Malkit , Brown Toby J.N. , Schofield Christopher J. , Baldwin Jack E. , Frère Jean-Marie
Published: July 2002

Isopenicillin N synthase (IPNS) from Aspergillus nidulans is a no‐heme iron(II)‐dependent oxygenase which catalyses, in a single reaction, the bicyclisation of δ‐(L‐α‐aminoadipoyl)‐L‐cysteinyl‐D‐valine into isopenicillin N, the precursor of all other penicillins, cephalosporins and cephamycins. The IPNS reaction can be followed directly and continuously by a new assay which monitors the absorbance increase at 235 nm characteristic of penicillin nucleus formation. Using this assay, the effects of influential factors affecting the in vitro IPNS enzymatic reaction were investigated. Even under optimal conditions, enzyme inactivation occurred during catalysis. Iron(II) depletion and product inhibition were not the cause of this phenomenon, the addition of antioxidants or reducing agents failed to slow down inactivation or reactivate the enzyme. Therefore, this phenomenon appears to be irreversible and is attributed to oxidative damage caused to the enzyme by reactive oxygen species generated in solution during catalysis. Nevertheless, the steady‐state kinetic parameters for the IPNS reaction were determined.
Genomic Exploration of the Hemiascomycetous Yeasts: 3. Methods and strategies used for sequence analysis and annotation
Authors: Tekaia Fredj , Blandin Gaëlle , Malpertuy Alain , Llorente Bertrand , Durrens Pascal , Toffano-Nioche Claire , Ozier-Kalogeropoulos Odile , Bon Elisabeth , Gaillardin Claude , Aigle Michel , Bolotin-Fukuhara Monique , Casarégola Serge , de Montigny Jacky , Lépingle Andrée , Neuvéglise Cécile , Potier Serge , Souciet Jean-Luc , Wésolowski-Louvel Micheline , Dujon Bernard
Published: July 2002

The primary analysis of the sequences for our Hemiascomycete random sequence tag (RST) project was performed using a combination of classical methods for sequence comparison and contig assembly, and of specifically written scripts and computer visualization routines. Comparisons were performed first against DNA and protein sequences fromSaccharomyces cerevisiae, then against protein sequences from other completely sequenced organisms and, finally, against protein sequences from all other organisms.Blastalignments were individually inspected to help recognize genes within our random genomic sequences despite the fact that only parts of them were available. For each yeast species, validated alignments were used to infer the proper genetic code, to determine codon usage preferences and to calculate their degree of sequence divergence withS. cerevisiae. The quality of each genomic library was monitored from contig analysis of the DNA sequences. Annotated sequences were submitted to the EMBL database, and the general annotation tables produced served as a basis for our comparative description of the evolution, redundancy and function of the Hemiascomycete genomes described in other articles of this issue.
Genomic Exploration of the Hemiascomycetous Yeasts: 10.Kluyveromyces thermotolerans
Authors: Malpertuy Alain , Llorente Bertrand , Blandin Gaëlle , Artiguenave François , Wincker Patrick , Dujon Bernard
Published: July 2002

A genomic exploration ofKluyveromyces thermotoleranswas performed by random sequence tag (RST) analysis. We sequenced 2653 RSTs corresponding to inserts sequenced from both ends. We performed a systematic comparison with a complete set of proteins fromSaccharomyces cerevisiae, other completely sequenced genomes and SwissProt. We identified six mitochondrial genes and 1358–1496 nuclear genes by comparison withS. cerevisiae. In addition, 25 genes were identified by comparison with other organisms. This corresponds to about 24% of the estimated gene content of this organism. A lower level of conservation is observed with orthologues to genes ofS. cerevisiaepreviously classified as orphans. Gene order was found to be conserved betweenS. cerevisiaeandK. thermotoleransin 56.5% of studied cases.
Furin and membrane type‐1 metalloproteinase mRNA levels and activation of metalloproteinase‐2 are associated with arterial remodeling
Authors: de Kleijn Dominique P.V. , Sluijter Joost P.G. , Smit Jenny , Velema Evelyn , Richard Wietske , Schoneveld Arjan H. , Pasterkamp Gerard , Borst Cornelius
Published: July 2002

Matrix metalloproteinase (MMP) activation is an essential feature of pathological and physiological arterial enlargement or shrinkage. Recently, furin‐activated membrane type‐1 MMP (MT1‐MMP) was identified as the in vivo activator of MMP2 in mice. Although arterial enlargement and shrinkage are important in several pathological processes, this proprotein convertase–MT1‐MMP axis has not been described during arterial remodeling. In rabbit femoral and carotid arteries, we report an increase in furin and MT1‐MMP mRNA levels before and at the onset of arterial remodeling followed by an increase in activated MMP2. This reveals the presence of the proprotein convertase–MT1‐MMP axis in flow‐induced arterial remodeling and identifies furin as a possible target for local intervention in pathological arterial remodeling.
A novel grass pollen allergen mimotope identified by phage display peptide library inhibits allergen–human IgE antibody interaction
Authors: Suphioglu Cenk , Schäppi George , Kenrick Josephine , Levy David , Davies Janet M. , O'Hehir Robyn E.
Published: July 2002

The aim of this study was to investigate the molecular basis of human IgE–allergen interaction by screening a phage‐displayed peptide library with an allergen‐specific human IgE‐mimicking monoclonal antibody (mAb). A mAb that reacted with major grass pollen allergens was successfully identified and shown to inhibit human IgE–allergen interaction. Biopanning of a phage‐displayed random peptide library with this mAb yielded a 12 amino acid long mimotope. A synthetic peptide based on this 12‐mer mimotope inhibited mAb and human IgE binding to grass pollen extracts. Our results indicate that such synthetic peptide mimotopes of allergens have potential as novel therapeutic agents.
Identification of Asp804 and Asp808 as Na+ and K+ coordinating residues in α‐subunit of renal Na,K‐ATPase
Authors: Pedersen Per Amstrup , Rasmussen Jakob H , Nielsen Jesper M , Jorgensen Peter L
Published: July 2002

Mutations to Asp804 and Asp808 in the α‐subunit almost abolish Na,K‐ATPase activity, but high‐affinity binding of [3H]ATP or [3H]ouabain at equilibrium and E1–E2 transitions are preserved. Titration of K+‐ion displacement of [3H]ATP or [3H]ouabain shows that the mutations interfere with occlusion of K+ in the E2[2K] conformation. Reduced phosphorylation levels or affinities for Na+ in presence of oligomycin indicate that Asp804 and Asp808 also contribute to coordination of Na+ in the E1P[3Na] form. Demonstration of alternate interactions of Na+ or K+ with Asp804 and Asp808 support the notion of cation binding in a ping‐pong sequence in catalytic models of Na,K‐pumping.
Sub‐mitochondrial localization of the catalytic subunit of pyruvate dehydrogenase phosphatase
Authors: Simonot Cédric , Lerme Fabienne , Louisot Pierre , Gateau-Roesch Odile
Published: July 2002

Using a specific antibody against the PDP catalytic subunit, PDPc, precise localization of this subunit in mitochondria was performed. Sub‐fractionation of purified mitochondria by controlled swelling processes led to the isolation of outer membranes, matrix space and inner membrane vesicles which were purified on a sucrose density gradient. In this study, we demonstrated that PDPc was not recovered as a soluble protein in the matrix space but was associated with the inner membrane. Moreover, Triton X‐114 phase partitioning performed on inner membranes showed that PDPc behaved both as a hydrophilic and as a hydrophobic protein, thus suggesting two different forms of this enzyme.
Mercuration of vanillyl‐alcohol oxidase from Penicillium simplicissimum generates inactive dimers
Authors: Fraaije Marco W , Mattevi Andrea , van Berkel Willem J.H
Published: July 2002

Vanillyl‐alcohol oxidase (EC 1.1.3.7) from Penicillium simplicissimum was modified with p‐mercuribenzoate. One cysteine residue reacts rapidly without loss of enzyme activity. Three sulfhydryl groups then react in an ‘all or none process’ involving enzyme inactivation and dissociation of the octamer into dimers. The inactivation reaction is slowed down in the presence of the competitive inhibitor isoeugenol and fully reversible by treatment of the modified enzyme with dithiothreitol. Vanillyl‐alcohol oxidase is more rapidly inactivated at low enzyme concentrations and protected from mercuration by antichaotropic salts. It is proposed that subunit dissociation accounts for the observed sensitivity of vanillyl‐alcohol oxidase crystals towards mercury compounds.
Plasma membrane content of insulin‐regulated glucose transporter in skeletal muscle of the male Otsuka Long‐Evans Tokushima Fatty rat, a model of non‐insulin‐dependent diabetes mellitus
Authors: Sato Takashi , Man Zhi-Wei , Toide Kiyotaka , Asahi Yoshihiko
Published: July 2002

The male Otsuka Long‐Evans Tokushima Fatty (OLETF) rat shows insulin resistance in skeletal muscle and visceral obesity. To obtain information on the mechanism of the insulin resistance in the diabetic rats, we examined the content of insulin‐regulated glucose transporter (GLUT4) in skeletal muscles. The results indicate that the total content of the transporter is significantly decreased (P<0.05) in muscles of the diabetic rats. Plasma membrane content of the GLUT4 protein in muscles of the diabetic rats was increased in the basal state as compared to control rats. Hyperinsulinemic clamps increased GLUT4 levels in the plasma membrane of control rats but failed to do so in the diabetic rats. The distribution of GLUT4 in OLETF rat is reminiscent of the characteristics of human non‐insulin‐dependent diabetes mellitus.
Vp165 and GLUT4 share similar vesicle pools along their trafficking pathways in rat adipose cells
Authors: Malide Daniela , St-Denis Jean-François , Keller Susanna R. , Cushman Samuel W.
Published: July 2002

vp165 (or gp160) is an aminopeptidase that has been identified as one of the major proteins of the GLUT4‐containing vesicles. In the present study we have determined the degree of co‐localization between vp165 and GLUT4 in rat adipose cells and used perturbation by wortmannin to assess the exocytic and endocytic steps along the translocation and recycling pathways of GLUT4 in the absence and presence of insulin. Western blots of subcellular membrane fractions demonstrate very similar distributions of vp165 and GLUT4. Confocal microscopy of whole cells provides direct evidence that these proteins share the same vesicle populations moving both towards and from the plasma membrane. These data are consistent with the presence of a distinct insulin‐sensitive compartment that sequesters both GLUT4 and vp165 and suggest similar trafficking routes through the recycling compartments.
P2X7 purinoceptor expression in Xenopus oocytes is not sufficient to produce a pore‐forming P2Z‐like phenotype
Authors: Petrou Steven , Ugur Mehmet , Drummond Robert M , Singer Joshua J , V. Walsh John
Published: July 2002

The purinergic rP2X7 receptor expressed in a number of heterologous systems not only functions as a cation channel but also gives rise to a P2Z‐like response, i.e. a reversible membrane permeabilization that allows the passage of molecules with molecular masses of ≥300 Da. We investigated the properties of rP2X7 receptors expressed in Xenopus oocytes. In two‐electrode voltage‐clamp experiments, ATP or BzATP caused inward currents that were abolished or greatly diminished when NMDG+ or choline+ replaced Na+ as the principal external cation. In fluorescent dye experiments, BzATP application did not result in entry of the fluorophore YO‐PRO‐12+. Thus, rP2X7 expression in Xenopus oocytes does not by itself give rise to the pore‐forming P2Z phenotype, suggesting that ancillary factors are involved.
Leptin inhibits glycogen synthesis in the isolated soleus muscle of obese (ob/ob) mice
Authors: Liu Yong-Ling , Emilsson Valur , Cawthorne Michael A.
Published: July 2002

The ob gene product, leptin, causes significant and dose‐dependent inhibition of basal and insulin‐stimulated glycogen synthesis in isolated soleus muscle from ob/ob mice, and a smaller, non‐significant inhibition in muscle from wild‐type mice. Leptin had no inhibitory effect on glycogen synthesis in soleus muscle from the diabetic (db/db) mice, which lack the functional leptin receptor. The full‐length leptin receptor (Ob‐Rb), is expressed in soleus muscle of both ob/ob and wild‐type mice, however with no detectable differences in expression level. These results suggest that hyperleptinaemia may attenuate insulin action on glucose storage in skeletal muscle.
Channel activators regulate ATP‐sensitive potassium channel (KIR6.1) expression in chick cardiomyocytes
Authors: Lu Changwan , Halvorsen Stanley W
Published: July 2002

ATP‐sensitive potassium channels (KATP) are widely expressed and yet little is known about the mechanisms regulating their expression. Here we report that expression of chick heart Kir6.1 is regulated by channel activators. Activation of KATP with either ATP depletion or pinacidil, up‐regulated Kir6.1 mRNA 1.8‐ to 2.4‐fold in cultured ventricular myocytes as measured by competitive PCR. Pinacidil treatment also increased Kir6.1 protein as detected using an antibody to Kir6.1. Glibenclamide, a KATP inhibitor, completely blocked the pinacidil‐induced increase in Kir6.1 levels. It appears that Kir6.1 is up‐regulated by an unknown signal transduction pathway initiated by KATP opening.
Comparative molecular analysis of Na+/H+ exchangers: a unified model for Na+/H+ antiport?
Authors: Dibrov Pavel , Fliegel Larry
Published: July 2002

Despite 30 years of study on Na+/H+ exchange, the molecular mechanisms of antiport remain obscure. Most challenging, the identity of amino acids involved in binding transported cations is still unknown. We review data examining the identity of residues that are involved in cation binding and translocation of prokaryotic and eukaryotic Na+/H+ antiporters. Several polar residues specifically distributed within or immediately adjacent to membrane spanning regions are implicated as being important. These key amino acids are conserved in prokaryotes and in some lower eukaryotic forms of the Na+/H+ antiporter, despite their being dispersed throughout the protein and despite an overall low similarity in the linear sequence of these Na+/H+ antiporters. We suggest that this conservation of isolated residues (together with distances between them) reflects a general physicochemical mechanism of cation binding by exchangers. The binding could be based on coordination of the substrate cation by a crown ether‐like cluster of polar atomic groups amino acids, as has been hypothesized by Boyer [1]. Traditional screening for the extended, highly conserved linear protein sequences might not be applicable when searching for functional domains of ion transporters. Three‐dimensional constellations of polar residues (3D‐motifs) may be evolutionary conserved rather than linear primary sequence.
Activation of a plant plasma membrane Ca2+ channel by TGα1, a heterotrimeric G protein α‐subunit homologue
Authors: Aharon Gilad S , Gelli Angie , Snedden Wayne A , Blumwald Eduardo
Published: July 2002

Wild‐type and GTPase‐deficient recombinant TGα1 were used along patch‐clamp techniques to study the role of heterotrimeric G proteins in the regulation of the hyperpolarized active tomato plasma membrane Ca2+ channel. Recombinant α‐subunits induced an increase in channel activity as shown by the increase in channel events and the mean open probability of the channel. Our results suggest a membrane‐delimited pathway involving heterotrimeric G proteins in Ca2+ channel activation.
Characterization of the 3′ end of the mouse SERCA 3 gene and tissue distribution of mRNA spliced variants1
Authors: Ozog Anne , Pouzet Bruno , Bobe Regis , Lompré Anne-Marie
Published: July 2002

The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) type 1 and 2 genes are alternatively spliced at their 3′ end. We hypothesized that similar mechanism may occur for SERCA 3. Two spliced variants were identified by RNase protection analysis. We then isolated and sequenced the 3′ end portion of the mouse SERCA 3 gene, and confirmed the presence of an alternative mRNA transcript by sequencing a cDNA fragment obtained by RT‐PCR. Tissue distribution of the alternatively spliced mRNAs was studied by RT‐PCR: SERCA 3b was the only isoform expressed in endothelial cells from aorta and heart and also was the major isoform in lung and kidney whereas SERCA 3a and 3b were coexpressed in trachea, intestine, thymus, spleen, and fetal liver.
The extracellular domain of CD4 receptor possesses a protein kinase activity
Authors: Yakubov Leonid A. , Kit Yury Y. , Richter Vladimir A. , Andreeva Anna Y. , Karamyshev Valery N. , Stein C.A. , Vlassov Valentin V.
Published: July 2002

The CD4 receptor of T‐helper cells is an essential participant in immune response formation and HIV infection. We report here that the extracellular domains of CD4 receptor can catalyze the phosphotransferase (kinase) reaction. Incubation of rsCD4 in solution with [γ‐32P]ATP results in the Ca2+‐dependent autophosphorylation of the protein presumably at a His residue because the reaction is prevented by the diethylpyrocarbonate treatment. The rsCD4 phosphorylates milk casein or human plasma proteins as a Ser/Thr protein kinase.
Characterization and partial purification of an oligopeptide elicitor receptor from parsley (Petroselinum crispum)
Authors: Nennstiel Dirk , Scheel Dierk , Nürnberger Thorsten
Published: July 2002

Parsley cells recognize the fungal phytopathogenPhytophthora sojaethrough a plasma membrane receptor. A 13 amino acid oligopeptide fragment (Pep‐13) of a 42 kDa fungal cell wall glycoprotein was shown to bind to the receptor and stimulate a complex defense response in cultured parsley cells. The Pep‐13 binding site solubilized from parsley microsomal membranes by non‐ionic detergents exhibited the same ligand affinity and ligand specificity as the membrane‐bound receptor. Chemical crosslinking and photoaffinity labeling assays with [125I]Pep‐13 revealed that a monomeric 100 kDa integral plasma membrane protein is sufficient for ligand binding and may thus constitute the ligand binding domain of the receptor. Ligand affinity chromatography of solubilized microsomal membrane protein on immobilized Pep‐13 yielded a 5000‐fold enrichment of specific receptor activity.
TPA and butyrate increase cell sensitivity to the vacuolating toxin of Helicobacter pylori
Authors: de Bernard Marina , Moschioni Monica , Papini Emanuele , Telford John L. , Rappuoli Rino , Montecucco Cesare
Published: July 2002

The Helicobacter pylori toxin VacA induces large membrane‐bound vacuolar compartments of late endosomal/lysosomal origin. Pre‐treatment of cells with TPA and butyrate enhances the toxin induced vacuolisation up to 20 times, depending on the cell line, whereas other differentiating factors such as DMSO, EGF, valeric and retinoic acid have no effect. The higher toxin sensitivity induced by TPA does not result from an increased surface binding or endocytosis. The effect of TPA is apparent after several hours from addition and is inhibited by a PKC specific inhibitor. These data suggest that expression of cellular proteins, other than the toxin receptor(s), influences the vacuolating activity of VacA and may contribute to the sensitivity of different cell lines. The present findings define the most sensitive in vitro assay of the activity of VacA.
Human D3 dopamine receptor in the medulloblastoma TE671 cell line: cross‐talk between D1 and D3 receptors
Authors: Levavi-Sivan Berta , Park Bae-Hang , Fuchs Sara , Fishburn C.Simone
Published: July 2002

In search of a cell line in which the D3 dopamine receptor is expressed endogenously, we found that the neuron‐derived human medulloblastoma cell line TE671 expresses the human D3 (hD3) and D1 (hD1) receptor, but neither the D2 or D4 receptors. Exposure of TE671 cells to the D3 agonist 7‐OH‐DPAT (DPAT), or to the D1 agonist SKF‐38393 (SKF) increased the expression of hD3 or hD1 mRNA, respectively. Moreover, whereas DPAT had no effect on hD1 mRNA levels, stimulating the cells with SKF caused an increase in both hD1 and hD3 transcript levels. These results suggest (i) that following ligand stimulation, hD3 and hD1 receptors are upregulated to enhance their own receptor expression, and (ii) that upregulation of hD1 receptor transcripts leads to a stimulation of the hD3 dopamine receptor transcripts.
Selective inhibition of human type 1 11β‐hydroxysteroid dehydrogenase by synthetic steroids and xenobiotics
Authors: Hult Malin , Jörnvall Hans , Oppermann Udo C.T
Published: July 2002

Functional analyses were performed with microsomal human 11β‐hydroxysteroid dehydrogenase type 1 overexpressed in the yeast Pichia pastoris. Cell extracts or microsomes from transformed strains displayed dehydrogenase and reductase activities, which were up to 10 times higher than in human liver microsomes, while for whole cells cortisone reduction but no dehydrogenase activity was observed. The synthetic glucocorticoids prednisolone and prednisone were efficiently metabolized by subcellular fractions, whereas no activity was observed with dexamethasone, budesonide and deflazacort. Inhibitors found to be effective towards the recombinant 11β‐hydroxysteroid dehydrogenase include synthetic steroids and xenobiotic compounds, revealing selective inhibition of the reaction direction, useful for development of specific inhibitors.
Hypoxia increases the association of 4E‐binding protein 1 with the initiation factor 4E in isolated rat hepatocytes
Authors: Tinton Sandrine A. , Buc-Calderon Pedro M.
Published: July 2002

Incubation of hepatocytes under hypoxia increases binding of translation initiation factor eIF‐4E to its inhibitory regulator 4E‐BP1, and this correlates with dephosphorylation of 4E‐BP1. Rapamycin induced the same effect in aerobic cells but no additive effect was observed when hypoxic cells were treated with rapamycin. This enhanced association of 4E‐BP1 with eIF‐4E might be mediated by mTOR. Nevertheless, only hypoxia produces a rapid inhibition of protein synthesis. Although hypoxia might be signalling via the rapamycin‐sensitive pathway by changing eIF‐4E availability, such a pathway is unlikely to be responsible for the depression in overall protein synthesis under hypoxia.
Modulation of α‐tropomyosin expression by α‐tocopherol in rat vascular smooth muscle cells
Authors: Aratri Elisabetta , Spycher Stefan E. , Breyer Isabel , Azzi Angelo
Published: July 2002

The effect of α‐tocopherol (vitamin E) on gene expression in rat vascular smooth muscle cells was studied by the differential display technique. One gene out of about 1000 genes analyzed, identified as α‐tropomyosin, showed an increased transcription level caused by α‐tocopherol treatment. Northern and Western blot analysis revealed a time‐dependent transient up‐regulation of the amount of mRNA (peak between 2 and 3 h) and protein (peak at 5 h) in α‐tocopherol‐treated cells. No effect was observed in cells treated with β‐tocopherol.
Daidzein and genistein but not their glucosides are absorbed from the rat stomach
Authors: Piskula Mariusz K , Yamakoshi Jun , Iwai Yukihiko
Published: July 2002

Absorption of isoflavone aglycones and glucosides was compared in rats. Daidzein, genistein, daidzin and genistin were orally administered at a dose of 7.9 μmol/kg in 25 mM Na2CO3 and next their metabolite concentration in blood plasma was monitored for 30 min. After isoflavone glucosides administration, their metabolites appeared in plasma with a few minutes delay as compared to aglycones, which suggested that aglycones, but not glucosides, were absorbed already in the rat stomach. This observation was confirmed when absorption site was restricted solely to the stomach and absorption was shown to be independent of the vehicle pH used for administration.
RNA helicase activity of Semliki Forest virus replicase protein NSP2
Authors: Gomez de Cedrón Marta , Ehsani Neda , Mikkola Marja L. , Garcı́a Juan Antonio , Kääriäinen Leevi
Published: July 2002

Semliki Forest virus replicase protein nsP2 shares sequence homology with several putative NTPases and RNA helicases. NsP2 has RNA‐dependent NTPase activity. Here we expressed polyhistidine‐tagged nsP2 in Escherichia coli, purified it by metal‐affinity chromatography, and used it in RNA helicase assays. RNA helicase CI of plum pox potyvirus was used as a positive control. Unwinding of α‐32P‐labelled partially double‐stranded RNA required nsP2, Mg2+ and NTPs. NsP2 with a mutation, K192N, in the NTP‐binding sequence GVPGSGK192SA could not unwind dsRNA and had no NTPase activity. This is the first demonstration of RNA helicase activity within the large alphavirus superfamily.
The acceptor substrate specificity of human β4‐galactosyltransferase V indicates its potential function in O‐glycosylation
Authors: van Die Irma , van Tetering Angelique , Schiphorst Wietske E.C.M , Sato Takeshi , Furukawa Kiyoshi , van den Eijnden Dirk H
Published: July 2002

In order to assess the function of the different human UDP‐Gal:GlcNAc β4‐galactosyltransferases, the cDNAs of two of them, β4‐GalT I and β4‐GalT V, were expressed in the baculovirus/insect cell expression system. The soluble recombinant enzymes produced were purified from the medium and used to determine their in vitro substrate specificities. The specific activity of the recombinant β4‐GalT V was more than 15 times lower than that of β4‐GalT I, using GlcNAcβ‐S‐pNP as an acceptor. Whereas β4‐GalT I efficiently acts on all substrates having a terminal β‐linked GlcNAc, β4‐GalT V appeared to be far more restricted in acceptor usage. β4‐GalT V acts with high preference on acceptors that contain the GlcNAcβ1→6GalNAc structural element, as found in O‐linked core 2‐, 4‐ and 6‐based glycans, but not on substrates related to N‐linked or blood group I‐active oligosaccharides. These results suggest that β4‐GalT V may function in the synthesis of lacNAc units on O‐linked chains, particularly in tissues which do not express β4‐GalT I, such as brain.
Extracellular matrix regulation of PTHrP and PTH/PTHrP receptor in a human breast cancer cell line
Authors: Luparello Claudio , Schilling Tobias , Cirincione Rosalia , Pucci-Minafra Ida
Published: July 2002

It was previously reported that 8701‐BC breast cancer cells express the gene for parathyroid hormone‐related peptide (PTHrP) and its cognate receptor (PTHrP‐R), and release immunoreactive PTHrP in the extracellular medium; it was also found that PTHrP, in turn, exerts a role on the proliferative and invasive behavior in vitro of the same cell line. On the other hand, evidence has been produced that adhesion of 8701‐BC cells onto different collagen substrates influences in various ways a number of phenotypic expressions, such as cell growth, motility, invasion of reconstituted basement membrane and production of lytic enzymes of the extracellular matrix (ECM). In light of these previous data, we have examined whether substrates of either reconstituted basement membrane or representative collagen components of the breast tumor stroma (type I, V and OF/LB) might (i) regulate the PTHrP promoter usage and mRNA splicing patterns, (ii) modulate quantitatively the extracellular release of immunoreactive PTHrP (iPTHrP), and (iii) affect the expression of PTHrP‐R. The results obtained give evidence that (i) 8701‐BC cells are able to utilize different start sites and mRNA splicing patterns for PTHrP transcription; (ii) ‘structural’ components of the stroma, such as collagens, are by themselves capable of controlling both the expression pattern of the PTHrP gene and the extent of extracellular release of iPTHrP, and (iii) PTHrP‐R expression can be up‐ or down‐regulated in response to the ECM substrate present. These data demonstrate that PTHrP and PTHrP‐R expression by 8701‐BC neoplastic cells can be modulated by ECM molecules, indirectly supporting the active participation of stromal collagen composition in the regulation of PTHrP‐controlled circuits which may play a role in carcinogenesis.
Association of Cbl with Fms and p85 in response to macrophage colony‐stimulating factor
Authors: Ota Jun , Sato Ken , Kimura Fumihiko , Wakimoto Naoki , Nakamura Yukitsugu , Nagata Naokazu , Suzu Shinya , Yamada Muneo , Shimamura Seiichi , Motoyoshi Kazuo
Published: July 2002

Tyrosine phosphorylation of Cbl and its association with signal‐transducing molecules in response to macrophage colony‐stimulating factor (M‐CSF) were analyzed by using cell lines which express the wild‐type and a mutant M‐CSF receptor, Fms. We found that in a clone, F723 TF‐1 cells expressing mutant Fms in which tyrosine 723 had been substituted with phenylalanine, the M‐CSF stimulation‐dependent association between Cbl and Fms was markedly impaired. However, phosphorylation of Cbl and its association with the p85 subunit of phosphatidylinositol 3‐kinase were induced in these mutant cells as seen in the wild‐type fms transfectant. These results suggest that phosphorylation of tyrosine 723 is particularly important for the recruitment of Cbl to the M‐CSF receptor, but is not required for the phosphorylation and binding of Cbl to signal‐transducing molecules such as p85.