bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of apoptosis and of interleukin‐1β secretion by 7β‐hydroxycholesterol and 7‐ketocholesterol: partial inhibition by Bcl‐2 overexpression
Authors: Lizard Gérard , Lemaire Stéphanie , Monier Serge , Gueldry Serge , Néel Dominique , Gambert Philippe
Published: July 2002

The oxysterols, 7β‐hydroxycholesterol and 7‐ketocholesterol, are involved in the cytotoxicity of oxidized LDL. To elucidate their molecular mechanisms, the human promonocytic leukemia cells U937 and U4 were used. U4 cells overexpressing Bcl‐2 were obtained by transfection of U937 cells. 7β‐hydroxycholesterol and 7‐ketocholesterol induced nuclear condensation and/or fragmentation, internucleosomal DNA fragmentation, and IL‐1β secretion, which were partially inhibited by Bcl‐2 overexpression. These findings underline that these oxysterols could constitute major risk factors in atherosclerosis by their cytotoxicity and their ability to induce IL‐1β release which might favor the recruitment of immunocompetent cells in the atherosclerotic plaque.
Molecular cloning and tissue distribution of an alternatively spliced variant of an A‐type K+ channel α‐subunit, Kv4.3 in the rat
Authors: Ohya Susumu , Tanaka Mamiko , Oku Takuma , Asai Yukari , Watanabe Minoru , Giles Wayne R , Imaizumi Yuji
Published: July 2002

We describe here (1) the heterogeneous expression of Ca2+‐independent transient (A‐type) K+ channel α‐subunits (Kv1.4, Kv3.3, Kv3.4, Kv4.2 and Kv4.3) in rat smooth muscle, heart and brain, (2) the molecular cloning and tissue distribution of a novel alternatively spliced variant of an A‐type K+ channel α‐subunit, Kv4.3, and (3) the functional expression of A‐type K+ channels in HEK293 cells by the transfection with the novel splice variant of Kv4.3. A cDNA encoding this splice variant was identified from rat vas deferens by RT‐PCR cloning. This cDNA clone contains a 1965 bp open reading frame that encodes for a protein of 655 amino acids. It has a 19 amino acid insertion in comparison with Kv4.3 previously reported in rat brain. RT‐PCR analyses showed that the mRNAs of this longer variant are abundantly expressed in a number of smooth muscles of the rat, and that the mRNAs of the previously reported clones are absent. The longer splice variant is very weakly expressed in brain, but is the major product in heart.
Purification and characterisation of p99, a nuclear modulator of protein phosphatase 1 activity
Authors: Kreivi Jan-Peter , Trinkle-Mulcahy Laura , Lyon Carol E , Morrice Nick A , Cohen Philip , Lamond Angus I
Published: July 2002

We have purified a form of protein phosphatase 1 (PP1) from HeLa cell nuclei, in which the phosphatase is complexed to a regulatory subunit termed p99. We report here the cloning and characterisation of the p99 component. p99 mRNA is widely expressed in human tissues and immunofluorescence analysis with anti‐p99 antibodies showed a punctate nucleoplasmic staining with additional accumulations within the nucleolus. The C‐terminus of p99 contains seven RGG RNA‐binding motifs, followed by eleven decapeptide repeats containing six or more of the following conserved residues (GHRPHEGPGG), and finally a putative zinc finger domain. Recombinant p99 suppresses the phosphorylase phosphatase activity of PP1 by >90% and the canonical PP1‐binding motif on p99 (residues 396–401) is unusual in that the phenylalanine residue is replaced by tryptophan.
Differential effects of suramin on protein kinase C isoenzymes. A novel tool for discriminating protein kinase C activities
Authors: Gschwendt Michael , Kittstein Walter , Johannes Franz-Josef
Published: July 2002

Suramin, a hexasulfonated naphthylurea, is known to induce differentiation and inhibit proliferation, angiogenesis, and development of tumors. It has also been shown to suppress the activity of the protein kinase C (PKC) isoenzymes α, β, and γ. Here we report on a differential effect of suramin on PKCμ and various PKC isoforms representing the cPKC, nPKC, and aPKC group of the PKC family. In the absence of any cofactors suramin activates all PKC isoforms in the order of aPKCζ≫PKCμ>cPKC, nPKCδ. As judged by the V
max/K
M ratios (0.5 for PKCμ and 2.2 for PKCζ) the substrate syntide 2 is phosphorylated by suramin‐activated PKCζ around four times more effectively than by suramin‐activated PKCμ. Suramin‐activated PKCμ behaves like that activated by phosphatidylserine and the phorbol ester TPA regarding autophosphorylation and differential inhibition by the PKC inhibitors Gö 6976 and Gö 6983. In the presence of activating cofactors, such as phosphatidylserine and TPA or cholesterol sulfate (for PKCζ), the activity of the aPKCζ is further stimulated, PKCμ is not significantly affected, and the cPKCs and the nPKCδ are strongly inhibited by suramin. The differential action of suramin on PKC isoenzymes might play a role in some of its biological effects, as for instance inhibition of proliferation and tumor development. Moreover, due to this property suramin will possibly be a valuable tool for discriminating the activities of PKC isoenzymes in vitro and in vivo.
Non‐peptide inhibitors of HCV serine proteinase
Authors: Kakiuchi Nobuko , Komoda Yasumasa , Komoda Keiko , Takeshita Norisue , Okada Satoshi , Tani Tadato , Shimotohno Kunitada
Published: July 2002

We screened a chemical library of 2000 compounds for inhibitors of hepatitis C virus (HCV) serine proteinase using an in vitro screening method measuring the hydrolysis of the peptide substrate. Three compounds were found to be the most potent inhibitors (IC50<10−5 M). Two of them had a similar structure, that of halogenated benzanilide, and were not inhibitory for common serine proteinases. They inhibited the enzyme non‐competitively with the substrate. Together with the result of the analogous compounds in the chemical library, the presumed structural requirements of the inhibition are pointed out.
Long terminal repeats of human endogenous retrovirus K family (HERV‐K) specifically bind host cell nuclear proteins
Authors: Akopov Sergey B , Nikolaev Lev G , Khil Pavel P , Lebedev Yuri B , Sverdlov Eugene D
Published: July 2002

Solitary long terminal repeats (LTRs) of the human endogenous retroviruses, scattered in several thousand copies throughout the human genome, are potentially capable of affecting the expression of closely located genes. To assess their regulatory potential, the LTR sequences of one of the most abundant HERV families (HERV‐K) were screened for the presence of binding sites for the host cell nuclear factors using mobility shift and UV‐crosslinking assays. It was shown that the LTR sequences of two subfamilies harbor a specific binding site for a complex consisting of at least three proteins, ERF1, ERF2 and ERF3 of 98, 91 and 88 kDa apparent molecular mass, respectively. This binding site is located in the 5′ region of the LTR U3 element. The preservation of the specific protein binding site in different HERV‐K LTR sequences suggests their possible role in regulation of nearby located genes.
NMR studies of the conformation of thiocellobiose bound to a β‐glucosidase from Streptomyces sp
Authors: Montero Esther , Vallmitjana Miquel , Pérez-Pons Josep A , Querol Enrique , Jiménez-Barbero Jesús , Cañada F.Javier
Published: July 2002

The conformation of 4‐thiocellobiose bound to β‐glucosidase from Streptomyces sp. has been studied by 1H‐NMR transferred nuclear Overhauser effect spectroscopy (TR‐NOE). Thiocellobiose behaves as an inhibitor of this glucosidase when cellobiose is used as substrate. NOE measurements and molecular mechanics calculations have also been performed to estimate the probability distribution of conformers of thiocellobiose when free in solution. Experimental data show that, in contrast with the natural O‐analogue, thiocellobiose presents three conformational families in the free state, namely syn, anti‐Ψ and anti‐Φ, whilst only one of them (syn) is recognized by the enzyme.
Oligomerization of protegrin‐1 in the presence of DPC micelles. A proton high‐resolution NMR study
Authors: Roumestand Christian , Louis Valérie , Aumelas André , Grassy Gérard , Calas Bernard , Chavanieu Alain
Published: July 2002

Protegrins are members of a family of five Cys‐rich naturally occurring cationic antimicrobial peptides. The NMR solution structure of protegrin‐1 (PG‐1) has been previously determined as a monomeric β‐hairpin both in water and in dimethylsulfoxide solution. Protegrins are bactericidal peptides but their mechanism of action is still unknown. In order to investigate the structural basis of their cytotoxicity, we studied the effect of lipid micelles on the structure of PG‐1. The NMR study reported in the present work indicates that PG‐1 adopts a dimeric structure when it binds to dodecylphosphocholine micelles. Moreover, the amide proton exchange study suggests the possibility of an association between several dimers.
Synergistic interaction of the cellulosome integrating protein (CipA) from Clostridium thermocellum with a cellulosomal endoglucanase
Authors: Ciruela Antonio , Gilbert Harry J , Ali Bassam R.S , Hazlewood Geoffrey P
Published: July 2002

Activity of a cellulosomal endoglucanase (endoglucanase E; EGE) from Clostridium thermocellum against two crystalline forms of cellulose was enhanced by combination with the cellulosome integrating protein (CipA), but CipA did not enhance EGE activity against amorphous cellulose, even though it was able to bind to it. Similarly, CipA added in trans to genetically truncated EGE that was unable to combine with it nevertheless enhanced EGE activity against crystalline cellulose. These results indicate that the CipA cellulose binding domain does not mediate an increase in activity solely by bringing the catalytic subunits of the cellulosome complex into intimate contact with the substrate.
Caspase‐mediated proteolysis during apoptosis: insights from apoptotic neutrophils
Authors: Sanghavi Darshak M , Thelen Marcus , Thornberry Nancy A , Casciola-Rosen Livia , Rosen Antony
Published: July 2002

Apoptosis is initiated by activation of caspases (interleukin 1β‐converting enzyme homologues), which cause coordinated cleavage of several death substrates that function in structural or homeostatic pathways. The relationship between substrate cleavage and apoptosis is not yet known, nor is it clear whether cleavage of specific substrates is a critical requirement for apoptosis. The human neutrophil provides novel insights into the roles of proteolysis of specific substrates during apoptosis, since only a subset of caspase substrates are present in mature neutrophils. Of the death substrates we screened, PARP, the nuclear mitotic apparatus protein (NuMA), the 70 kDa subunit of the U1 small ribonucleoprotein (U1‐70kDa) and the catalytic subunit of DNA‐dependent protein kinase (DNA‐PKcs) were not detected in non‐apoptotic neutrophils; in contrast, lamin B and fodrin were present in amounts similar to those found in other cells. Caspase‐3 activity was absent in freshly isolated neutrophils, but was detected when neutrophils were aged in vitro, coincident with the onset of morphologic and biochemical apoptosis. The absence of PARP, NuMA, U1‐70kDa and DNA‐PKcs in non‐apoptotic neutrophils suggests that these are not critical anti‐apoptotic proteins, and that their fragments are not required components of the neutrophil apoptotic pathway. These studies highlight the conserved role of caspase activation in the apoptotic mechanism, and focus attention on several conserved structural substrates as potential transducers of the proteolytic signal in apoptosis.
Mutations in the proteolytic domain of Escherichia coli protease Lon impair the ATPase activity of the enzyme
Authors: Starkova Natalie N , Koroleva Ekaterina P , Rumsh Lev D , Ginodman Lev M , Rotanova Tatyana V
Published: July 2002

Conserved residues of the proteolytic domain of Escherichia coli protease Lon, putative members of the classic catalytic triad (H665, H667, D676, and D743) were identified by comparison of amino acid sequences of Lon proteases. Mutant enzymes containing substitutions D676N, D743N, H665Y, and H667Y were obtained by site‐directed mutagenesis. The mutant D743N retained the adenosine triphosphate (ATP)‐dependent proteolytic activity, thereby indicating that D743 does not belong to the catalytic site. Simultaneously, the mutants D676N, H665Y, and H667Y lost the capacity for hydrolysis of protein substrates. The ATPase activity of these three mutants was decreased by more than an order of magnitude, which suggests a close spatial location of the ATPase and proteolytic active sites and their tight interaction in the process of protein degradation.
Inducible expression of the GLT‐1 glutamate transporter in a CHO cell line selected for low endogenous glutamate uptake
Authors: Levy Line M , Attwell David , Hoover Frank , Ash John F , Bjørås Magnar , Danbolt Niels Christian
Published: July 2002

Inducible expression of the mammalian glial cell glutamate transporter GLT‐1 has been established in a CHO cell line selected for low endogenous Na+‐dependent glutamate uptake by [3H]aspartate suicide selection. Culturing the cells in doxycycline‐containing medium, to activate GLT‐1 expression via the Tet‐On system, increased uptake of the GLT‐1 substrate d‐aspartate 280‐fold, and increased cell size. Applying glutamate to whole‐cell clamped, doxycycline‐treated cells evoked a transporter‐mediated current with characteristics appropriate for GLT‐1. This cell line provides a useful tool for further examination of the electrical, biochemical and pharmacological properties of GLT‐1, the most abundant glutamate transporter in the brain.
Presence of leptin receptors in rat small intestine and leptin effect on sugar absorption
Authors: Lostao M.Pilar , Urdaneta Elena , Martı́nez-Ansó Eduardo , Barber Ana , Martı́nez J.Alfredo
Published: July 2002

Leptin is involved in food intake and thermogenesis regulation. Since leptin receptor expression has been found in several tissues including small intestine, a possible role of leptin in sugar absorption by the intestine was investigated. Leptin inhibited d‐galactose uptake by rat small intestinal rings 33% after 5 min of incubation. The inhibition increased to 56% after 30 min. However, neither at 5 min nor at 30 min did leptin prevent intracellular galactose accumulation. This leptin effect was accompanied by a decrease of the active sugar transport apparent V
max (20 vs. 4.8 μmol/g wet weight 5 min) and apparent K
m (15.8 vs. 5.3 mM) without any change in the phlorizin‐resistant component. On the other hand, immunohistochemical experiments using anti‐leptin monoclonal antibodies recognized leptin receptors in the plasma membrane of immune cells located in the lamina propria. These results indicate for the first time that leptin has a rapid inhibitory effect on sugar absorption and demonstrate the presence of leptin receptors in the intestinal mucosa.
ABI1 of Arabidopsis is a protein serine/threonine phosphatase highly regulated by the proton and magnesium ion concentration1
Authors: Leube M.P , Grill E , Amrhein N
Published: July 2002

The plant hormone abscisic acid (ABA) mediates various responses such as stomatal closure, maintenance of seed dormancy, and inhibition of plant growth. All three responses are regulated by the ABI1 gene product. The ABI1 protein (ABI1p) has been characterized as a protein serine/threonine phosphatase of type 2C that is highly affected in its activity by changes in the proton and magnesium ion concentrations. In the ABA‐insensitive mutant abi1 of Arabidopsis thaliana a single amino acid exchange in the primary structure results in both a dominant insensitive phenotype and a strongly reduced protein phosphatase activity in vitro by possibly impairing metal ion coordination.
Diffusion‐controlled DNA recognition by an unfolded, monomeric bZIP transcription factor
Authors: Berger Christine , Piubelli Luciano , Haditsch Ursula , Rudolf Bosshard Hans
Published: July 2002

Basic leucine zipper (bZIP) transcription factors are dimers that recognize mainly palindromic DNA sites. It has been assumed that bZIP factors have to form a dimer in order to bind to their target DNA. We find that DNA binding of both monomeric and dimeric bZIP transcription factor GCN4 is diffusion‐limited and that, therefore, the rate of dimerization of the bZIP domain does not affect the rate of DNA recognition and GCN4 need not dimerize in order to bind to its specific DNA site. The results have implications for the mechanism by which bZIP transcription factors find their target sites for transcriptional regulation.
Origin of octameric creatine kinases
Authors: Ellington W.Ross , Roux Kenneth , Pineda Agustin O
Published: July 2002

Mitochondrial creatine kinase (MiCK) occurs primarily as an octameric form localized in the mitochondrial intermembrane compartment in vertebrate tissues and echinoderm spermatozoa (both deuterostome groups). The octameric quaternary structure is thought to play important functional and enzyme targeting roles. We have found that the spermatozoa of the protostome polychaete Chaetopterus variopedatus contain three distinct isoenzymes of creatine kinase (CK) termed CK1, CK2 and CK3. CK3 appears to be present only in the sperm head/midpiece complex where mitochondria are restricted and has a subunit relative molecular mass (M
r) of 43.4 kDa. Gel permeation chromatography using Superdex 200HR showed that CK3 has a native M
r of 344.9 kDa indicating that this enzyme exists as an octamer. Electron micrographs of negatively stained CK3 preparations show structures which are virtually identical to those that have been seen for octameric vertebrate MiCK. The above observations show that CK3 from C. variopedatus displays great similarities to MiCKs from vertebrates and echinoderms. Octamerization of CK is not an advanced feature. The evolution of octameric subunit association is ancient and occurred prior to the divergence of protostomes and deuterostomes.
Yeast putative transcription factors involved in salt tolerance
Authors: Mendizabal Iratxe , Rios Gabino , Mulet Jose M , Serrano Ramón , de Larrinoa Iñigo F
Published: July 2002

Four putative yeast transcription factors (Hal6–9p) have been identified which upon overexpression in multicopy plasmids increase sodium and lithium tolerance. This effect is mediated, at least in part, by increased expression of the Ena1p Na+/Li+ extrusion pump. Hal6p and Hal7p are bZIP proteins and their gene disruptions affected neither salt tolerance nor ENA1 expression. Hal8p and Hal9p are putative zinc fingers and their gene disruptions decreased both salt tolerance and ENA1 expression. Therefore, Hal8p and Hal9p, but not Hal6p and Hal7p, qualify as transcriptional activators of ENA1 under physiological conditions. Hal8p seems to mediate the calcineurin‐dependent part of ENA1 expression.
Structural plasticity of the feline leukaemia virus fusion peptide: a circular dichroism study
Authors: Davies Sarah M.A , Kelly Sharon M , Price Nicholas C , Bradshaw Jeremy P
Published: July 2002

The secondary structure of the feline leukaemia virus (FeLV) fusion peptide was investigated using circular dichroism (CD). Our results show that this peptide can readily flip between random, α‐helical and β‐sheet conformations, depending upon its environment. The CD spectrum changes from one characteristic of random coil to predominantly β‐sheet type, and finally to that showing the characteristics of α‐helical structure on moving from an aqueous solvent, through several increasingly hydrophobic systems, to a highly hydrophobic solvent. Electron microscopy confirmed the presence of β structure. We propose that the structural plasticity demonstrated here is crucial to the ability of the fusion peptide to perturb lipid bilayers, and thus promote membrane fusion.
Intercellular communication between vascular smooth muscle and endothelial cells mediated by heparin‐binding epidermal growth factor‐like growth factor and vascular endothelial growth factor
Authors: Abramovitch Rinat , Neeman Michal , Reich Reuven , Stein Ilan , Keshet Eli , Abraham Judith , Solomon Arie , Marikovsky Moshe
Published: July 2002

Heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF), a potent mitogen and migration factor for vascular smooth muscle cells (SMC), promoted neovascularization in vivo in the rabbit cornea. MRI demonstrated quantitatively the angiogenic effect of HB‐EGF when introduced subcutaneously into nude mice. HB‐EGF is not directly mitogenic to endothelial cells but it induced the migration of bovine endothelial cells and release of endothelial cell mitogenic activity from bovine vascular SMC. This mitogenic activity was specifically blocked by neutralizing anti‐vascular endothelial growth factor (VEGF) antibodies. In contrast, EGF or transforming growth factor‐α (TGF‐α) had almost no effect on release of endothelial mitogenicity from SMC. In addition, RT‐PCR analysis demonstrated that VEGF165 mRNA levels were increased in vascular SMC 4–10‐fold by 0.35–2 nM of HB‐EGF, respectively. Our data suggest that HB‐EGF, as a mediator of intercellular communication, may play a new important role in supporting wound healing, tumor progression and atherosclerosis by stimulating angiogenesis.
Identification and location on syndecan‐1 core protein of the epitopes of B‐B2 and B‐B4 monoclonal antibodies
Authors: Dore Jean-Michel , Morard Florence , Vita Natalio , Wijdenes John
Published: July 2002

Using a phage display peptide library, we characterized the epitope of two monoclonal antibodies reacting with syndecan‐1: B‐B2 and B‐B4. The identified epitopes QDIT, for B‐B2, and LPEV, for B‐B4, were found to align with residues 36–39 and 90–93 of the mature protein, respectively. In contrast to B‐B4, the B‐B2 epitope is close to a potential glycosaminoglycan attachment site. Since syndecan‐1 is heavily glycosylated and post‐translational modifications are cell type specific, these results might explain the differences observed in the reactivity pattern of B‐B2 and B‐B4 and suggest that these monoclonal antibodies are useful probes to study cell surface exposed syndecan‐1.
The mongoose acetylcholine receptor α‐subunit: analysis of glycosylation and α‐bungarotoxin binding
Authors: Asher Orna , Jensen Bo S , Lupu-Meiri Monica , Oron Yoram , Fuchs Sara
Published: July 2002

The mongoose AChR α‐subunit has been cloned and shown to be highly homologous to other AChR α‐subunits, with only six differences in amino acid residues at positions that are conserved in animal species that bind α‐bungarotoxin (α‐BTX). Four of these six substitutions cluster in the ligand binding site, and one of them, Asn‐187, forms a consensus N‐glycosylation site. The mongoose glycosylated α‐subunit has a higher apparent molecular mass than that of the rat glycosylated α‐subunit, probably resulting from the additional glycosylation at Asn‐187 of the mongoose subunit. The in vitro translated mongoose α‐subunit, in a glycosylated or non‐glycosylated form, does not bind α‐BTX, indicating that lack of α‐BTX binding can be achieved also in the absence of glycosylation.
The regulation of phosphorylation of τ in SY5Y neuroblastoma cells: the role of protein phosphatases
Authors: Tanaka Toshihisa , Zhong Jun , Iqbal Khalid , Trenkner Ekkhart , Grundke-Iqbal Inge
Published: July 2002

In Alzheimer disease brain the microtubule associated protein (MAP) τ is abnormally hyperphosphorylated. The role of protein phosphatases (PP) in the regulation of phosphorylation of τ was studied in undifferentiated SY5Y cells. In cells treated with 10 nM okadaic acid (OA), a PP‐2A/PP‐1 inhibitor, the PP‐1 and ‐2A activities decreased by 60% and 100% respectively and the activities of MAPKs, cdc2 kinase and cdk5, but not of GSK‐3, increased. OA increased the phosphorylation of τ at Thr‐231/Ser‐235 and Ser‐396/404, but not at Ser‐262/356 or Ser‐199/202. An increase in tyrosinated/detyrosinated tubulin ratio, a decrease in the microtubule binding activities of τ, MAP1b and MAP2, and cell death were observed. Treatment with 1 μm taxol partially inhibited the cell death. These data suggest (1) that OA induced hyperphosphorylation of τ is probably the result of activated MAPK and cdks in addition to decreased PP‐2A and PP‐1 activities and (2) that in SY5Y cells the OA induced cell death is associated with a decrease in stable microtubules.
Interaction between an intraluminal loop peptide of the inositol 1,4,5‐trisphosphate receptor and the near N‐terminal peptide of chromogranin A
Authors: Yoo Seung Hyun , Lewis Marc S
Published: July 2002

The near N‐terminal region of chromogranin A (CGA) has been shown to be the secretory vesicle membrane binding region, and tetrameric chromogranin A has been demonstrated to bind four molecules of an intraluminal loop peptide of the inositol 1,4,5‐trisphosphate (IP3) receptor. It was therefore necessary to determine whether the conserved near N‐terminal region of CGA interacts with the intraluminal loop region of the IP3 receptor. In the present study, we found that the proposed anchor region of CGA, the conserved near N‐terminal region, does indeed interact with the intraluminal loop region of the IP3 receptor at the intravesicular pH of 5.5, further strengthening the case for the potential interaction between tetrameric chromogranins and tetrameric IP3 receptors in the cell.
Cross‐linking and mutational analysis of the oligomerization state of the cytokine macrophage migration inhibitory factor (MIF)
Authors: Mischke Ralf , Kleemann Robert , Brunner Herwig , Bernhagen Jürgen
Published: July 2002

The structure of the cytokine MIF has been investigated by X‐ray crystallography, NMR, and biochemical methods with conflicting results regarding the structural and functional oligomerization state of this protein. Determination of the oligomeric state(s) is important for understanding more precisely the molecular mechanism of MIF action. To address this issue, we performed cross‐linking of human and mouse MIF and selected mutants by various methods and analyzed the oligomerization by SDS‐PAGE and gel filtration. MIF was found to form a mixture of monomeric, dimeric, and trimeric states at physiological concentrations, with the monomer and dimer representing the major species. Similar results were obtained when the carboxy‐truncated mutants MIF(1–104) and MIF(1–109) were examined, indicating that the C‐terminus of MIF is not critical for trimer stabilization. Cross‐linking analysis of the isosteric Cys→Ser mutants C56S and C80S of human MIF resulted in a similar oligomer distribution, whereas substitution of Cys59 led to a significant reduction in the dimeric and trimeric forms, indicating that the hydrophobic region around Cys59 is important for the oligomerization of MIF. Together, our data argue that physiological MIF solutions contain a mixture of monomers, dimers, and trimers.
Multi‐antennary Galβ1→4GlcNAc and Galβ1→3GalNAc clusters as important ligands for a lectin isolated from the sponge Geodia cydonium
Authors: Wu June H , Song Shuh-Chyung , Chen Yuen-Yuen , Tsai Ming-Chi , Kabat Elvin A , Wu Albert M
Published: July 2002

Based on the present and previous results, it is proposed that tri‐antennary Galβ1→4GlcNAc and Galβ1→3GalNAc clusters, in addition to GalNAcα1→3GalNAc and GalNAcα1→3Gal, are also important ligands for binding; and sialic acid of glycoprotein does interfere with binding.
Release of matrix proteins from mitochondria to cytosol during the prereplicative phase of liver regeneration
Authors: Greco M , Moro L , Pellecchia G , Di Pede S , Guerrieri F
Published: July 2002

70% partial hepatectomy (PH) in the rat causes a release, into the cytosolic fraction, of mitochondrial matrix proteins, namely the mitochondrial isoform of aspartate aminotransferase (mAAT) and malate dehydrogenase (MDH), during the first 24 h after PH, when no growth of the residual liver is observed. After this time interval, the weight of the liver starts to increase and the normal weight is reached at 96 h after PH. This proliferative phase is characterized by a progressive recovery of the normal levels of intramitochondrial activities of mAAT and MDH. Mitochondria isolated at 24 h after PH show a membrane permeabilization to sucrose accompanied by a release of matrix enzymes; both are blocked by cyclosporin A. These results suggest an alteration of mitochondrial membrane integrity, during the prereplicative phase of liver regeneration, with the occurrence of an increased permeability that allows the passage into the cytosol of matrix enzymes.
The interacting RNA polymerase II subunits, hRPB11 and hRPB3, are coordinately expressed in adult human tissues and down‐regulated by doxorubicin
Authors: Fanciulli Maurizio , Bruno Tiziana , Di Padova Monica , De Angelis Roberta , Lovari Sarah , Floridi Aristide , Passananti Claudio
Published: July 2002

We previously isolated the human RPB11 cDNA, encoding the 13.3 kDa subunit of RNA polymerase II, and demonstrated that expression of this subunit is modulated by doxorubicin. Using hRPB11 as bait in a yeast two‐hybrid system, two cDNA variants encoding a second RNA polymerase II subunit, hRPB3, have now been isolated and characterized. These two hRPB3 mRNA species differed in 3′ UTR region length, the longer transcript containing the AU‐rich sequence motif that mediates mRNA degradation. Both hRPB11 and hRPB3 transcripts share a similar pattern of distribution in human adult tissues, with particularly high levels in both heart and skeletal muscle, and the expression of both is down‐regulated by doxorubicin as found previously for the hRPB11 subunit. Taken together, these findings suggest that the interaction between hRPB3 and hRPB11 is fundamental for their function and that this heterodimer is involved in doxorubicin toxicity.
An intrinsically stable antibody scFv fragment can tolerate the loss of both disulfide bonds and fold correctly
Authors: Wörn Arne , Plückthun Andreas
Published: July 2002

A fully functional cysteine‐free derivative of the intrinsically stable anti‐HER2 scFv fragment hu4D5–8 was generated by replacing the disulfide forming cysteine residues inVHandVLwith the amino acid combination valine‐alanine in both domains. The antigen binding properties, determined by ELISA and BIAcore measurements, were not affected by removal of the disulfide bonds. The thermodynamic stability of the disulfide‐containing scFv of 8.1 kcal/mol is decreased upon complete reduction of both disulfides to 2.7 kcal/mol, while that of the valine‐alanine variant is somewhat higher (about 3.8 kcal/mol). Our results suggest that, in principle, a disulfide‐free fully functional derivative of any scFv can be obtained, as long as the corresponding disulfide‐containing scFv has a high enough thermodynamic stability.
Residues unique to the pro‐hormone convertase PC2 modulate its autoactivation, binding to 7B2 and enzymatic activity
Authors: Benjannet Suzanne , Mamarbachi Aida M , Hamelin Josée , Savaria Diane , Munzer Jon Scott , Chrétien Michel , Seidah Nabil G
Published: July 2002

The prohormone convertase PC2 is one of the major subtilisin/kexin‐like enzymes responsible for the formation of small bioactive peptides in neural and endocrine cells. This convertase is unique among the members of the subtilisin/kexin‐like mammalian serine proteinase family in that it undergoes zymogen processing of its inactive precursor proPC2 late along the secretory pathway and requires the help of a PC2‐specific binding protein known as 7B2. We hypothesized that some of these unique properties of PC2 are dictated by the presence of PC2‐specific amino acids, which in the six other known mammalian convertases are otherwise conserved but distinct. Accordingly, six sites were identified within the catalytic segment of PC2. Herein we report on the site‐directed mutagenesis of Tyr194 and of the oxyanion hole Asp309 and the consequences of such mutations on the cellular expression and enzyme activity of PC2. The data show that the Y194D mutation markedly increases the ex vivo ability of PC2 to process proopiomelanocortin (POMC) into β‐endorphin in cells devoid of 7B2, e.g. BSC40 cells. In these cells, expression of native PC2 does not result in the secretion of measurable in vitro activity against a pentapeptide fluorogenic substrate. In contrast, secreted Y194D‐PC2 exhibited significant enzymatic activity, even in the absence of 7B2. Based on co‐immunoprecipitations and Western blots, binding assays indicate that Tyr194 participates in the interaction of PC2 with 7B2, and that the oxyanion hole Asp309 is critical for the binding of proPC2 with pro7B2.
Gene‐encoded peptide antibiotics and innate immunity
Authors: Barra Donatella , Simmaco Maurizio , Boman Hans G
Published: July 2002

Gene‐encoded peptide antibiotics have been isolated from plants, animals and microbes. Their protective role has been related to innate immunity, which has gradually become accepted across the biomedical community. The evidence for the immune function of peptide antibiotics has been convincingly demonstrated by a combination of both in vitro and in vivo data for plants and insects, but for vertebrates in vivo data are scarce. Using frogs as model systems, it has been shown that the genes for antibacterial peptides are down‐regulated by glucocorticoids, while IκBα is clearly up‐regulated. Experimental infections with frog bacteria have shown that the normal capacity to control the natural flora is lost after treatment with glucocorticoids. A low‐specificity immune mechanism is cost‐effective, something that may have been of importance during animal evolution.
TSG‐6 interacts with hyaluronan and aggrecan in a pH‐dependent manner via a common functional element: implications for its regulation in inflamed cartilage
Authors: Parkar Ashfaq A , Kahmann Jan D , Howat Sarah L.T , Bayliss Michael T , Day Anthony J
Published: July 2002

Cartilage matrix is stabilised by the interactions of proteins with hyaluronan (HA). We compare the pH dependences of HA binding by aggrecan, link protein and TSG‐6. Aggrecan and link protein exhibit maximal binding across a wide pH range (6.0–8.0). TSG‐6, a protein that is only produced during inflammation, binds maximally at about pH 6.0 but shows a dramatic loss of function with increasing pH. TSG‐6 also interacts with aggrecan, with a similar pH dependence, and this can be inhibited by HA. Thus, a common binding surface on TSG‐6 may be involved in HA and aggrecan binding. We propose that TSG‐6 is involved in matrix dissociation and that this is regulated by pH gradients in cartilage.
Analysis of functional domains of Wilson disease protein (ATP7B) in Saccharomyces cerevisiae
Authors: Iida Masatake , Terada Kunihiko , Sambongi Yoshihiro , Wakabayashi Tokumitsu , Miura Naoyuki , Koyama Kenji , Futai Masamitsu , Sugiyama Toshihiro
Published: July 2002

Wilson disease is a genetic disorder of copper metabolism characterized by the toxic accumulation of copper in the liver. The ATP7B gene, which encodes a copper transporting P‐type ATPase, is defective in patients with Wilson disease. To investigate the function of ATP7B, wild type or mutated ATP7B cDNA was introduced into a yeast strain lacking the CCC2 gene (Δccc2), the yeast homologue of ATP7B. Wild type and the H1069Q mutant could rescue Δccc2, however, the N1270S mutant could not, reflecting phenotypic variability of Wilson disease. In addition, the mutant containing only the sixth copper binding domain could rescue Δccc2, indicating its functional importance.
On the origin of the `35‐μs kinetics' of P680+⋅ reduction in photosystem II with an intact water oxidising complex
Authors: Christen G , Reifarth F , Renger G
Published: July 2002

The origin of the `35‐μs kinetics' of P680+⋅ reduction in photosystem II (PS II) with an intact water oxidising complex has been analysed by comparative measurements of laser flash induced changes of the 830‐nm absorption and the relative quantum yield of chlorophyll (Chl) fluorescence. The latter parameter was monitored at a time resolution of 500 ns by using newly developed home built equipment [Reifarth, F., Christen, G. and Renger, G. (1997) Photosynth. Res. 51, 231–242]. It was found that: (i) the amplitudes of the unresolved ns‐kinetics of both 830‐nm absorption changes and the rise of fluorescence yield exhibit virtually the same period four oscillation pattern when dark adapted samples are excited with a train of saturating laser flashes; (ii) the corresponding oscillation patterns of the normalised extent of the 35‐μs kinetics under identical excitation conditions are strikingly different with maxima after the 3rd and 5th flash for the 830‐nm absorption changes vs. pronounced maxima after the 4th and 8th flash for the rise of the fluorescence yield. The period four oscillations unambiguously show that the `35‐μs kinetics' of P680+⋅ reduction are characteristic for reactions in PS II entities with an intact water oxidising complex. However, the disparity of the oscillation patterns of (ii) indicates that in contrast to the ns components of P680+⋅ reduction the 35‐μs kinetics do not reflect exclusively an electron transfer from YZ to P680+⋅. It is inferred that a more complex reaction takes place which comprises at least two processes: (a) P680+⋅ reduction by YZ and (b) coupled and/or competing reaction(s) which give rise to additional changes of the chlorophyll fluorescence yield.
Insights into the structure of hepatocyte growth factor/scatter factor (HGF/SF) and implications for receptor activation
Authors: Chirgadze Dimitri Y , Hepple Jonathan , Byrd R.Andrew , Sowdhamini R , Blundell Tom L , Gherardi Ermanno
Published: July 2002

The modular structure of HGF/SF offers a reductionist or `divide and rule' approach to the analysis of structure and function. Domain deletion experiments have established that the N domain, kringle 1 and kringle 2 are essential for HGF/SF activity and that truncated variants containing the N domain and kringle 1 (NK1) or kringles 1 and 2 (NK2) can exhibit partial agonistic or antagonistic activity depending on target cells. Comparative modelling has been used to predict the 3D structures of the six domains of HGF/SF. More recently, NMR methods have shown that the N domain has a novel fold, the charge distribution of which suggests a heparin binding site. Crystals of NK1 indicate the relationship of this domain to the kringle 1, offering further insights into the mechanism of domain interactions and receptor activation.
A calcium pump at the higher plant nuclear envelope?
Authors: Downie Louise , Priddle John , Hawes Chris , Evans David E
Published: July 2002

Evidence for a Ca2+‐pump at the nuclear envelope (NE) in plant cells has been obtained using confocal and electron microscope immunocytochemistry and antibodies raised to a plant homologue of the mammalian SERCA pump. This is the first evidence suggesting an NE Ca2+‐pump in plants. In addition to being localised with the NE in interphase, the antigen was localised to membrane derived from the NE and associated ER during mitosis, correlating with known Ca2+‐pools. The work suggests that a SERCA pump is present at the NE of plant as well as animal cells.
The structural basis for substrate recognition and control by protein kinases1
Authors: Johnson Louise N , Lowe Edward D , Noble Martin E.M , Owen David J
Published: July 2002

Protein kinases catalyse phospho transfer reactions from ATP to serine, threonine or tyrosine residues in target substrates and provide key mechanisms for control of cellular signalling processes. The crystal structures of 12 protein kinases are now known. These include structures of kinases in the active state in ternary complexes with ATP (or analogues) and inhibitor or peptide substrates (e.g. cyclic AMP dependent protein kinase, phosphorylase kinase and insulin receptor tyrosine kinase); kinases in both active and inactive states (e.g. CDK2/cyclin A, insulin receptor tyrosine kinase and MAPK); kinases in the active state (e.g. casein kinase 1, Lck); and kinases in inactive states (e.g. twitchin kinase, calcium calmodulin kinase 1, FGF receptor kinase, c‐Src and Hck). This paper summarises the detailed information obtained with active phosphorylase kinase ternary complex and reviews the results with reference to other kinase structures for insights into mechanisms for substrate recognition and control.
The complete genome of Bacillus subtilis: from sequence annotation to data management and analysis
Authors: Moszer Ivan
Published: July 2002

The completion of the entire 4.2‐Mb genome sequence of the Gram‐positive bacterium Bacillus subtilis has been a milestone for biological studies on this model organism. This paper describes bioinformatics work related to this joint European and Japanese project: methods and strategies for gene annotation and detection of sequencing errors, using an integrated cooperative computer environment (Imagene); construction of a specialized database for data management and a WWW server for data retrieval (SubtiList); DNA sequence analysis, yielding striking results on oligonucleotide bias, repeated sequences, and codon usage, all landmarks of evolutionary events shaping the B. subtilis genome.
A heat shock‐related protein, p20, plays an inhibitory role in platelet activation
Authors: Matsuno Hiroyuki , Kozawa Osamu , Niwa Masayuki , Usui Akihiko , Ito Hidenori , Uematsu Toshihiko , Kato Kanefusa
Published: July 2002

Some low molecular mass heat shock proteins (HSPs) appear to act as molecular chaperones, but their exact physiological roles have not been fully elucidated. We reported previously that a 20‐kDa protein (p20), which is classified as a low molecular mass HSP, is present at high levels in skeletal and smooth muscles. In the present study, we investigated a physiological role of p20 on platelet function in vitro and ex vivo. p20 inhibited platelet aggregation using human platelets dose‐dependently induced by botrocetin. On the other hand, HSP27, the other type of low molecular mass HSP, did not affect platelet aggregation. When p20 (300 μg/kg) was injected intravenously as a bolus in hamsters, platelet aggregation ex vivo induced by botrocetin was also significantly inhibited. In order to further investigate the inhibitory effect by p20 on platelet activation, we performed platelet aggregation induced by thrombin or ADP using human platelets. p20 markedly prevented platelet aggregation induced by thrombin, but not ADP. These findings suggest that p20 can act intercellularly to regulate platelet functions. Our results may provide the basis for a novel defensive system to thrombus formation.
AtPUMP: an Arabidopsis gene encoding a plant uncoupling mitochondrial protein
Authors: Maia Ivan G. , Benedetti Celso E. , Leite Adilson , Turcinelli Silvia R. , Vercesi Anibal E. , Arruda Paulo
Published: July 2002

A cDNA clone (AtPUMP) encoding a plant uncoupling mitochondrial protein was isolated from Arabidopsis thaliana. The cDNA contains an open reading frame of 921 nucleotides encoding 306 amino acids (predicted molecular weight 32 708). The predicted polypeptide is 81% identical and 89% similar to the potato UCP‐like protein, and includes an energy transfer protein motif common to mitochondrial transporters. The AtPUMP gene exists as a single copy in the Arabidopsis genome. The corresponding transcript was expressed in all tissues and was strongly induced by cold treatment. We suggest that the putative AtPUMP protein may play a role in heat‐requiring physiological events in Arabidopsis.
Valine substituted winter flounder `antifreeze': preservation of ice growth hysteresis
Authors: Haymet A.D.J , Ward Leanne G , Harding Margaret M , Knight Charles A
Published: July 2002

Three mutant polypeptides of the type I 37‐residue winter flounder `antifreeze' protein have been synthesized. All four threonine residues in the native peptide were been mutated to serine, valine and glycine respectively and two additional salt bridges were incorporated into the sequences in order to improve aqueous solubility. The peptides were analyzed by nanoliter osmometry, the `ice hemisphere' test, the `crystal habit' test, measurement of ice growth hysteresis and CD spectroscopy. While the valine and serine mutants retain the α‐helical structure, only the valine mutant retains `antifreeze' activity similar to that of the native protein. These data show that the threonine hydroxyl groups do not play a crucial role in the accumulation of the native `antifreeze' protein at the ice/water interface and the inhibition of ice growth below the equilibrium melting temperature.
Gulonolactone oxidase activity‐dependent intravesicular glutathione oxidation in rat liver microsomes
Authors: Puskás Ferenc , Braun László , Csala Miklós , Kardon Tamás , Marcolongo Paola , Benedetti Angelo , Mandl József , Bánhegyi Gábor
Published: July 2002

The orientation of gulonolactone oxidase activity was investigated in rat liver microsomes. Ascorbate formation upon gulonolactone addition resulted in higher intravesicular than extravesicular ascorbate concentrations in native microsomal vesicles. The intraluminal ascorbate accumulation could be prevented or the accumulated ascorbate could be released by permeabilising the vesicles with the pore‐forming alamethicin. The formation of the other product of the enzyme, hydrogen peroxide caused the preferential oxidation of intraluminal glutathione in glutathione‐loaded microsomes. In conclusion, these results suggest that the orientation of the active site of gulonolactone oxidase is intraluminal and/or the enzyme releases its products towards the lumen of the endoplasmic reticulum.
Decrease in mitochondrial energy coupling by thyroid hormones: a physiological effect rather than a pathological hyperthyroidism consequence
Authors: Bobyleva V , Pazienza T.L , Maseroli R , Tomasi A , Salvioli S , Cossarizza A , Franceschi C , Skulachev V.P
Published: July 2002

The effect of the in vivo thyroid status on mitochondrial membrane potential (ΔΨm) in isolated rat hepatocytes was studies by means of a cytofluorimetric technique and the ΔΨm‐specific probe JC‐1. It is shown that the ΔΨm level decreases in the order hypothyroid>euthyroid>hyperthyroid. Polarographic measurement of the hepatocyte respiratory rates revealed an opposite trend of values: the highest respiratory rate in hepatocytes from hyperthyroid animals, the lowest in those from hypothyroid ones. This means that mitochondrial energy coupling is highest in hypothyroid hepatocytes and lowest in hyperthyroid hepatocytes. 6‐Ketocholestanol added to hepatocytes failed to counterbalance the uncoupling effect of thyroid hormones on ΔΨm and respiration rate. Under the same conditions, 6‐ketocholestanol appeared to be effective in recoupling of respiration uncoupled by low concentrations of the artificial protonophore FCCP. The mechanism and possible physiological functions of the thyroid hormone‐induced decrease in mitochondrial energy coupling are discussed.
RNase P RNA from Prochlorococcus marinus: contribution of substrate domains to recognition by a cyanobacterial ribozyme
Authors: Hess Wolfgang R , Fingerhut Christiane , Schön Astrid
Published: July 2002

The molecular organisation of the Prochlorococcus marinus rnpB gene and the catalytic activity of the encoded RNA were characterised. Kinetic parameters for several pre‐tRNA substrates were comparable to those from other eubacterial RNase P RNAs, although unusually high cation concentrations were required. The CCA‐end of pre‐tRNAs is essential for efficient turnover despite the lack of the canonical binding motif in P. marinus RNase P RNA. A trnR gene is located only 38 nt upstream the rnpB 5′ end on the complementary strand. This arrangement resembles those in the plastids of Cyanophora and Porphyra but not in any other bacterium.