bluuebunny/crossref_metadata_2025 huggingface dataset:
Efficient CD4 binding and immunosuppressive properties of the 13B8.2 monoclonal antibody are displayed by its CDR‐H1‐derived peptide CB11
Authors: Bès Cédric , Briant-Longuet Laurence , Cerruti Martine , DeBerardinis Piergiuseppe , Devauchelle Gérard , Devaux Christian , Granier Claude , Chardès Thierry
Published: July 2002

A systematic exploration of the VH2/Vκ12–13 variable domains of the anti‐CD4 monoclonal antibody (mAb) 13B8.2 was performed by the Spot method to screen for paratope‐derived peptides (PDPs) demonstrating CD4 binding ability. Nine peptides, named CB1 to CB9, were identified, synthesized in a cyclic and soluble form and tested for binding to recombinant soluble CD4. Among them, CB1, CB2 and CB8 showed high anti‐CD4 activity. Competition studies for CD4 binding indicated that PDPs CB1, CB8, and the parental mAb 13B8.2 recognized the same complementarity determining region (CDR)3‐like loop region. PDP CB1 was shown to mimic the biological properties of 13B8.2 mAb in two independent cellular assays, demonstrating inhibitory activities in the micromolar range on antigen presentation and human immunodeficiency virus promoter activation. Our results indicate that the bioactive CDR‐H1 PDP CB1 has retained a significant part of the parental 13B8.2 mAb properties and might be a lead for the design of anti‐CD4 peptidomimetics of clinical interest.
Unusual pKa of the carboxylate at the putative catalytic position of the thermophilic F1‐ATPase β subunit determined by 13C‐NMR
Authors: Tozawa Kaeko , Ohbuchi Hiroshi , Yagi Hiromasa , Amano Toyoki , Matsui Tadashi , Yoshida Masasuke , Akutsu Hideo
Published: July 2002

Glutamic acid‐190 in the β subunit of F1‐ATPase from thermophilic Bacillus PS‐3 (TF1) was reporte to be essential for the ATPase activity. The mutant TF1β subunit in which Glu‐190 had been substituted by cysteine was carboxymethylated with 13C‐labeled monoiodoacetic acid. The pK
a value of the carboxymethylene group at the 190 position was determined as 5.6 ± 0.4 by 13C‐NMR. On the basis of this value, the pK
a of the carboxylate of Glu‐190 of the TF1β subunit was estimated to be 6.8 ± 0.5. The unusually high pK
a could play a role in the catalytic mechanism of F1‐ATPase.
Formation of bacteriophage MS2 infectious units in a cell‐free translation system
Authors: L. Katanaev Vladimir , S. Spirin Alexander , Reuss Matthias , Siemann Martin
Published: July 2002

We show that a simple cell‐free translation system from Escherichia coli, programmed with phage MS2 RNA, is able to infect F+ E. coli cells. The plaques appearing on the E. coli host strain are morphologically indistinguishable from those derived from normal phage MS2 infection. This effect is strictly translation‐dependent, since an incomplete translation system or the system inhibited by antibiotics leads to no infection. The cellfree based infection is maximal under conditions favouring the highest synthesis of maturation protein (one of the four phage‐encoded proteins). The infection is abolished when RNase A or trypsin treatment is included before addition of cells. Similarly, due to RNA and maturation protein degradation, the continued incubation of the translation mixture under protein synthesis conditions significantly decreases infectivity. These findings suggest the formation of ‘minimal infectious units’, simple complexes of MS2 RNA and maturation protein. Here we describe the first example of bacteriophage infectious unit formation directly performed in a cell‐free translation system. A possible application of this phenomenon might be the construction of newly designed RNA vector delivery systems and, moreover, could be an approach for molecular evolution studies.
Occurrence of an acyl‐CoA:1‐acylglycerophosphorylcholine acyltransferase in plant mitochondria
Authors: Testet Eric , Bessoule Jean-Jacques , Mongrand Sébastien , Guillot-Salomon Thérèse , Cantrel Catherine , Cassagne Claude
Published: July 2002

In the presence of oleoyl‐CoA, purified and intact mitochondria from potato tuber formed phosphatidylcholine from labeled lysophosphatidylcholine. The labeled oleoyl moiety of the acyl‐CoA was also incorporated in the absence of exogenous lysolipids, such incorporation being largely increased by the addition of exogenous lysophosphatidylcholine. In the presence of various other lysophospholipids, no synthesis of the corresponding phospholipids was observed, suggesting a high specificity of the acyltransferase towards the acyl acceptor. This enzyme was chiefly located in the outer membrane of mitochondria. These results indicate that any acylglycerophosphorylcholine transferred from the endoplasmic reticulum to mitochondria may be acylated to phosphatidylcholine.
Diacylglycerol mediates the thrombin‐induced, protein kinase C and Ca2+ independent activation of the Na+/H+ exchanger in platelets
Authors: Cavallini Lucia , Coassin Mariagrazia , Alexandre Adolfo
Published: July 2002

Treatment of aspirinated platelets with the electroneutral K+/H+ exchanger nigericin induces a decrease in intraplatelet pH as measured with the intracellular fluorescent indicator BCECF. Under these conditions, the proton permeability of the plasma membrane is unaffected. The addition of thrombin induces a rapid partial recovery of pHi, which is completely abolished by the Na+/H+ exchanger inhibitor NHA. The effect is also evident in the presence of the PKC inhibitors GF 109203X or staurosporine and in the absence of both external (EGTA‐chelated) and internal (BAPTA‐chelated) Ca2+. This makes the thrombin‐induced activation of the exchanger independent of the involvement of the hitherto described activators, namely PKC and the increase in [Ca2+]i, as well of the recently reported activator arachidonic acid [Cavallini, L., Coassin, M., Borean, A., and Alexandre, A. (1996) Biochem. J. 319, 567–574], whose production requires a high [Ca2+]i. The thrombin‐dependent recovery of pHi is prevented by the phospholipase C inhibitor ET 18 O‐CH3 and is mimicked by the addition of the permeable diglyceride dioctanoyl glycerol (DiC8) exogenously supplied. The effect of thrombin and DiC8 is unaffected by inhibition of diacylglycerol lipase and diacylglycerol kinase. These experiments identify diglyceride as a novel activator of the Na+/H+ exchanger in platelets.
Yeast RAS2 mutations modulating the ras‐guanine exchange factor interaction
Authors: Hermann-Le Denmat Sylvie , Jacquet Michel
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Distinct distal and proximal p53‐binding sites in the MCK promoter govern the transcriptional response to p53
Authors: Jackson Paul , Yardley Gina
Published: July 2002

We have investigated the functional importance of nucleotide sequence differences between proximal (P‐) and distal (D‐) p53‐binding elements in the MCK promoter. P‐ and D‐elements normally co‐operate to permit synergistic promoter activation by p53. Interestingly, we find that P‐elements cannot co‐operate with each other. In contrast, co‐operation between D‐binding sites results in levels of p53‐induced transcription far higher than those obtained by co‐operation between P‐ and D‐elements. These studies imply that distinct D‐ and P‐p53‐binding sites in the MCK promoter may dictate the promoter response to p53.
Reversible inhibition of sheep liver sorbitol dehydrogenase by the antidiabetogenic drug 2‐hydroxymethyl‐4‐(4‐N,N‐dimethylaminosulfonyl‐1‐piperazino) pyrimidine
Authors: Lindstad Rune I. , McKinley-McKee John S.
Published: July 2002

The mechanism of the inhibition of sheep liver sorbitol dehydrogenase by the novel antidiabetogenic drug 2‐hydroxymethyl‐4‐(4‐N,N‐dimethylaminosulfonyl‐1‐piperazino)pyrimidine has been investigated by steady‐state kinetics over the range pH 5–10. The pyrimidine derivative exhibits mixed inhibition with respect to sorbitol, fructose and coenzyme, due to the formation of enzyme‐inhibitor and enzyme‐NAD(H)‐inhibitor complexes. The formation of each of the binary and ternary complexes is inhibited by protonation and deprotonation of groups which, in the enzyme‐inhibitor complex, have pK values of 6.6 and 8.0, respectively.
Induction of glutathione synthetase by 1,10‐phenanthroline
Authors: Sun Yi
Published: July 2002

The differential display (DD) was employed to identify the gene(s) responsible for 1,10‐phenanthroline (OP)‐induced apoptosis in murine tumor cells (Sun, Y., Bian, J., Wang, Y. and Jacobs, C. (1997) Oncogene 14, 385–393 [1]). An OP‐inducible gene was isolated which encodes mouse glutathione synthetase (GSS). The GSS mRNA level began to increase 6 h post OP treatment and remained at a high level thereafter up to 24 h tested. Induction of GSS was found not to be associated with p53 activation. No significant induction of DNA fragmentation was detected in two murine tumor lines upon GSS transfection. This is the first observation indicating that GSS is inducible rather specifically by a metal chelator and that induction of GSS, however, is not sufficient to induce apoptosis. It may merely reflect a cellular response to OP‐induced redox disturbance.
Thermodynamic characterization of the binding of dCMP to the Asn229Asp mutant of thymidylate synthase
Authors: Téllez-Sanz Ramiro , Bernier-Villamor Vı́ctor , Garcı́a-Fuentes Luis , González-Pacanowska Dolores , Barón Carmen
Published: July 2002

Isothermal titration microcalorimetry and equilibrium dialysis have been used to characterize the binding of 2′‐deoxycytidine 5′‐monophosphate (dCMP) to the Asn229Asp mutant of Lactobacillus casei recombinant thymidylate synthase at pH 7.4 over a temperature range of 15°C to 35°C. Equilibrium dialysis analysis shows that dCMP binds to two sites in the dimer of both wild‐type and mutant thymidylate synthase. A concomitant net uptake of protons with binding of dCMP to both enzymes, was detected carrying out calorimetric experiments in various buffer systems with different heats of ionization. The change in protonation for binding of dCMP to wild‐type enzyme is lower than that obtained for binding of this nucleotide to TS N229D, which suggests that the pK value of Asp‐229 is increased upon dCMP binding to the mutant enzyme. At 25°C, although the binding of dCMP to wild‐type and N229D TS is favoured by both enthalpy and entropy changes, the enthalpy change is more negative for the mutant protein. Thus, the substitution of Asn 229 for Asp results in a higher affinity of TS for dCMP due to a more favourable enthalpic contribution. The Gibbs energy change of binding of dCMP to the mutant enzyme is weakly temperature‐dependent, because of the enthalpy‐entropy compensation arising from a negative heat capacity change of binding equal to −0.83±0.02 kJ K−1 per mol of dCMP bound.
Identification of a biological inactive complex form of pokeweed antiviral protein
Authors: Desvoyes Bénédicte , Poyet Jean-Luc , Schlick Jean-Luc , Adami Pascale , Jouvenot Michèle , Dulieu Philippe
Published: July 2002

Pokeweed antiviral protein (PAP) inactivates both eukaryotic and prokaryotic ribosomes via a specific depurination of rRNA. The sensitivity of pokeweed ribosomes to PAP implies the existence of a mechanism to protect the plant. Using monoclonal antibodies specific to PAP, a protein complex (PAPi) which contained PAP was identified in leaf extract. In this complex, the enzymatic activity of the toxin was strongly inhibited. This protein complex had a pI lower than that of PAP and was separated from free PAP by a preparative native gel electrophoresis. PAPi had an apparent molecular mass of 57 kDa and was dissociated by heating for 5 min at 80°C or by treatment by alkaline or acidic pH or by 7 M urea. The other components involved in the complex remain unknown.
A simple method for determining kinetic constants of complexing inactivation at identical enzyme and inhibitor concentrations
Authors: Wang Ming-Hua , Zhao Kang-Yuan
Published: July 2002

The kinetics of complexing inactivation at identical enzyme and inhibitor concentrations were analyzed and the equations of product generation were derived when the free enzyme concentration is great, larger or smaller than the dissociation constant of inhibitor, K
I. The kinetic constants can be obtained by fitting the derived equations to the progress curve. Numerical examples show that this method is valid and gives satisfactory results.
Differential expression of mRNAs for endopeptidases in phenotypically modulated (`dedifferentiated') human articular chondrocytes
Authors: Kostoulas Georgios , Lang Angela , Trueb Beat , Baici Antonio
Published: July 2002

Human articular chondrocytes modulated away from their original phenotype by serial subcultures in monolayer differentially express mRNAs for endopeptidases. The mRNAs for the cathepsins B and L are extremely low in differentiated cells, but are soon expressed in parallel with the loss of the differentiated state. In contrast, the mRNA for collagenase‐1 is strongly expressed by differentiated chondrocytes and declines rapidly following phenotypic modulation. The mRNA for stromelysin‐1 and the tissue inhibitor of metalloproteinases‐2 is high and does not appreciably change after modulation. Chondrocyte activation induced by alteration of its original phenotype leads to the expression of endopeptidases in a way that markedly differs from that induced by cytokines. The results are relevant to cartilage catabolism in osteoarthritis and suggest a prominent role of fibroblastic metaplasia on the part of the chondrocytes as a mechanism of expressing catabolic endopeptidases.
Proteolytic processing of presenilin‐1 (PS‐1) is not associated with Alzheimer's disease with or without PS‐1 mutations
Authors: Okochi Masayasu , Ishii Kazuhiko , Usami Mihoko , Sahara Naruhiko , Kametani Fuyuki , Tanaka Kikuko , Fraser Peter E , Ikeda Masaki , Saunders Ann M , Hendriks Lydia , Shoji Shin-Ichi , Nee Linda E , Martin Jean-Jacques , Van Broeckhoven Christine , St. George-Hyslop Peter H , Roses Allen D , Mori Hiroshi
Published: July 2002

Cerebral presenilin‐1 protein (PS‐1) is normally composed of the amino‐terminal fragment (NTF) with M
r 28 kDa and the carboxy‐terminal fragment (CTF) with 18 kDa. We analyzed human PS‐1 in brains with early‐onset familial Alzheimer's disease (FAD) with and without PS‐1 mutations to study whether mutated PS‐1 was abnormally metabolized. Cerebral PS‐1 were found to be cleaved into two fragments of NTF and CTF independently of the occurrence of PS‐1 mutation in human brains. A small portion of PS‐1 was recently found to suffer another processing by caspase‐3, an apoptosis‐related cysteine protease. In contrast to the recent finding that the Volga‐German mutation on presenilin‐2 (PS‐2) affects the increasing caspase‐3 PS‐2 fragment, the PS‐1 mutation did not cause a significant change in PS‐1 fragmentation. We conclude that PS‐1 fragmentation and other (probably caspase‐3‐mediated) digestion following apoptosis occur independently of PS‐1 mutations.
Detection and cloning of unique integration sites of retrotransposon, intracisternal A‐particle element in the genome of acute myeloid leukemia cells in mice1
Authors: Ishihara H , Tanaka I
Published: July 2002

We previously found retrotransposition of the intracisternal A‐particle (IAP) element in the genome of acute myeloid leukemia (AML) cells induced by X‐irradiation of C3H/He mice (FEBS 16333). To analyze the occurrence of the IAP‐mediated retrotransposition in AML cells, we compared integration sites of the IAP element by polymerase chain reaction (PCR) in the genomes of five AML strains derived from different C3H mice. Unique PCR products were found in all of the above independent leukemia cells, whereas no such products were detected in normal cells. Results of cloning, sequencing and Southern analyses showed that the PCR products were derived from novel integration sites of the IAP element in the genome. The data suggest that IAP‐mediated retrotransposition occurs frequently in radiation‐induced AML cells from C3H/He mice.
N‐tail translocation of mature β‐lactamase across the Escherichia coli cytoplasmic membrane
Authors: Mitsopoulos Costas , Hashemzadeh-Bonehi Lida , Broome-Smith Jenny K
Published: July 2002

Mature β‐lactamase was attached to the N‐terminus of human glycophorin C, an N‐out membrane protein lacking a cleavable signal peptide (an N‐tail membrane protein). When synthesised in Escherichia coli more than 30% of the intact mature β‐lactamase‐glycophorin C molecules assembled N‐out, C‐in into the cytoplasmic membrane. The N‐tail translocated β‐lactamase folded into an enzymatically active form, but it was more susceptible to proteolysis than the equivalent portion of β‐lactamase‐glycophorin C synthesised with an N‐terminal signal peptide. Its translocation was virtually abolished when the N‐out domain of glycophorin C was truncated or when the basic residues C‐terminally flanking the glycophorin C membrane‐spanning segment were replaced with neutral ones.
Action of acetylxylan esterase from Trichoderma reesei on acetylated methyl glycosides
Authors: Biely P , Côté G.L , Kremnický L , Greene R.V , Tenkanen M
Published: July 2002

Substrate specificity of purified acetylxylan esterase (AcXE) from Trichoderma reesei was investigated on partially and fully acetylated methyl glycopyranosides. Methyl 2,3,4‐tri‐O‐acetyl‐β‐d‐xylopyranoside was deacetylated at positions 2 and 3, yielding methyl 4‐O‐acetyl‐β‐d‐xylopyranoside in almost 90% yield. Methyl 2,3‐di‐O‐acetyl β‐d‐xylopyranoside was deacetylated at a rate similar to the fully acetylated derivative. The other two diacetates (2,4‐ and 3,4‐), which have a free hydroxyl group at either position 3 or 2, were deacetylated one order of magnitude more rapidly. Thus the second acetyl group is rapidly released from position 3 or 2 after the first acetyl group is removed from position 2 or 3. The results strongly imply that in degradation of partially acetylated β‐1,4‐linked xylans, the enzyme deacetylates monoacetylated xylopyranosyl residues more readily than di‐O‐acetylated residues. The T. reesei AcXE attacked acetylated methyl β‐d‐glucopyranosides and β‐d‐mannopyranosides in a manner similar to the xylopyranosides.
Modifications of proteoglycans extracted from monolayer cultures of young and senescent human skin fibroblasts
Authors: Passi Alberto , Albertini Riccardo , Campagnari Francesco , De Luca Giancarlo
Published: July 2002

Proteoglycans (PGs) were extracted from culture monolayers of human skin fibroblasts (HFs) at early and late passages. Total PGs from senescent cells had markedly reduced abilities to bind type I collagen and hyaluronic acid, but retained normal binding properties with fibronectin and laminin. The constituent polysaccharides of PGs were comparatively characterised. PGs recovered from young and senescent HF cultures had equivalent total polyanionic charges and similar size distributions of the glycosaminoglycan chains. This applied to both types of polysaccharide chains found in PGs, namely the galactosaminoglycuronans (GalN‐GAGs) and the glucosaminoglycuronans (GlcN‐GAGs). However, senescent HFs produced a greater proportion of PGs containing GlcN‐GAG chains and increased the sulphation of the remainding PG fraction with GalN‐GAG moieties, yielding a major gain of C6‐sulphate groups in the galactosamine residues.
Calcium binding to the first EGF‐like module of human factor IX in a recombinant fragment containing residues 1–85
Authors: Persson Kristina E.M , Astermark Jan , Björk Ingemar , Stenflo Johan
Published: July 2002

The first EGF‐like module of human coagulation factor IX contains a single functionally important calcium ion binding site. We have now shown the dissociation constant for this site to be approximately 160 μM in a recombinant protein fragment consisting of residues 1–85 in human fIX. This represents a ≈10‐fold increase in affinity as compared with the isolated EGF module (residues 46–85). The Gla module (here with Glu instead of Gla) thus increases the affinity of the EGF module calcium ion binding site. Each of two mutations, V46E and Q50E, made to investigate whether the extra negative charge would increase the affinity of the calcium binding site manifested a negligible increase in affinity.
Hydrogen peroxide is not released following reaction of cyanide with several catalytically important derivatives of cytochrome c oxidase
Authors: Fabian Marian , Palmer Graham
Published: July 2002

We have looked for the production of hydrogen peroxide following reaction of oxidized cytochrome c oxidase and two oxy derivatives (compounds P and F) with cyanide. In each case the final product was the cyanide adduct of cytochrome c oxidase. In no case release of hydrogen peroxide was detected, as gauged by the scopoletin plus horse radish peroxidase assay. The simplest conclusion is that none of these forms of the enzyme contains intact hydrogen peroxide.
Functional heterogeneity of transducin α subunits
Authors: Neubert Thomas A , Hurley James B
Published: July 2002

The N‐terminal glycine of transducin α subunits is acylated by lauroyl (C12:0), myristoyl (C14:0), (cis‐Δ5)‐tetradecaenoyl (C14:1) or (cis,cis‐Δ5,Δ8)‐tetradecadienoyl (C14:2) fatty acyl groups. We examined functional heterogeneity of transducin by sequentially eluting it from bleached outer segments using increasing concentrations of GTP then identifying the N‐terminal acyl groups on the eluted α subunits. C14:2 acylated transducin eluted at low GTP concentrations followed by C12:0, C14:1 and C14:0 transducin at higher GTP concentrations. This suggests functional heterogeneity in the different forms of transducin α subunits.
Overexpression, purification, crystallization and preliminary X‐ray diffraction analysis of the pMV158‐encoded plasmid transcriptional repressor protein CopG
Authors: Gomis-Rüth F.Xavier , Solà Marı́a , Pérez-Luque Rosa , Acebo Paloma , Alda M.Teresa , González Ana , Espinosa Manuel , del Solar Gloria , Coll Miquel
Published: July 2002

Plasmid pMV158 encodes a 45 amino acid transcriptional repressor, CopG, which is involved in copy number control. A new procedure for overproduction and purification of the protein has been developed. The CopG protein thus obtained retained its ability to specifically bind to DNA and to repress its own promoter. Purified CopG protein has been crystallized using the sitting‐drop vapor diffusion method. The crystals, belonging to orthorhombic space group C2221 (cell constants a=67.2 Å, b=102.5 Å, c=40.2 Å), were obtained from a solution containing methylpentanediol, benzamidine and sodium chloride, buffered to pH 6.7. Complete diffraction data up to 1.6 Å resolution have been collected. Considerations about the Matthews parameter account for the most likely presence of three molecules in the asymmetric unit (2.27 Å3/Da).
Minimal tRNASer and tRNASec substrates for human seryl‐tRNA synthetase: contribution of tRNA domains to serylation and tertiary structure
Authors: Heckl Martha , Busch Kristina , Gross Hans J
Published: July 2002

The recognition process of tRNASer and tRNASec by human seryl‐tRNA synthetase (SerRS) was studied using T7 transcripts representing defined regions of human tRNASer or tRNASec and the influence of the tRNA elements on serylation and tertiary structure was elucidated. The anticodon arms of both tRNAs showed no contribution to serylation in contrast to the acceptor stems and the long extra arms. D and T arms were only involved in formation of the L‐shaped tRNA structure, not in the recognition process between tRNAs and SerRS. This is the first report of microhelices adapted from human tRNAs being aminoacylated by their homologous synthetase.
Structural and dynamic helix geometry alterations induced by mismatch base pairs in double‐helical RNA
Authors: Vogtherr Martin , Schübel Harald , Limmer Stefan
Published: July 2002

A ribooligonucleotide microhelix derived from the acceptor stem of Escherichia coli tRNAAla having a C3‐A70 mismatch in place of the G3‐U70 wobble pair in the wild‐type tRNAAla, and a sequence variant with a regular U3‐A70 base pair have been investigated by NMR. In vivo, suppressor tRNAAla variants with C3‐A70 (as well as several other) mismatch pairs are substrates for alanyl‐tRNA synthetase (ARS), supporting the hypothesis of an `indirect' recognition of the identity element 3‐70 mismatch pair via structural modifications caused by the mispair in comparison to canonical A‐RNA helices. It is demonstrated that the C‐A mismatch likewise induces helix geometry alterations, in particular with respect to base stacking in the vicinity of the mismatch. However, with reference to the `wild‐type' G3‐U70 microhelix, destacking in the C3‐A70 acceptor stem duplex occurs in the opposite direction from the mismatch pair. Therefore it is concluded that the locally enhanced conformational flexibility or dynamics associated with the structural changes induced by the mismatch pairs could be an essential prerequisite for optimal adaptation of the tRNAAla acceptor stem to the contact region of the ARS.
Regulation of the type II oncostatin M receptor expression in lung‐derived epithelial cells
Authors: Cichy Joanna , Rose-John Stefan , Puré Ellen
Published: July 2002

Oncostatin M (OSM) is a potent modulator of human lung‐derived epithelial cell function. This cytokine binds two distinct receptor complexes: type I OSM receptor which is also a functional receptor for leukemia inhibitory factor (LIF), and type II OSM‐specific receptor. The role of these two distinct receptors in mediating the response of individual cell types to OSM has not been delineated. In contrast to LIF, OSM induces synthesis of α1‐antichymotrypsin and α1‐antiproteinase inhibitor in lung‐derived epithelial cells. The differential responsiveness to LIF and OSM suggested that the response of lung epithelial cells to OSM may be mediated by the OSM‐specific receptor. Therefore, we characterized lung‐derived epithelial cells for the expression of type II OSM receptor mRNAs, and the regulation of the mRNAs encoding the components of the OSM‐specific receptor by cytokines and dexamethasone.
Src phosphorylates EAST, a novel EGF receptor‐associated protein
Authors: Lohi Olli , Lehto Veli-Pekka
Published: July 2002

We have recently found and characterized EAST, a novel EGF receptor‐associated protein with SH3 and TAM domains. In this study we show that EAST becomes phosphorylated by Src kinase. This, in conjunction with our earlier observations on the close association between EAST and the endocytic machinery, suggests that EAST could be involved in Src‐dependent effects on EGF receptor endocytosis.
The distance between the 3′‐pyrimidine‐rich tract and the AUG codon modulates internal initiation of translation of hepatitis A virus RNA
Authors: Silveira Carneiro Jane , Bucci M , Equestre M , Pagnotti P , Pierangeli Alessandra , Pérez Bercoff R
Published: July 2002

Protein synthesis directed by hepatitis A virus (HAV) RNA is mediated by a mechanism involving the recognition of internal sequences. Two in‐frame AUG codons initiate the long open reading frame (positions 734–736 and 740–742). The extra‐cistronic region extending between the uncapped 5′‐end and the ORF contains two pyrimidine‐rich tracts (PRTs): one 12 nucleotides in length in the close vicinity of the initiator AUG, and a longer one between bases 94 and 140. In order to study the relative contribution of these elements to the process of internal initiation of translation, cDNA representations of the 5′‐terminal extra‐cistronic region of HAV RNA were inserted in the intergenic region of the bi‐cistronic plasmid pSV‐GH/CAT, between the genes encoding the human growth hormone (GH) and the bacterial enzyme chloramphenicol acetyltransferase (CAT), and following transfection of COS‐1 cells, the transient expression of both genes was quantified. The importance of the 3′‐PRT appeared to be strongly influenced by the length of the ‘spacer’ sequence extending between this structure and the translation initiation site: placed 45 nucleotides upstream from the initiator codon of a reporter gene, its integrity was stringently required for initiation to occur. Bringing the length of the ‘spacer’ back to its actual size in HAV RNA (i.e. 11 or 17 nt) reduced considerably the overall rate of internal initiation of translation, and the relative contribution to this process of the 3′‐PRT became marginal. Concomitantly, the importance of the functional domains previously identified in the 5′‐PRT fluctuated: while integrity of domain 100–106 was always stringently required for initiation to occur, the activity of domain 113–118 paralleled that of the 3′‐PRT, and the opposite applied to domain 121–126, whose contribution became relevant only after switching off the 3′‐PRT. Systematic mutations introduced in the ‘spacer’ sequences suggest that the length of this region may be responsible for the down regulation of translation of HAV RNA and, possibly, for its lengthy replication cycle.
Expression of a p16INK4a‐specific ribozyme downmodulates p16INK4a abundance and accelerates cell proliferation
Authors: Nylandsted Jesper , Rohde Mikkel , Bartek Jiri , Strauss Michael
Published: July 2002

The p16INK4a tumor suppressor negatively regulates progression through the G1 phase of the mammalian cell cycle. To mimic the downmodulation of p16INK4a commonly seen in cancer, we designed and characterized a hammerhead ribozyme against exon E1α of the murine p16INK4a transcript. Stable expression of the ribozyme in murine erythroleukemia (MEL) cells reduced the endogenous p16INK4a protein by more than 70% and significantly accelerated cell cycle progression. The specificity and efficiency of our new ribozyme suggest its possible application in elucidating the role of p16INK4a in fundamental biological processes including homeostatic tissue renewal, protection against oncogenic transformation, and cellular senescence.
Loss of activation of Gs but not Gi following expression of an α2A‐adrenoceptor‐Gi1α fusion protein
Authors: Sautel Martine , Milligan Graeme
Published: July 2002

Both the porcine α2A‐adrenoceptor and a fusion protein between this receptor and a pertussis toxin‐resistant form of Gi1α were stably expressed in Rat‐1 fibroblasts. The agonist UK14304 mediated a biphasic regulation of adenylyl cyclase activity via the isolated receptor with inhibition of the enzyme activity at low concentrations of the compound which was subsequently reversed at higher concentrations. By contrast, stimulation of the fusion protein with this agonist could only produce inhibition of enzyme activity. This inhibition was produced by activation of endogenous Gi rather than the fused α subunit of Gi1, as pertussis toxin treatment obliterated inhibitory regulation of adenylyl cyclase via the fusion construct. Pertussis toxin treatment potentiated stimulation of adenylyl cyclase via the isolated receptor but such treatment was unable to uncover capacity of the fusion protein to produce such an effect.
Impaired glucose sensing by intrahepatic, muscarinic nerves for an insulin‐stimulated hepatic glucose uptake in streptozotocin‐diabetic rats
Authors: Stümpel Frank , Scholtka Bettina , Jungermann Kurt
Published: July 2002

Insulin‐induced net hepatic glucose uptake depends on the sensing by muscarinic, intrahepatic nerves of a glucose concentration gradient between portal vein and hepatic artery. The function of these intrahepatic nerves was examined in streptozotocin‐diabetic rats. In the presence of the glucose gradient insulin induced net glucose uptake in isolated perfused livers from control and acutely diabetic but not from chronically diabetic animals. The neurotransmitter acetylcholine still mimicked the existence of the gradient, excluding a metabolic impairment of livers of chronically diabetic animals. The impairment of the intrahepatic nerves due to diabetic neuropathy could contribute to postprandial hyperglycemia in diabetes mellitus.
A novel photointermediate of octopus rhodopsin activates its G‐protein
Authors: Nakagawa Masashi , Kikkawa Satoshi , Tominaga Kayo , Tsugi Naoko , Tsuda Motoyuki
Published: July 2002

The photointermediate of octopus rhodopsin responsible for G‐protein activation was examined by a GTPγS‐binding assay in a reconstituted system with purified rhodopsin and photoreceptor G‐protein. When octopus rhodopsin alone was incubated in the dark after illumination, its ability to stimulate GTPγS‐binding by the G‐protein decreased in a time‐dependent manner. We associate this decay with the decay of a novel photointermediate, transient acid metarhodopsin, which lies between mesorhodopsin and acid metarhodopsin. Spectroscopic evidence for its existence was suggested by its effects on the turbidity of the vesicles. These results suggest that the transient acid metarhodopsin, not the stable final photoproduct, acid metarhodopsin, activates a G‐protein in octopus photoreceptors.
Identification of a Torpedo homolog of Sam68 that interacts with the synapse organizing protein rapsyn
Authors: Fung Eric T. , Lanahan Anthony , Worley Paul , Huganir Richard L.
Published: July 2002

Nicotinic acetylcholine receptors (nAChRs) are initially expressed diffusely on the surface of myotubes and, in response to neuronally derived factors, cluster at the endplate to a final concentration of approximately 10 000/μm2. The synaptic peripheral membrane protein rapsyn has been shown to mediate clustering of nAChRs in several systems. Here we describe the use of the yeast two‐hybrid system to identify proteins that can interact with rapsyn. One of the clones we have identified is a Torpedo californica homolog of the Src‐associated in mitosis protein (Sam68). We further show that Sam68, like rapsyn, is localized at the neuromuscular junction.
Developmental changes of gene expression in heme metabolic enzymes in rat placenta
Authors: Ihara Naomi , Akagi Reiko , Ejiri Kohei , Kudo Takafumi , Furuyama Kazumichi , Fujita Hiroyoshi
Published: July 2002

Transcription levels of the non‐specific δ‐aminolevulinate synthase (ALAS‐N) and heme oxygenase‐1 (HO‐1) in the placenta at the terminal stage of pregnancy were comparable to those in the female adult liver and in the spleen, respectively. Immunohistochemical studies demonstrated that both enzymes were exclusively expressed in the trophoblast. During gestation, transcript of ALAS‐N slightly increased, while HO‐1 mRNA significantly decreased. Induced acute fetal hypoxia resulted in an increase in ALAS‐N mRNA and in a decrease in HO‐1 mRNA. These findings indicate that placental heme metabolism is influenced by the oxygen supply.
2‐Chloro‐1,4‐dimethoxybenzene as a mediator of lignin peroxidase catalyzed oxidations
Authors: Teunissen Pauline J.M , Field Jim A
Published: July 2002

Poly R‐478, 4‐methoxymandelic acid and oxalic acid were oxidized by lignin peroxidase (LiP) in the presence of the fungal metabolite 2‐chloro‐1,4‐dimethoxybenzene (2Cl‐14DMB), whereas no oxidation occurred in the absence of 2Cl‐14DMB. These substrates clearly inhibited the consumption of 2Cl‐14DMB and the formation of 2‐chloro‐1,4‐benzoquinone from 2Cl‐14DMB by LiP. The results suggest that 2Cl‐14DMB can replace the function of veratryl alcohol as a redox mediator in lignin peroxidase catalyzed oxidations.
Photochemical localization of the semotiadil binding region within the cardiac Ca2+ channel α1 subunit. Comparison with the skeletal muscle counterpart
Authors: Ii Nariaki , Kuniyasu Akihiko , Kawahara Kohichi , Shibano Toshiro , Schwartz Arnold , Nakayama Hitoshi
Published: July 2002

We have previously identified the binding region of a new Ca2+ antagonist semotiadil in the skeletal muscle Ca2+ channel. To the same semotiadil derivatives, the cardiac counterpart showed distinct and different binding characteristics: semotiadil and its photoaffinity analog D51‐4700 inhibited [3H]PN200‐110 binding to cardiac membrane preparations with IC50 values of 13–20 μM, which are 10 times higher than those in skeletal muscle. Hill slopes of the binding inhibition were 0.94–1.0 for the cardiac channels compared to 0.63–0.67 for the skeletal muscle channels. A possible explanation for the difference is that the semotiadil binding site is differently conferred in cardiac and skeletal muscle Ca2+ channels. To reveal this within the primary structure, photoaffinity labeling of cardiac membranes was employed. [3H]D51‐4700 was photoincorporated in several polypeptides but only the α1 subunit of the Ca2+ channel was photolabeled in a specific manner. Antibody mapping of the [3H]D51‐4700‐labeled α1 subunit with several anti‐peptide antibodies revealed that the labeled site was located solely in a peptide fragment between Cys1461 and Lys1529. This region encompasses the labeled site of skeletal muscle, but contains several non‐identical amino acid residues, which may participate in expressing different binding characteristics between the two muscle type Ca2+ channels.
Active site mutants of human herpesvirus‐6 proteinase
Authors: Tigue Natalie J , Kay John
Published: July 2002

Amino acid residues thought to comprise the catalytic triad of HHV‐6 proteinase were changed by site‐directed mutagenesis in the precursor form of the proteinase. By monitoring the ability of each mutant proteinase precursor to undergo autoprocessing, Ser116, His46 and His135 were identified as catalytically crucial. An attempt was made to mimic the catalytic triad arrangement of archetypal serine proteinases by replacement of the second histidine, His135, by an Asp. Instead of increasing the autoprocessing ability of the His135Asp mutant HHV‐6 proteinase precursor, this mutation had a detrimental effect since the precursor persisted predominantly in its unprocessed form.
Mapping of T7 RNA polymerase active site with novel reagents – oligonucleotides with reactive dialdehyde groups
Authors: Tunitskaya Vera L , Rusakova Ekaterina E , Memelova Lyudmila V , Kochetkov Sergey N , Van Aerschot Arthur , Herdewijn Piet , Efimtseva Ekaterina V , Ermolinsky Boris S , Mikhailov Sergey N
Published: July 2002

Oligonucleotides of a novel type containing 2′‐O‐β‐ribofuranosyl‐cytidine were synthesized and further oxidized to yield T7 consensus promoters with dialdehyde groups. Both types of oligonucleotides were tested as templates, inhibitors, and affinity reagents for T7 RNA polymerase and its mutants. All oligonucleotides tested retained high affinity towards the enzyme. Wild‐type T7 RNA polymerase and most of the mutants did not react irreversibly with oxidized oligonucleotides. Affinity labeling was observed only with the promoter‐containing dialdehyde group in position (+2) of the coding chain and one of the mutants tested, namely Y639K. These results allowed us to propose the close proximity of residue 639 and the initiation region of the promoter within initiation complex. We suggest the oligonucleotides so modified may be of general value for the study of protein‐nucleic acid interactions.
A unique DNase activity shares the active site with ATPase activity of the RecA/Rad51 homologue (Pk‐REC) from a hyperthermophilic archaeon
Authors: Rashid Naeem , Morikawa Masaaki , Kanaya Shigenori , Atomi Haruyuki , Imanaka Tadayuki
Published: July 2002

A RecA/Rad51 homologue from Pyrococcus kodakaraensis KOD1 (Pk‐REC) is the smallest protein among various RecA/Rad51 homologues. Nevertheless, Pk‐Rec is a super multifunctional protein and shows a deoxyribonuclease activity. This deoxyribonuclease activity was inhibited by 3 mM or more ATP, suggesting that the catalytic centers of the ATPase and deoxyribonuclease activities are overlapped. To examine whether these two enzymatic activities share the same active site, a number of site‐directed mutations were introduced into Pk‐REC and the ATPase and deoxyribonuclease activities of the mutant proteins were determined. The mutant enzyme in which double mutations Lys‐33 to Ala and Thr‐34 to Ala were introduced, fully lost both of these activities, indicating that Lys‐33 and/or Thr‐34 are important for both ATPase and deoxyribonuclease activities. The mutation of Asp‐112 to Ala slightly and almost equally reduced both ATPase and deoxyribonuclease activities. In addition, the mutation of Glu‐54 to Gln did not seriously affect the ATPase, deoxyribonuclease, and UV tolerant activities. These results strongly suggest that the active sites of the ATPase and deoxyribonuclease activities of Pk‐REC are common. It is noted that unlike Glu‐96 in Escherichia coli RecA, which has been proposed to be a catalytic residue for the ATPase activity, the corresponding residual Glu‐54 in Pk‐REC is not involved in the catalytic function of the protein.
Domain organization of flagellar hook protein from Salmonella typhimurium
Authors: Uedaira Hatsuho , Morii Hisayuki , Ishimura Miyuki , Taniguchi Hisaaki , Namba Keiichi , Vonderviszt Ferenc
Published: July 2002

Hook forms a universal joint, which mediates the torque of the flagellar motor to the outer helical filaments. Domain organization of hook protein from Salmonella typhimurium was investigated by exploring thermal denaturation properties of its proteolytic fragments. The most stable part of hook protein involves residues 148 to 355 and consists of two domains, as revealed by deconvolution analysis of the calorimetric melting profiles. Residues 72–147 and 356–370 form another domain, while the terminal regions of the molecule, residues 1–71 and 371–403, avoid a compact tertiary structure in the monomeric state. These folding domains were assigned to the morphological domains of hook subunits known from EM image reconstructions, revealing the overall folding of hook protein in its filamentous state.
Eukaryotic expression of enzymatically active human immunodeficiency virus type 1 reverse transcriptase
Authors: Isaguliants Maria G , Pokrovskaya Katja , Kashuba Vladimir I , Pokholok Dmitrii , Hinkula Jorma , Wahren Britta , Kochetkov Sergey N
Published: July 2002

Reverse transcriptase of human immunodeficiency virus type 1 is a vitalenzyme in the HIV‐1 replication cycle and an attractive target of attempts to arrest a primary viral infection. We designed a vector for eukaryotic expression of the 66 kDa subunit of reverse transcriptase under the control of the immediate early cytomegalovirus promoter. Efficient transient expression of the 66 kDa subunit of reverse transcriptase was achieved in a variety of cells. Immunostaining of the transfected cells revealed the cytoplasmatic localization of reverse transcriptase. Reverse transcriptase activity was detected in all transfected cell lines. Injection of this plasmid encoding the 66 kDa subunit of reverse transcriptase into mice resulted in strong reverse transcriptase‐specific immune responses indicating that the 66 kDa subunit of reverse transcriptase is expressed in vivo. Sera from DNA‐immunized mice inhibited reverse transcription in vitro.
Biochemical analysis of interleukin‐2 receptor β chain phosphorylation by p56lck
Authors: Delespine-Carmagnat Maryvonnick , Bouvier Ghislaine , Allée Guillaume , Fagard Remi , Bertoglio Jacques
Published: July 2002

Tyrosine phosphorylation of multiple proteins, including the receptor itself, is an initial event in IL‐2 signaling and leads to recruitment of SH2 or PTB domain‐containing proteins to the receptor. In this study, we have used subdomains of the IL‐2 receptor β chain (IL‐2Rβ) expressed in Escherichia coli as GST fusion proteins to identify the tyrosine residues that could be phosphorylated by p56lck, one of the critical tyrosine kinases activated by IL‐2. We report that recombinant p56lck phosphorylates in vitro tyrosine residues within the IL‐2Rβ chain but not those within the IL‐2Rγ chain. p56lck phosphorylates tyrosine residues 355, 358 and 361 but not 338 of the IL‐2Rβ chain acidic subdomain. Interestingly, phosphorylation of Tyr‐358 appears to require the presence of either Tyr‐355 or Tyr‐361. p56lck also phosphorylates very efficiently the two tyrosines present in the IL‐2Rβ chain C‐terminal region, Tyr‐392 and Tyr‐510. We also investigated the association of p56lck with the IL‐2Rβ chain which was found to depend on a short stretch of the IL‐2Rβ chain acidic subdomain, and to be independent of the presence of its tyrosine residues.
A potential NES of the Epstein‐Barr virus nuclear antigen 1 (EBNA1) does not confer shuttling
Authors: Fischer Nicole , Voß Marc D. , Mueller-Lantzsch Nikolaus , Grässer Friedrich A.
Published: July 2002

The Epstein‐Barr virus nuclear antigen 1 (EBNA1) is a multifunctional protein involved in the replication and maintenance of the viral episome. We identified a potential Rev‐like nuclear export signal (NES) which, however, does not confer the export of EBNA1. In the yeast two‐hybrid system EBNA1 does not bind to the nuclear exporter Crm1p. In spite of the RNA‐binding ability of EBNA1 and its structural homologies to RNA binding proteins like hnRNP U and/or A1, EBNA1 does not shuttle to the cytoplasm in heterokaryon analysis. We propose the function of the RNA binding of EBNA1 in retaining RNAs to the nucleus.
The cleaved presequence is not required for import of subunit 6 of the cytochrome bc1 complex into yeast mitochondria or assembly into the complex*
Authors: DeLabre Marie Laure , Nett Jürgen H , Trumpower Bernard L
Published: July 2002

Subunit 6 of the yeast cytochrome bc
1 complex contains a 25 amino acid presequence that is not present in the mature form of the protein in the bc
1 complex. The presequence of subunit 6 is atypical of presequences responsible for targeting proteins to mitochondria. Whereas mitochondrial targeting sequences rarely contain acidic residues and typically contain basic residues that can potentially form an amphiphilic structure, the presequence of subunit 6 contains only one basic amino acid and is enriched in acidic amino acids. If the 25 amino acid presequence is deleted, subunit 6 is imported into mitochondria and assembled into the cytochrome bc
1 complex and the activity of the bc
1 complex is identical to that from a wild‐type yeast strain. However, if the C‐terminal 45 amino acids are truncated from the protein, subunit 6 is not present in the mitochondria and the activity of the bc
1 complex is diminished by half, identical to that of the bc
1 complex from a yeast strain in which the QCR6 gene is deleted. These results indicate that the presequence of subunit 6 is not required for targeting to mitochondria or assembly of the subunit into the bc
1 complex and that information necessary for targeting and import into mitochondria may be present in the C‐terminus of the protein.
Synechococcus mutants resistant to an enamine mechanism inhibitor of glutamate‐1‐semialdehyde aminotransferase
Authors: Bishop Konrad , Gough Kevin , Mahoney Sonia , Smith Arnold , Rogers Lyndon
Published: July 2002

An enamine mechanism‐based inactivator of mammalian δ‐aminobutyric acid aminotransferase, 4‐amino 5‐fluoropentanoic acid is a potent inhibitor of cell growth and pigment formation in the cyanobacterium Synechococcus PCC 6301. It was demonstrated that 4‐amino 5‐fluoropentanoic acid inhibits the aminolaevulinate synthesis at glutamate 1‐semialdehyde aminotransferase and that in the mutant obtained by exposing cells to 40 μM 4‐amino 5‐fluoropentanoic acid, this enzyme was insensitive to the inhibitor. The specific activity of glutamate 1‐semialdehyde aminotransferase in cell extracts was lower in the mutant, although the cell growth rate was unaffected. The decrease in sensitivity to 4‐amino 5‐fluoropentanoic acid in the mutant is due to a structural gene mutation, a single base change in the hemL gene resulting in a S162T substitution in the gene product.
Permutation of modules or secondary structure units creates proteins with basal enzymatic properties
Authors: Tsuji Toru , Kobayashi Kensei , Yanagawa Hiroshi
Published: July 2002

The RNase activity of barnase mutants obtained by the permutation of modules or secondary structure units was investigated. Four of the 45 mutants had weak but distinct RNase activity, and they had unique optimum pHs and temperatures like natural enzymes. One of the active mutants had an ordered conformation, but the others did not. An active mutant having disordered conformation formed an ordered conformation in the presence of GMP, which is an inhibitor of this mutant. These results indicate that the amino acid sequences derived from barnase have sufficient plasticity to be rearranged into different proteins with basal enzymatic properties.