bluuebunny/crossref_metadata_2025 huggingface dataset:
The proteasome/multicatalytic—multifunctional proteinase In vivo function in the ubiquitin‐dependent N‐end rule pathway of protein degradation in eukaryotes
Authors: Richter-Ruoff Birgit , Heinemeyer Wolfgang , Wolf Dieter H.
Published: July 2002

Proteinase yscE, the proteasome/multicatalytic—multifunctional proteinase of yeast had been shown to function in stress response and in the degradation of ubiquitinated proteins [(1991) EMBO J. 10, 555–562]. A well‐defined set of proteins degraded via ubiquitin‐mediated proteolysis are the substrates of the N‐end rule pathway [(1986) Science 234, 179–186; (1989) Science 243, 1576–1583]. We show that mutants defective in the chymotryptic activity of proteinase yscE fail to degrade substrates of the N‐end rule pathway. This gives further proof of the proteasome being a central catalyst in ubiquitin‐mediated proteolysis.
Interaction of mannose‐6‐phosphate with the hysteretic transition in glucose‐6‐phosphate hydrolysis in intact liver microsomes
Authors: Vidal Hubert , Berteloot Alfred , Larue Marie-Josée , St-Denis Jean-François , van de Werve Gérald
Published: July 2002

We showed previously that glucose‐6‐phosphatase activity was characterised in intact liver microsomes by a hysteretic transition between a rapid and a slower catalytic form of the enzyme. We have now further investigated the substrate specificity of these two kinetic forms. It was found that the pre‐incubation of intact microsomes with mannose‐6‐phosphate or glucose‐6‐phosphate (50 μM for 30 s) suppressed the burst in glucose‐6‐phosphatase activity, that the hysteretic transition was reversible and that mannose‐6‐phosphate inhibited glucose‐6‐phosphate hydrolysis during the first seconds of incubation, but not anymore after the burst. Our results indicate (i) that mannose‐6‐phosphate is recognised by the enzyme and can promote the hysteretic transition and (ii) that the transient phase is part of the catalytic mechanism itself.
Efficient thyroid hormone formation by in vitro iodination of a segment of rat thyroglobulin fused to Staphylococcal protein A
Authors: Asunción Miryam , Ingrassia Rosaria , Escribano Julio , Martin Uly , Méndez Enrique , Di Lauro Roberto , Lamas Luis
Published: July 2002

A polypeptide of 224 amino acids from the C terminus of rat thyroglobulin fused to Staphylococcal protein A (TgC 224), containing 3 tyrosines which have been shown to be hormonogenic in vivo (Tyr‐2555, ‐2569 and ‐2748), forms thyroid hormones with relatively high efficiency upon in vitro enzymatic iodination using, most likely, the hormonogenic Tyr‐2555 and Tyr‐2569. Acetylcholinesterase, which has sequence and structural homology with the C terminus of the thyroglobulin molecule and bovine serum albumin, used as control proteins, formed thyroid hormones with lower efficiency. These results validate our experimental approach to define the structural requirements for thyroid hormone formation using thyroglobulin fragments.
α‐Amylases and approaches leading to their enhanced stability
Authors: Janeček Štefan , Baláž Štefan
Published: July 2002

The recent state of the knowledge of properties and structure of α‐amylases is reviewed with the aim of elucidation the basis for their stabilization. Three principal ways for obtaining stable α‐amylases (isolation of enzymes from extremophiles, production of extremophilic enzymes in mesophiles, and modification of mesophilic enzymes) are discussed separately. Detailed experimental examples are given for modification approaches.
A novel class of nucleolar RNAs from Tetrahymena
Authors: Nielsen Henrik , Ørum Henrik , Engberg Jan
Published: July 2002

We describe a family of at least four nucleolar RNAs (snoRNAs) from the ciliate, Tetrahymena. The snoRNAs are 120–140 nucleotides long, moderately AU‐rich and contain no modified nucleotides. Their 5′ ends are blocked by a cap of unknown nature. The snoRNAs can be folded into similar secondary structures consisting of two hairpins separated by a single‐stranded AU‐rich spacer. The sequences and secondary structures show no extensive sequence or secondary structure resemblance to any other small RNAs in the public databases.
Membrane inserted APP fragments containing the βA4 sequence of Alzheimer's disease do not aggregate
Authors: Dyrks Thomas , Dyrks Elke , Masters Colin , Beyreuther Konrad
Published: July 2002

Previously we have shown that the COOH‐terminal fragment (A4CT) of the Alzheimer amyloid protein precursor (APP), which at the NH2‐terminus carries the sequence of the amyloid βA4 protein, forms highly insoluble aggregates [EMBO J. (1988) 7, 949–957]. Here we report that aggregation is prevented if A4CT is expressed in vitro with a signal sequence at the NH2‐terminus (SPA4CT) under conditions which allow membrane insertion. Aggregates from SPA4CT are obtained after removal of membranes by chloroform/methanol extraction or heating.
Protein dynamics An overview on flash‐photolysis over broad temperature ranges
Authors: Di Iorio Ernesto E.
Published: July 2002

Ligand binding kinetics to heme‐proteins between 40 and 300 K point to a regulatory role of protein dynamics. A protein‐specific susceptibility of the heme‐iron reactivity to dynamic fluctuations emerges from the distribution of reaction enthalpies derived from flash‐photolysis measurements below ca. 180 K; we quantify it in terms of ‘intramolecular viscosity’, postulating that narrow low‐temperature enthalpy distributions correspond to low internal viscosity and vice versa. The thermal evolution of ligand binding kinetics suggests, with other results, an interplay between high‐frequency transitions of the amino acid side chains and low‐frequency collective motions as a possible regulatory mechanism of protein dynamics.
Synthesis and characterization of a 25‐residue rubredoxin(II)‐like metalloprotein and its valine‐leucine mutant
Authors: Christensen Hans E.M. , Hammerstad-Pedersen Jan M. , Holm Arne , Roepstorff Peter , Ulstrup Jens , Vorm Ole , Østergård Søren
Published: July 2002

An iron‐sulfur metalloprotein containing the 5–12 and 35–50 residues of Desulfovlbrio gigas rubredoxin has been synthesized by Fmoc solid phase peptide synthesis and subsequent peptide folding. A Gly links the two residue chains between Val‐5 and Glu‐50. Sybyl Tripos structure optimization indicates only minor structural changes of the folded synthetic protein compared to the similar residue positions in the native protein. The UV‐VIS spectrum of the reduced synthetic protein is very similar to that or native D. gigas rubredoxin and the molecular mass determined by laser mass spectrometry has the expected value (± 2D). No metal is transferred to the gas phase by the laser beam merely by mixing the peptide and iron(II), substantiating that the folding procedure is a necessary pre‐requisite for protein formation. The Val → Leu41 chemical mutant has also been synthesized and behaves in a closely similar fashion.
Site‐directed mutation makes rabbit calcyclin dimer
Authors: Ando Yuhko , Watanabe Masato , Akatsuka Hajime , Tokumitsu Hiroshi , Hidaka Hiroyoshi
Published: July 2002

Unlike human, rat and mouse calcyclin, purified rabbit calcyclin did not form a dimer on Tricine SDS‐PAGE under non‐reduced conditions. Based on the internal peptide sequence of rabbit calcyclin, we isolated and sequenced a cDNA clone encoding calcyclin. The sequence of this clone (pCaiC) is 629 bp long and codes 90 amino acid residues of a protein with a molecular mass of 10,153 Da. By Northern blot analysis, a major band of 0.9 kbp and a minor band of 2.6 kbp were detected in the lung. The recombinant calcyclin mutated serine at the third position to cysteine was expressed in E. coli and made dimer formation under non‐reduced conditions on SDS‐PAGE. Whether or not this type of mutation which prevents dimer formation of calcyclin plays a physiological role in the rabbit lung is the subject of an ongoing study.
Evidence that deprotonation of Serine‐55 is responsible for the pH‐dependence of the parvalbumin Eu3+7F0→5D0 Spectrum
Authors: Henzl Michael T. , Treviño Claudia L. , Dvoráková Lenka , Boschi Jeanne M.
Published: July 2002

The Eu(III) 7F0→5D0 excitation spectra of the parvalbumins are highly pH‐dependent. Below pH 6.0, they exhibit a sharp, partially resolved doublet centered near 5,795 Å. However, as the pH is raised, the spectrum becomes increasingly dominated by a much broader signal near 5,784 Å. This behavior has been traced to the Eu(III) ion bound at the CD site, but the identity of the moiety undergoing deprotonation remains uncertain. Site‐specific mutagenesis studies on the parvalbumin‐like protein known as oncomodulin now suggest that the species in question is a liganding serine hydroxyl group. Specifically, replacement of serine‐55 by aspartate (the residue present at the corresponding position in the EF site) affords a protein that retains two functional lanthanide binding sites, but fails to undergo the pH‐dependent spectral alteration. By contrast, replacement of aspartate‐59 by glycine (the corresponding EF site residue) fails to abolish the pH‐dependent behavior.
Biosynthesis of bacillomycin D by Bacillus subtilis Evidence for amino acid‐activating enzymes by the use of affinity chromatography
Authors: Besson Françoise , Michel Georges
Published: July 2002

Bacillomycin D is an antifungal lipopeptide produced by B. subtilis. The formation of the peptidyl bonds of bacillomycin D occurs non‐ribosomally, as demonstrated by the use of chloramphenicol, an inhibitor of protein biosynthesis. Amino acid‐activating enzymes were found in B. subtilis cell lysates purified by affinity chromatography on a gel containing l‐Pro, an amino acid of bacillomycin D. Presence of ATP during this purification increases the binding of enzymatic proteins and their activity. An enzyme, with an apparent molecular weight of 230 kDa, catalyzed ATP‐PPi exchange reactions, which were mediated by specific amino acids, corresponding to a partial sequence of bacillomycin D.
Internucleosomal chromatin degradation in myeloma and B‐hybridoma cell cultures
Authors: Sokolova Irina A. , Volgin Andrei U. , Makarova Natalia V. , Volgina Veronica V. , Shishkin Sergei S. , Khodarev Nikolai N.
Published: July 2002

The activity of Ca/Mg‐dependent endonuclease (CME) is strongly inhibited in myeloma X‐63.Ag8.653 and B‐hybridoma MLC‐1c is compared with mouse splenocytes. Nevertheless, pronounced internucleosomal chromatin degradation occurs in both cell lines during long‐term cultivation without passing. In isolated cell nuclei of X‐63 the activation of CME, which precedes chromatin fragmentation in vivo and loss of cell viability, is revealed. The time—course of CME activation is opposite to cell proliferation and is not accompanied by alterations in enzyme quantity. The results suggest that cell death of X‐63 and MLC‐1c occurs via apoptosis, and involves the mechanisms controlling the activation and/or interaction of CME with chromatin.
Genomic structure of the gene for copper/zinc‐superoxide dismutase in rice
Authors: Sakamoto Atsushi , Okumura Takekazu , Ohsuga Hiroyuki , Tanaka Kunisuke
Published: July 2002

We have isolated and determined the nucleotide sequence of the structural gene (sodB) coding for one of the copper/zinc‐superoxide dismutase (Cu/Zn‐SOD) isozymes from rice plants (Oryza sativa L.). The sodB gene is split into 8 exons spread about 2 kb of the chromosomal DNA, with the coding sequence beginning in the 2nd and ending in the 8th. Although none of sodB introns are inserted into similar positions in Cu/Zn‐SOD genes from other sources, four of six introns in the protein‐coding region are located at discriminating positions within structural domains of the protein. Genomic Southern analysis indicated that cytosolic Cu/Zn‐SOD is coded for by 2–3 genes in the rice genome.
Identification of a small Na+/H+ exchanger‐like message in the rabbit myocardium
Authors: Dyck Jason R.B. , Lopaschuk Gary D. , Fliegel Larry
Published: July 2002

We examined the Na+/H+ exchanger message in isolated perfused rabbit hearts using Northern blot analysis with cDNA encoding for the rabbit cardiac Na+/H+ exchanger. A cDNA probe from the coding region of the rabbit myocardial Na+/H+ exchanger hybridized to mRNA of 5 kb under high stringency, and to a second 3.8 kb mRNA species under low stringency. When Northern blots were re‐probed with a section of the 3′‐untranslated region of the cDNA, the 5 kb message was apparent while the smaller 3.8 kb message was not. If isolated working rabbit hearts were subjected to ischemia we observed increases in the 3.8 kb message. Overall, the results show that a 3.8 kb mRNA product, which is homologous to the amiloride sensitive Na+/H+ exchanger, exists in the myocardium and increases during ischemia in the myocardium.
Effect of perturbing diacylglycerol metabolism on cytosolic free Ca2+ oscillations induced in single hepatocytes
Authors: Sanchez-Bueno Antonio , Cobbold Peter H.
Published: July 2002

Single rat hepatocytes microinjected with aequorin show free Ca oscillations when stimulated with Ca2+‐mobilizing hormones. We show here that an inhibitor of diacylglycerol kinase (R 59 022) and an analogue of native diacylglycerol (diC8) inhibit free Ca oscillations induced by phenylephrine and vasopressin. These results agree with a negative feedback effect of protein kinase C on free Ca oscillations.
Intrinsic activity of precursor forms of HIV‐1 proteinase
Authors: Phylip Lowri H. , Mills John S. , Parten Benne F. , Dunn Ben M. , Kay John
Published: July 2002

The wild‐type ‐Phe*Pro‐ bond located at the N‐terminus of the mature aspartic proteinase of HIV‐1 was replaced by ‐Ile‐Pro‐ or ‐Val‐Pro‐. By this means, processing at this cleavage junction was prevented and so, extended or precursor forms of HIV‐proteinase were generated. These constructs were expressed in Escherichia coli, purified therefrom, and their specificity, activity at different pH values and susceptibility to the potent inhibitor, Ro31‐8959, was assessed. A hitherto unobserved cleavage junction (at ∼Ala‐Phe*Leu‐Gln∼) in the frame‐shift region of the gag‐pol viral penome was identified and confirmed by demonstrating cleavage of a synthetic peptide corresponding to this region. The implications for viral replication of self‐processing at neutral pH by proteinase whilst still present (in a precursor form) as a component of the polyprotein are considered; such reactions, however, are still blocked even at pH values as high as 8.0 by Ro31‐8959.
Comparison of phospholipase D activity in vasopressin‐ and phorbol ester‐stimulated fibroblasts
Authors: Huang Chunfa , Wykle Robert L. , Cabot Myles C.
Published: July 2002

Phospholipase D (PLD) activation by vasopressin (VP) was compared to activation by TPA in REF52 cells prelabeled with [3H]glycerol and [14C]myristic acid. Upon VP‐treatment, the formation of [3H] and [14c]phosphatidic acid (PA) and phosphatidylethanol (PEt) was accompanied by the loss of radioactivity from PC and PI. However, upon TPA‐treatment, radioactivity was lost from PC only. No significant changes of phosphatidylethanolamine and phosphatidylserine were detected in the same samples. The inclusion of 5μM staurosporine for 10 min diminished the production of [3H]PEt and [14C]PEt by 27% and 53% in VP‐treated cells, and by 100% and 75% in TPA‐treated cells, respectively. Adding 1 mM EGTA to chelate extracellular Ca2+ inhibited [3H]PEt by approximately 31% and [14C]PEt by 17% after VP‐stimulation. In contrast, EGTA had no effeet on TPA‐stimulation. The data suggest that REF52 cells contain dual PLD activities. The first is stimulated only by VP, requires Ca2+ and hydrolyzes PI. The second is stimulated by both TPA and VP, activated by protein kinase C and hydrolyzes PC.
Identification of a protein from Saccharomyces cerevisiae with E2F‐like DNA‐binding and transactivating properties
Authors: Mai Bernard , Lipp Martin
Published: July 2002

The promoter of the human proto‐oncogene MYC has been the first cellular target shown to be subject to regulation by the E2F transcription factor. E2F also has binding sites in other promoters regulated by cell proliferation and during the cell cycle. We have analyzed Saccharomyces cerevisiae for the presence of an E2F‐analogous protein. GAL1‐based promoter constructs carrying the E2F binding site of the MYC or the adenovirus E2 promoter showed transcriptional activity in yeast cells. A DNA‐binding factor, designated YE2F, binds specifically to the E2F consensus sequence and was partially purified from yeast extracts. YE2F showed identical contact points within the MYC binding site as authentic E2F protein from mammalian cells. The results suggest the existence of an E2F‐like protein in the yeast S. cerevisae.
The catalytic domain of human hepatitis delta virus RNA
Authors: Lee Bao-Shiang , Wu Huey-Nan , Huang Tai-Huang
Published: July 2002

We have obtained and analyzed the 600 MHz proton NMR spectra of a 74‐mer RNA derived from the catalytic domain of hepatitis delta virus genomic RNA (HDV RNA) to determine its secondary structure. Deconvolution of the NMR spectrum obtained at 32°C indicates that part of the 74‐mer RNA molecule may exist in multiple conformations in equilibrium. The major conformer contains two A‐U base pairs and 14 ± 2 G‐C base pairs. It appears to contain no standard G‐U base pairs. Our NMR melting study suggests that this conformer has at least two stem‐loop regions. One of the regions has been identified to be a tetra‐loop. We have assigned five imino proton resonances of the tetraloop stem. Our data is consistent with the pseudoknot model of Perrotta and Been.
Stimulatory effect of the maize HMGa protein on reporter gene expression in maize protoplasts
Authors: Grasser Klaus D. , Hetz Winfried , Griess Eike A. , Feix Günter
Published: July 2002

The high mobility group (HMG) proteins represent a class of chromosomal non‐histone proteins with an assumed influence on transcription. In this context, the effect of the maize HMGa protein on reporter gene expression was examined. Transient co‐transformation experiments in maize protoplasts with plasmid constructs directing the synthesis of the maize HMGa protein and with a luciferase reporter plasmid demonstrated a stimulatory effect of the HMGa protein on the reporter gene expression. Additional experiments with HMGa deletion constructs indicated that the HMG‐Box DNA‐binding motif is important for the observed effect, while the acidic carboxy‐terminal domain of the HMGa protein appears to be dispensable.
Cell‐specific expression of Gq/11 protein and mRNA in rat seminiferous tubules
Authors: Haugen Trine B. , Paulssen Ruth H. , Hansson Vidar
Published: July 2002

As part of a study to elucidate the involvement of G proteins in signal transduction in testicular cells, we have examined the cellular localization of Gq/11 within the seminiferous tubules. The somatic cells (Sertoli cells, peritubular cells) contain high amounts of both Gq/11α mRNA and immunoreactive protein. In contrast, very low levels of these G proteins and the corresponding mRNAs are present in the germ cells (pachythene spermatocytes, round spermalids). Thus, in the germ cells, receptor‐regulated inositol phospholipid hydrolysis is not likely to be regulated via Gp/11, but rather through the Go protein, which has been previously shown to be abundant in rat germ cells. Since the somatic cells are nearly devoid of Go, the Gpp(NH)p‐stimulated phospholipase C in these cells is probably regulated by Gq and/or g11.
Assembly of eukaryotic class III (N‐out, C‐in) membrane proteins into the Escherichia coli cytoplasmic membrane
Authors: Hennessey Emma S. , Hashemzadeh-Bonehi Lily , Hunt Lesley A. , Broome-Smith Jenny K.
Published: July 2002

Class III membrane proteins lack cleavable signal peptides but adopt an N‐out, C‐in topology with respect to their native membranes. We have analysed the fate of two eukaryotic class III plasma membrane proteins, human erythrocyte glycophorin C and influenza A virus M2 protein, in Escherichia coli. The N‐terminal domains of both proteins were efficiently localised to the extracytoplasmic side of the bacterial cytoplasmic membrane. When β‐lactamase was fused to the C‐terminus of glycophorin C it was localised to the cytoplasm, and protease treatment of spheroplasts caused a reduction in size of the fusion protein consistent with glycophorin C adopting its native topology in E. coli.
Two proteins involved in kinetoplast compaction
Authors: Tittawella Ivor , Carlsson Lena , Thornell Lars-Eric
Published: July 2002

The kinetoplast is the genome of the single mitochondrion of trypanosomatid Protozoa, and contains up to 30% of total cellular DNA in a network of catenated AT‐rich rings. EM studies show that the kinetoplast is organized into a compact, disc‐shaped structure in vivo, but little is known about proteins involved in its architecture. Defining such proteins would be useful to understand the molecular biology of this unusual organelle and to design compounds to contain parasite growth. We show here that two proteins, p1 and p2 of M
r ~ 22 and ~ 21 kDa, respectively, from the trypanosomatid Crithidia fasciculata can compact kDNA networks efficiently in vitro, the first such demonstration with purified trypanosome proteins. We show that these proteins are localized exclusively in the parasite's kinetoplast. Our data thus define two proteins potentially involved in kinetoplast organization in vivo.
Isolation of two members of the rat MAP kinase kinase gene family
Authors: Otsu Masayuki , Terada Yasuhiko , Okayama Hiroto
Published: July 2002

Mitogen‐activated protein (MAP) kinase kinase (MAPKK) is a recently characterized activator of MAP kinase (MAPK), and is considered to be regulated by a protooncogene product c‐Raf‐1. It is, however, unclear whether the signals originating from c‐Raf‐1 utilize this phosphorylation cascade to lead to oncogenesis. To clarify this point, we isolated rat MAPKK cDNAs, and identified two distinct cDNAs encoding MAPKK and a highly related kinase, both with molecular weights of 4̃5 kDa (MEK1 and MEK2). Genomic Southern blot analyses suggested that MAPKK. may form a large gene family.
AT(D)PMg‐induced dissociation of the α3β3 complex of the F1‐ATPase from a thermophilic Bacillus PS3 into α1β1 heterodimers is prevented by mutation β(Y341C)
Authors: Kaibara Chitose , Odaka Masafumi , Hisabori Toru , Yoshida Masasuke
Published: July 2002

AT(D)PMg induces dissociation of the α3β3 complex of F1‐ATPase from a thermophilic Bacillus strain, PS3, into the α1β1 heterodimers [(1991) Biochim. Biophys. Acta 1056, 279‐284] but the location of the AT(D)PMg binding site responsible is not known. From the analysis of AT(D)PMg binding properties of the isolated mutant β subunit, β(y341c) and the stability of the α3β(y341c)3 complex in the presence of AT(D)PMg, we conclude that binding of AT(D)PMg to the Tyr‐341 site of the β subunit(s) in the α3β3 complex triggers the dissociation of the α3β3 complex into the α1β1 heterodimers.
Prenylated protein methyltransferase of rat cerebellum is developmentally co‐expressed with its substrates
Authors: Paz A. , Ben-Baruch G. , Marciano D. , Egozi Y. , Haklai R. , Kloog Y.
Published: July 2002

High levels of prenylated protein methyltransferase are expressed in the developing rat cerebellum and are responsible for methylation of endogenous G‐proteins and 50–52 kDa synaptosomal proteins. Enzyme activity in cerebellar synaptosomes of 3 week postnatal rats is 2‐fold higher than that found in adult rat cerebellum. A 10‐fold rise in activity occurs at the end of the second and during the third postnatal weeks, followed by a subsequent decline. Expression of the enzymes' substrates follows the same pattern. The high methyltransferase activity in 3‐week‐old cerebellum coincides with the period of granule cell migration and synaptogenesis, suggesting a regulatory role for the enzyme and its substrates in cerebellar ontogenesis.
Characterisation of ATP binding inhibition to the sarcoplasmic reticulum Ca2+‐ATPase by thapsigargin
Authors: DeJesus Florence , Girardet Jean-Luc , Dupont Yves
Published: July 2002

The inhibition of Ca2+‐ATPase of sarcoplasmic reticulum by thapsigargin has been reported to be associated with a suppression of calcium binding to the high affinity transport sites. We report here that thapsigargin also acts as an inhibitor of ATP binding by reducing its apparent affinity by about two orders of magnitude. This inhibition is non‐competitive indicating that thapsigargin does not bind to the ATP binding site. This is confirmed by the fact that thapsigargin binding to the Ca2+‐ATPase does not affect the binding of 2',3'‐O‐(2,4,6‐trinitrocyclohexadienylidene)‐ATP (TNP‐ATP).
Different ratios in 20 S proteasomes and regulatory subunit complexes in two isoforms of the 26 S proteasome purified from rabbit skeletal muscle
Authors: Sawada Hitoshi , Muto Kazuko , Fujimuro Masahiro , Akaishi Takahiro , Sawada Michiko Takagi , Yokosawa Hideyoshi , Goldberg Alfred L.
Published: July 2002

A ubiquitin/ATP‐dependent proteinase complex (26 S proteasome) was highly purified from rabbit skeletal muscle. The purified 26 S proteasome easily dissociated into a 20 S proteasome and a regulatory subunit complex on non‐denaturing PAGE. By using cleavable and non‐cleavable cross‐linkers, it was revealed that the 26 S proteasome exists in two isoforms: one (D complex) consists of the 20 S proteasome and the regulatory subunit complex in the ratio of one to two, while the other (C complex) exists in an equal molar ratio. Molecular masses of the former and the latter isoforms were estimated to be 1,700 kDa and 1,400 kDa, respectively, by gel filtration, and 2,400 kDa and 1,400 kDa, respectively, by Ferguson plot analysis. Furthermore, both isoforms efficiently hydrolyzed Suc‐Leu‐Leu‐Val‐Tyr‐MCA and ubiquitin‐conjugated [125I]lysozyme. These results suggest that the D and C complexes are active proteinase complexes, most probably corresponding to the dumbbell‐like and mushroom‐like (or space capsule‐like) molecules, respectively.
Identification of DNA binding proteins in vaccinia virus by DNA‐protein crosslinking
Authors: Chkheidze A.N. , Prikhod'ko G.G. , Karpov V.L. , Vassilenko S.K. , Mirzabekov A.D.
Published: July 2002

DNA binding proteins of vaccinia virus (VV) virions, strain LIVP, have been studied by their covalent crosslinking to DNA, using two‐dimensional gel retardation electrophoresis of crosslinked DNA and proteins as well as the ‘protein image’ hybridization assay. Five proteins with molecular masses of 16, 25, 27, 41 and 54 kDa, respectively, associated with all analysed DNA sequences, including early and late genes and their promoters, have been identified.
Use of substituted and tandem‐repeated peptides to probe the relevance of the highly conserved RGD tripeptide in the immune response against foot‐and‐mouth disease virus
Authors: Novella Isabel S. , Borrego Belén , Mateu Mauricio G. , Domingo Esteban , Giralt Ernest , Andreu David
Published: July 2002

Antigenic site A of foot‐and‐mouth disease virus (FMDV) is an exposed, mobile loop which includes a central, highly conserved Arg‐Gly‐Asp tripeptide (RGD, VP1 residues 141–143 in serotype C) thought to be part of the cell attachment site. We have analyzed the contribution of RGD to the interaction of site A with antibodies by incorporating selected amino acid replacements at RGD into synthetic peptides representing site A, and analyzing the reactivity of substituted peptides with site A‐specific monoclonal antibodies (MAbs). Replacement of Arg‐141, Gly‐142 or Asp‐143 by alanine resulted in the loss of one, three and five epitopes, respectively, out of seven epitopes probed. Other replacements resulted in the loss of even larger numbers of epitopes, suggesting that the amino acids of the RGD region are either directly involved in interaction with antibodies or that they exert an important influence on the interaction of surrounding residues with antibodies. Thus, we explored the ability of tandem repeats of the RGDL sequence (corresponding to FMDV C‐S8cl) to evoke neutralizing antibodies in rabbits and guinea pigs. Neutralizing activity was generally low but with a broad specificity for different FMDV serotypes and variants. Significant decreases in neutralizing titers were observed with boosting, suggesting a possible suppression of those anti‐peptide antibodies which may also be directed to cellular RGD sequences. The results point to an involvement of RGD in the antigenic structure of site A, and open the possibility that broadly neutralizing antibodies might be induced by tandem repeats of the critical, conserved domain.
Uteroglobin, an apically secreted protein of the uterine epithelium, is secreted non‐polarized from MDCK cells and mainly basolaterally from Caco‐2 cell
Authors: Vogel Lotte Katrine , Suske Guntram , Beato Miguel , Norén Ove , Sjöström Hans
Published: July 2002

A complete cDNA encoding rabbit Uteroglobin was constructed and expressed in MDCK and Caco‐2 cells. The MDCK cells secrete Uteroglobin in approximately equal amounts to the apical and the basolateral side, whereas the Caco‐2 cells secrete Uteroglobin mainly to the basolateral side. Both MDCK and Caco‐2 cells thus secrete Uteroglobin in a non‐sorted manner. It has, however, previously been shown that Uteroglobin is secreted exclusively at the apical membrane in primary cell culture of endometrial epithelial cells [S.K. Mani et al. (1991) Endocrinology 128, 1563‐1573]. This suggests that either the endometrial epithelium has an apical default pathway or recognises a sorting signal not recognised by MDCK cells and Caco‐2 cells. Our data thus show that a soluble molecule can be secreted at the apical, the basolateral or both membranes depending on the cell type.
Acylphosphatase synergizes with progesterone during maturation of Xenopus laevis oocytes
Authors: Dolfi Fabrizio , Carnero Amancio , Cuadrado Antonio , Ramponi Giampietro , Lacal Juan Carlos
Published: July 2002

Xenopus laevis oocytes are physiologically arrested in the G2/M phase border of the first meiotic division. A number of different stimuli can trigger off the re‐entry into the cell cycle as a consequence of activation of either membrane‐dependent or ‐independent intracellular signals. This system has been widely used to study signal transduction mechanisms induced by hormones. Among those more intensively researched, special attention has been devoted to elucidate the mechanism of activation induced by progesterone. However, despite intense efforts to understand the intracellular signalling mechanism of progesterone, a clear notion of the most relevant events involved in this process has not yet been elucidated. We provide evidence that acylphosphatase, an enzyme responsible for the regulation of membrane pumps in eukaryotic cells, synergizes with progesterone for induction of oocyte maturation. We deduced that this synergism may be related to the regulation of intracellular Ca2+ levels for several reasons: (1) maturation of oocytes by extracellular Ca2+ is blocked by acylphosphatase; (2) both progesterone and acylphosphatase drastically reduced Ca2+ uptake; (3) progesterone‐induced maturation does not depend on a rise in intracellular Ca2+, since microinjection of EGTA, a calcium chelator, does not affect maturation induced by progesterone.
A gene homologous to the reg gene is expressed in the human pancreas
Authors: Bartoli Catherine , Gharib Bouchra , Giorgi Dominique , Sansonetti Alice , Dagorn Jean-Charles , Bergé-Lefranc Jean-Louis
Published: July 2002

We have determined the nucleotide sequence of reg1 a human genomic DNA fragment homologous to the reg gene which is expressed in the exocrine pancreas and regenerating islets. Sequence comparisons of reg and reg1 suggested similar exonintron organisation. Based on this assumption, specific oligonucleotides for reg1 exons were used to demonstrate expression of the reg1 gene in pancreas and liver. The proteins encoded by reg and reg1 comprise 166 amino acids and differ by 22 amino acids only.
Pre‐translational regulation of lipid synthesizing enzymes and surfactant proteins in fetal rat lung in explant culture
Authors: Fraslon C. , Batenburg J.J.
Published: July 2002

In hormone‐free explant cultures of 18‐day fetal rat lung the levels of the mRNAs for fatty acid synthase, ATP citrate lyase and surfactant proteins A, B, and C, increased as they do in vivo. Also CTP:phosphocholine cytidylyltransferase mRNA increased spontaneously. Unlike in vivo, malic enzyme mRNA increased drastically in cultured explants. Culture with dexamethasone increased the abundance of fatty acid synthase and surfactant protein mRNAs, but considerably depressed that of malic enzyme mRNA. Dexamethasone had little effect on CTP:phosphocholine cytidylyltransferase mRNA, supporting the concept that the increased activity of this enzyme caused by glucocorticoid is due to increased fatty acid synthesis.
An exon‐trapping system with a newly constructed trapping vector pEXT2; its application to the proximal region of the human chromosome 21 long arm
Authors: Ozawa Naoki , Kano Takeshi , Taga Chiharu , Hattori Masahira , Sakaki Yoshiyuki , Suzuki Harukazu
Published: July 2002

We have developed an exon‐trapping system with a newly constructed trapping vector containing multiple cloning sites (designated pEXT2). The system revealed high sensitivity for trapping a control exon from several hundred kbp of DNA. We have applied the system to the cosmid clones located on human chromosome 21pll–q21, and identified two fragments highly homologous to neurofibromatosis 1 (NF1) gene and a clearly transcribed fragment hybridized with ~1.6 kb RNA from human brain and human glioblastoma A 172 cell.
A human T lymphotropic virus type I (HTLV‐I) long terminal repeat‐directed antisense c‐myc construct with an Epstein‐Barr virus replicon vector inhibits cell growth in a HTLV‐I‐transformed human T cell line
Authors: Fujita Masatoshi , Shiku Hiroshi
Published: July 2002

A panel of EB virus replicon‐based vectors was constructed to examine the relative utility of four distinct eukaryotic promoters for high‐level gene expression in a HTLV‐I‐transformed human T cell line, HUT102. We found that HTLV‐I LTR, which is trans‐activated by the viral tax protein, was most suited for EBV vector‐based stable gene expression in it. We prepared a HTLV‐I LTR‐directed antisense c‐myc construct with an EBV vector. This antisense plasmid suppressed c‐myc expression and inhibited growth of HUT102 cells in vitro with unaltered expression of tax. Non‐specific plasmid toxicity was excluded by showing that the antisense construct had little effect on growth and c‐myc expression of HTLV‐I‐negative Jurkat T cells, in which the viral LTR is expected to be less active. Our results indicate that c‐myc may play an important role in the deregulated growth of HTLV‐I‐transformed T cells.
Estradiol up‐regulates the stimulatory GTP‐binding protein expression in the MCF‐7 human mammary carcinoma cell line
Authors: Yasutomo Yoshiro , Shimada Nobuko , Kimura Narimichi , Nagata Naokazu
Published: July 2002

The effect of estradiol treatment of the human mammary carcinoma cell MCF‐7 on the adenylyl cyclase system was examined. Treatment with 10 nM estradiol for 72 h increased the basal level ofcAMP, and isoproterenol‐, PGE2‐ or calcitonin‐stimulated cAMP production. Estradiol also increased the response to cholera toxin but did not alter the response to forskolin. No significant change in growth rate was observed during the 72 h of estradiol treatment. In MCF‐7 cell membranes the responsiveness to isoproterenol, PGE2, or cholera toxin was also enhanced by estradiol treatment. The cholera toxin‐catalyzed ADP‐ribosylation of gsoe in MCF‐7 cell membranes was significantly increased by 72 h of treatment with estradiol. Consistent with this observation, the level of Gsα immunoreactivity was increased in the estradiol‐treated cell membranes. On the other hand, pertussis toxin did not change the responsiveness to isoproterenol, PGE2 or calcitonin in either control or estradiol‐treated cells. In addition, ADP‐ribosylation with pertussis toxin also did not reveal any change in Gi. These results clearly indicate that Gs expression is under the control of estradiol, and that this effect may contribute to the increased sensitivity of hormone‐stimulated adenylyl cyclase activities in MCF‐7 cells.
Age‐dependent deamidation of αB‐crystallin
Authors: Groenen Patricia J.T.A. , van Dongen Maria J.P. , Voorter Christina E.M. , Bloemendal Hans , de Jong Wilfried W.
Published: July 2002

Bovine and human αB‐crystallin undergo deamidation upon aging in the lens. In bovine αB‐crystallin, the specific site of dearnidation has been identified by peptide mapping after tryptic digestion. Asn‐146 was found to be subject to deamidation, whereas the only other asparagine residue, at position 78, is not affected. Asn‐146 is flanked at the carboxylic side by a glyeyl residue. Yet, the rate of in vivo deamidation is low. In vitro studies reveal that the deamidation is accompanied by significant racemization, indicating that the deamidation proceeds via formation of a succinimide intermediate.
Isolation of human cardiac troponin T and localization of epitopes recognized by monoclonal antibodies to cardiac troponin T
Authors: Katrukha A.G. , Bogatcheva N.V. , Gusev N.B.
Published: July 2002

Human cardiac troponin T has been isolated and its properties compared with those of rabbit skeletal and bovine cardiac troponin T. Seven monoclonal antibodies to troponin T have been obtained. Two antibodies cross‐reacted with both cardiac and skeletal troponin T and recognized epitopes located between residues 98–177 of bovine cardiac troponin T. Five other antibodies were specific for cardiac troponin T and recognized antigenic determinants located between residues 180–258 of bovine cardiac troponin T. Localization of antigenic determinants in the central part of troponin T seems to be due to the high hydrophilicity and flexibility of this part of the molecule. The monoclonal antibodies thus obtained may be used for diagnosing various types of human heart diseases.
A new spectral intermediate in cyanide binding with the oxidized cytochrome c oxidase
Authors: Berka Vladimir , Vygodina Tatiana , Musatov Andrey , Nicholls P. , Konstantmov Alexander A.
Published: July 2002

Reaction of cyanide with oxidized cytochrome c oxidase at a low concentration of the ligand and pH > 8 reveals an initial phase, not reported earlier, associated with a small blue shift of the absorption spectrum, which is followed by a conventional red shift of the heme a
3
3+. The initial blue shift resembles the spectral changes induced under the same conditions by low concentrations of azide and it is not observed in the presence of 0.3 mM azide. It is suggested that, similarly to NO, cyanide and HN3 cannot only bind to heme a
3 but to Cu2+
B as well, perturbing the spectrum of a
3+
3 indirectly. A rapid binding to Cu2+
B could provide the long‐sought intermediate in the cyanide reaction with heme a
3+
3, the existence of which is implied by the Michaelis‐Menten type kinetics of the latter process.
Mosquito DNA polymerase ε
Authors: Lee Seung-Koo , Fuchs Morton S.
Published: July 2002

The mosquito homolog of mammalian DNA polymerase ϵ, formerly known as a proliferating cell nuclear antigen (PCNA)‐independent form of DNA polymerase δ, has been purified from mosquito larval extracts. The polymerase ϵ was separated from DNA polymerase α by chromatography on hydroxylapatite, and the enzyme was subsequently purified on single‐stranded DNA agarose, followed by a 5' AMP‐agarose chromatography step. The purified polymerase exhibits an intrinsic 3'‐5' exonuclease activity and shows high activity using an oligo‐primed DNA template. Neither human nor Drosophila PCNA stimulated this polymerase activity. Additional immunochemical and biochemical evidence indicates that this enzyme is distinct from DNA polymerase α.
The in vivo assembly and function of the N‐ and C‐terminal halves of the Tn 10‐encoded TetA protein in Escherichia coli
Authors: Yamaguchi Akihito , Someya Yuichi , Sawai Tetsuo
Published: July 2002

The tetA gene was cut into its N‐ and C‐terminal halves at the central EcoRI site and the two halves were subcloned individually or together under a separate lac promoter/operator. The expression of the C‐terminal half was detected with a C‐terminal‐specific antibody. The amount of the N‐terminal half in the cytoplasmic membrane was not affected by the presence of the C‐terminal half. In contrast, the amount of the C‐terminal half in the membrane was increased in the presence of the N‐terminal half, indicating that the N‐terminal half helps the stable folding of the C‐terminal half in the membrane. Each half individually showed no tetracycline transport activity, however, when both halves were expressed together, the resultant complex showed about 40% of the tetracycline transport activity of the wild‐type per number of the C‐terminals of TetA protein in the membrane.
Application of poly(A)+RNA patterns method for searching of differentially expressed genes
Authors: Chenchik Alexander A. , Diachenko Ludmila B. , Beabealashvilli Robert Sh.
Published: July 2002

Poly(A)+RNA composition differences for normal, fetal and cirrhotic human liver before and after retinoic acid‐induced differentiation of the F9 embryonal carcinoma cell line were analyzed by a novel poly(A)+RNA patterns method. The method is based on the polyacrylamide gel electrophoretic analysis of short cDNA termination products, synthesized by reverse transcriptase using poly(A)+RNA as a template, a set of short 5'‐end labeled primers, three natural and one terminator deoxyribonucleotide. A number of known differentially expressed genes and some unknown ones were then identified by direct sequencing of the differentially represented bands excised from a gel and searching a complementary mRNA target sites in GenBank database.
Augmentation of human natural killer cells by splenopentin analogs
Authors: Rastogi A. , Singh V.K. , Biswas S. , Haq W. , Mathur K.B. , Agarwal S.S.
Published: July 2002

Splenopentin, Arg‐Lys‐Glu‐Val‐Tyr (SP‐5) and its synthetic analogs; Arg‐d‐Lys‐Glu‐Val‐Tyr (pentapeptide 1), Lys‐Lys‐Glu‐Val‐Tyr (2), d‐Lys‐Lys‐Glu‐Val‐Tyr (3), Arg‐Lys‐Gly‐Val‐Tyr (4), and Arg‐Lys‐Gln‐Val‐Tyr (5) have been examined for augmentation of human natural killer (NK) cell activity and human T‐cell transformation response. Pentapeptides 2 and 3 were found to significantly augment the in vitro human NK cell activity. However, none of them had any effect on lymphocyte proliferative responses.
Growth hormone, but not insulin‐like growth factor I, induces a serum protease activity for insulin‐like growth factor binding protein‐3 in hypophysectomized rats in vivo
Authors: Rutishauser Jonas , Schmid Christoph , Hauri Christina , Froesch E.Rudolf , Zapf Jürgen
Published: July 2002

Insulin‐like growth factor binding proteins (IGFBPs) modulate IGF action. Proteolytic cleavage of IGFBPs yields lower molecular forms with reduced ability to bind IGFs, thereby increasing IGF bioavailability. In serum from normal adult rats, we found a proteolytic activity for IGFBP‐3, presumably a cation‐dependent serine protease. It is lacking in serum from hypophysectomized rats and restored by infusion of growth hormone (GH), but not IGF I. Thus, IGF I does not appear to mediate the GH effect on IGFBP‐3 proteolysis. Rather, GH seems to modulate IGF action indirectly via alteration of IGFBP‐3 structure.
Conversion of GalNAcβ(1–4)GlcNAcβ‐OMe into GalNAcβ(1–4)‐[Fucα(1–3)]GlcNAcβ‐OMe using human milk α3/4‐fucosyltransferase synthesis of a novel terminal element in glycoprotein glycans
Authors: Bergwerff Aldert A. , van Kuik J.Albert , Schiphorst Wietske E.C.M. , Koeleman Carolien A.M. , van den Eijnden Dirk H. , Kamerling Johannis P. , Vliegenthart Johannes F.G.
Published: July 2002

Incubation of GalNAcβ(1–4)GlcNAcβ‐OMe with GDP‐Fuc in the presence of human milk α3/4‐fucosyltransferase resulted in the formation of GalNAcβ(1–4)[Fucα(1–3)]GlcNAcβ‐OMe. Under conditions that led to complete α3‐fucosylation of Galβ(1–4)GlcNAcβ‐OEt, GalNAcβ(1–4)GlcNAcβ‐OMe was fucosylated for more than 85%. For the identification of the isolated fucosylated products one‐ and two‐ dimensional 1H‐NMR spectroscopy was applied. In vacuo molecular dynamics simulations of Galβ(1–4)[Fucα(1–3)]GlcNAcβ‐OEt and GalNAcβ(1–4)[Fucα(1–3)]GlcNAcβ‐OMe using the CHARMm based force field CHEAT, demonstrated only small differences between the conformations of these compounds. This illustrates the minor conformational influence of the substituent at C‐2′, i.e. a hydroxyl function versus a N‐acetyl group.
Long‐term effects of thrombin require sustained activation of the functional thrombin receptor
Authors: Zacharias Ute , He Ci-Jiang , Nguyen Geneviève , Sraer Jean Daniel , Rondeau Eric
Published: July 2002

Thrombin is a potent activator of human glomerular epithelial cells (HGEC). Here we compare short‐term and long‐term effects of thrombin and thrombin receptor agonist peptide (TRAP) which selectively activates the functional thrombin receptor. TRAP, as thrombin, increases intracellular free Ca2+ concentration and acts synergistically with growth factors possessing tyrosine kinase receptors on DNA synthesis. Thrombin induces synthesis of proteins of the fibrinolytic system and cell proliferation if it is present for at least 8 h. TRAP alone does not stimulate protein synthesis and is not mitogenic. However, in the presence of the aminopeptidase inhibitor amastatin all long‐term effects of thrombin can be fully mimicked by TRAP. In conclusion, different effects of thrombin and TRAP may be related to the degradation of TRAP by cellular ectoenzymes. The recently cloned thrombin receptor accounts for early intracellular signals and long‐term cellular effects that require sustained activation of this receptor.
Microinjection of acylphosphatase blocks Xenopus laevis oocytes maturation induced by ras‐p21
Authors: Dolfi Fabrizio , Carnero Amancio , Ramponi Giampietro , Lacal Juan Carlos
Published: July 2002

ras proteins induce germinal vesicle breakdown (GVBD) when microinjected into Xenopus laevis oocytes. The mechanism of action is still unresolved, although several hypotheses have been proposed. Acylphosphatase is a cytosolic enzyme that specifically catalyses the hydrolysis of the carboxylphosphate bond of acylphosphate for the removal of acylphosphate residues of various membrane pumps. A direct effect of acylphosphatase on the regulation of ionic balance of a cell by interaction with ionic membrane pumps has been proposed. We have analyzed the effect of microinjecting acylphosphatase, by itself or along with ras‐p21 proteins or progesterone, into oocytes. The enzyme alone is unable to induce GVBD, but increases oocyte maturation induced by progesterone. By contrast, acylphosphatase blocked GVBD induced by microinjection of oncogenic ras‐p21. These data suggest that acylphosphatase acts synergistically or antagonistically with factors involved in proliferating signals by altering the intracellular ionic conditions of the cell, conforming the hypothesis that the intracellular ionic condition of the cell is important in the induction of proliferating signals, and that its perturbation may have a serious effect on signal transduction.
Protein kinase C isoforms in murine erythroleukemia cells and their involvement in the differentiation process
Authors: Patrone M. , Pessino A. , Passalacqua M. , Sparatore B. , Melloni E. , Pontremoli S.
Published: July 2002

In addition to α, δ and ε‐protein kinase C, murine erythroleukemia cells contain ζ‐PKC and also a c‐PKC isoform, named α1, which shows cross‐reactivity with an anti‐α‐PKC antipeptide antibody. In a C44 MEL cell clone, characterized by a high rate of differentiation, both c‐PKC forms are expressed at a level higher than that of the N23 MEL cell clone which differentiates at a low rate and contains higher levels of ε‐PKC and particularly of the δ‐PKC isozyme. In the course of MEL cell differentiation, δ‐PKC in N23 cells and α1‐PKC in C44 cells are rapidly down‐regulated and the overall process is almost completed before cell commitment. Of the other three PKC isozymes present in both clones, only α‐PKC is down‐regulated to a significant extent. It is proposed that modulation of the signal delivered by each PKC isozyme is one of the biochemical mechanisms involved in MEL cell differentiation.