bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional characterization of the promoter region of the mouse protein kinase C γ gene
Authors: Takanaga Hiromi , Mukai Hideyuki , Shimakawa Masaki , Konishi Hiroaki , Kikkawa Ushio , Koizumi Tamio , Ono Yoshitaka
Published: April 2003

Promoter activity of protein kinase C (PKC) γ gene was analysed by chloramphenicol acetyltransferase (CAT) assay using extracts from the cells transfected with various fusion constructs containing the 5′‐flanking region of the mouse PKC γ gene and CAT gene. Transient expression experiments in PC12 cells revealed that the upstream region of 87 bp from the transcriptional initiation site was sufficient for promoter activity. The region containing nucleotides 87 upstream from the transcriptional initiation site was shown to silence CAT activity in Balb/c3T3 cells, in which mRNA of PKC γ was not detected, suggesting that this region might contain a transcriptional regulatory element for the cell type‐specific expression of the PKC γ gene.
Isolation and characterisation of plant defensins from seeds of Asteraceae, Fabaceae, Hippocastanaceae and Saxifragaceae
Authors: Osborn Rupert W. , De Samblanx Genoveva W. , Thevissen Karin , Goderis Inge , Torrekens Sophie , Van Leuven Fred , Attenborough Sheila , Rees Sarah B. , Broekaert Willem F.
Published: April 2003

From seeds of Aesculus hippocastanum, Clitoria ternatea, Dahlia merckii and Heuchera sanguinea five antifungal proteins were isolated and shown to be homologous to plant defensins previously characterised from radish seeds and γ‐thionins from Poaceae seeds. Based on the spectrum of their antimicriobial activity and the morphological distortions they induce on fungi the peptides can be divided into two classes. The peptides did not inhibit any of three different α‐amylases.
Identification and localization of the first glutaredoxin in leaves of a higher plant
Authors: Morell S. , Follmann H. , Häberlein I.
Published: April 2003

Glutaredoxin(thioltransferase) has been identified and purified to homogeneity from spinach leaves. Its cytosolic localization was demonstrated by chromatographic and immunological analysis of extracts from isolated spinach chloroplasts and mitochondria, respectively. Spinach glutaredoxin shows a significant crossreactivity with antibodies raised against E. coli glutaredoxin and possesses a specific thioltransferase activity comparable to that of the E. coli protein. Minor thioltransferase activities (less than 10% of total leaf activity) have been observed in spinach chloroplasts which are probably due to the presence of trypsin inhibitor and thioredoxins (TRf and TRm).
The effect on actin ATPase of phalloidin and tetramethylrhodamine phalloidin
Authors: Pinaev George , Schutt Clarence E. , Lindberg Uno
Published: April 2003

Actin polymerization has been studied in the absence of excess nucleotide. Using G‐actin ATP monomers, it was shown that mechanical shearing stimulates ATP hydrolysis. The procedures used enabled the detection of differential effects of phalloidin and tetramethylrhodamine‐phalloidin, on the Pi‐release step of the actin ATPase. It is concluded that tetramethylrhodamine, in contrast to phalloidin, accelerates Pi‐release from actin filaments.
Effects of the selective protein kinase C inhibitor Ro 31‐7549 on human angiotensin II receptor desensitisation and intracellular calcium release
Authors: Barker Stewart , Kapas Supriya , Fluck Richard J. , Clark Adrian J.L.
Published: April 2003

The mechanism underlying type I angiotensin II (Ang II) receptor (AT1 receptor) desensitisation is unknown. Structural features suggest it may be a substrate for protein kinase C (PKC). The effects of a selective PKC inhibitor, Ro 31‐7549, on receptor desensitisation were investigated in CHO cells expressing the human AT1 receptor. Desensitisation was demonstrated with respect to the calcium response to Ang II in Fura‐2 ‐loaded cells. Ro 31‐7549 had no effect on desensitisation. However, pretreatment with Ro 31‐7549 caused a dose‐dependent reduction in calcium release from intracellular stores. PKC may therefore act at a locus distal from the receptor itself.
The membrane proximal region of the cytoplasmic domain of the growth hormone receptor is involved in the activation of Stat 3
Authors: Sotiropoulos Athanassia , Moutoussamy Soraya , Binart Nadine , Kelly Paul A. , Finidori Joëlle
Published: April 2003

Growth hormone receptor (GHR) signaling involves activation of the Janus Kinases (Jak) and of Stat proteins (signal transducers and activators of transcription). Growth hormone (GH) induces transcriptional activation of c‐fos gene and the c‐sis inducible element (SIE) of its promoter was shown to bind the Stat proteins. Using cells co‐transfected with GHR and Stat 3 expression vectors, we directly demonstrate that GH induces tyrosine phosphorylation of Stat 3 and its binding to the SIE probe. We showed, using mutant forms of GHR, that only the cytoplasmic membrane proximal domain of the receptor, including a conserved proline rich region (box 1), is required for this effect.
Leucine‐58 in the putative 5th helical region of human interleukin (IL)‐6 is important for activation of the IL‐6 signal transducer, gp130
Authors: de Hon Floris D. , Bos Hanny Klaasse , Ebeling Saskia B. , Grötzinger Joachim , Kurapkat Günther , Rose-John Stefan , Aarden Lucien A. , Brakenhoff Just.P.J.
Published: April 2003

A model of the tertiary structure of human IL‐6, derived from the crystal‐structure of granulocyte‐colony stimulating factor, reveals a 5th helical region in the loop between the first and second α‐helix. To investigate the importance of this region for biological activity of IL‐6, residues Glu‐52, Ser‐53, Ser‐54, Lys‐55, Glu‐56, Leu‐58 and Glu‐60 were individually replaced by alanine. IL‐6·Leu‐58Ala displayed a 5‐fold reduced biological activity on the IL‐6‐responsive human cell lines XG‐1 and A375. This reduction in bioactivity was shown to be due to a decreased capacity of the mutant protein to trigger IL‐6 receptor‐α‐chain‐dependent binding to the IL‐6 signal transducer, gp130.
Cleavage points of rabbit skeletal myosin light chains selectively modified in situ by limited proteolysis: structural characteristics of the neoformed isozymes
Authors: Burgat Jean-Marc , Ghelis Charis , Cardinaud Robert
Published: April 2003

The functional significance of myosin light chains in vertebrate striated muscle is an issue of interest and myosin species selectivity modified by papain or trypsin in their LC1 and LC2 light chains are potentially useful for further investigation. We therefore determined the cleavage sites resulting in the (T)‐LC1′, (P)‐LC1′ and (T)‐LC2′ species. Sequence analysis of (T)LC1′ indicated that the cleavage point in LC1 is at Lys7. Under appropriate conditions papain rapidly cleaves a short N‐terminal segment from myosin light chain 1 and produces a new isozyme specifically modified in its essential light chain 1. The cleavage occurred at either Ala11, Ala12 or Ala13, the Ala11 cleavage being the most frequent. Trypsin was used to produce a myosin species with a regulatory light chain 2 specifically truncated of a short N‐terminal segment. The cleavage was specific at Arg8 with no indication of other significant cleavage sites in this LC2. The effects of trypsin and papain on myosin light chains are different, indicating different proteolytic specificities. None of these modifications, including (CT)‐LC2″ cleavage at Phe19, changed the K+‐EDTA‐ and Ca2+‐ATPase activities of monomeric myosin significantly, indicating that LC1 and LC2 N‐termini have little or no direct influence on the active site. An electric birefringence study also showed that these modified species retained their average shape and flexibility. These observations are essential in showing that the role of light chain extremities is expressed only in the presence of a minimum of structural organization (filament or acto‐myosin complex).
Cannabinoids enhance human B‐cell growth at low nanomolar concentrations
Authors: Derocq J.-M. , Ségui M. , Marchand J. , Le Fur G. , Casellas P.
Published: April 2003

This study examined the effect of cannabinoid ligands on human tonsillar B‐cells activated either through cross‐linking of surface immunoglobulins or ligation of the CD40 antigen. The two synthetic cannabinoids, CP55,940 and WIN55212‐2, as well as Δ9‐tetrahydrocannabinol (THC), the psychoactive component of marijuana, caused a dose‐dependent increase of B‐cell proliferation and displayed EC50 at low nanomolar concentrations. This cannabinoid‐induced enhancing activity was inhibited by pertussis toxin which suggested a G‐protein‐coupled receptor process. In addition, the absence of antagonistic effect of SR141716A, a specific CB1 receptor antagonist, together with the demonstration that human B‐cells displayed large amount of CB2 receptor mRNAs, led us to assume that the growth enhancing activity observed on B‐cells at very low concentrations of cannabinoids could be mediated through the CB2 receptor.
The role of ATP hydrolysis in the function of the chaperonin GroEL: dynamic complex formation with GroES
Authors: Kawata Yasushi , Hongo Kunihiro , Nosaka Koji , Furutsu Yoshinobu , Mizobata Tomohiro , Nagai Jun
Published: April 2003

In order to understand the role of ATP hydrolysis of the chaperonin GroEL during protein folding, we have studied GroEL‐GroES complex formation in the presence of ATP or ADP by using capillary electrophoresis and surface plasmon resonance. Capillary electrophoresis analysis showed that the GroEL 14‐mer and GroES 7‐mer formed a 1:1 complex in the presence of ATP. In the presence of ADP, both the association and dissociation rates of the complex were slower by about one order of magnitude than the rates in the presence of ATP at 25°C. The implications of such a stable complex on the overall mechanism of chaperonin function are discussed.
Cyclophilin‐A is a zinc‐dependent DNA binding protein in macrophages
Authors: Krummrei Ulrike , Bang Renate , Schmidtchen Robert , Brune Kay , Bang Holger
Published: April 2003

The association of cyclosporin A (CsA) immunosuppression with inhibition of transcription factor‐dependent lymphokine gene activation formed the basis of our decision to investigate nuclear‐associated Cyp isoforms. Immunofluorescence microscopy of mouse macrophages cell line with a monoclonal antibody mAb7F1 raised against CypA shows a co‐localisation of CypA in the nucleus and in the cytosol. Nuclear CypA binds to DNA in a zinc ion‐dependent manner, in contrast to recombinant CypB. Peptidyl‐prolyl cisltrans isomerase (PPIase) activity of nuclear CypA is inhibited by zinc ions. The zinc inhibited CypA does not bind cyclosporin A (CsA). We suggest that nuclear Cyp in complex with zinc ions recognizes DNA sequences and is involved in transcription modulating processes.
The primary structure of carboxypeptidase S3 from Penicillium janthinellum IBT 3991
Authors: Svendsen Ib , Day Estera Szwajcer
Published: April 2003

The complete amino acid sequence of penicillopeptidase S3, a serine carboxypeptidase isolated from Penicillium janthinellum IBT 3991, has been determined. The enzyme consists of 481 amino acids arranged in a single polypeptide chain. Six glycosylation sites were established in positions 41, 218, 256, 326, 384 and 392. The molecule contains six cysteinyl residues among which disulfide bridges was established between Cys‐71‐Cys‐333 and Cys‐233‐Cys‐289. Carboxypeptidase S3 is homologous to carboxypeptidase PEPF (or carboxypeptidase I) from Aspergillus niger (67% identical positions). It is proposed that these enzymes form a separate sub‐family among the serine carboxypeptidases.
Conformational changes in mutant lysozymes detected with monoclonal antibody
Authors: Kato Akio , Shimizu Toshiaki , Saga Shinichiro
Published: April 2003

A monoclonal antibody (mAb) against hen egg white lysozyme (HEWL) with the exquisitely sensitive specificity to native conformation was prepared to detect the conformational changes in mutant lysozymes constructed by genetic modification in a yeast expression system. The binding of mAb with lysozyme was decreased both by denaturation with heat and guanidine‐HCl, corresponding to the denaturation curves of lysozyme. These results demonstrate that mAb is a powerful probe to monitor the conformational changes in the lysozyme molecule. By using this probe, the conformational change of various mutant lysozymes was detected. A good correlation was observed between the binding with mAb and the ΔG (Gibbs free energy change), reflecting the conformational stability of wild‐type and seven mutant lysozymes. This result suggests that a monoclonal antibody with the specificity for native conformation can be used as a powerful prove of protein conformation.
The derivation of the oxygen atoms of the 131‐oxo and 3‐acetyl groups of bacteriochlorophyll a from water in Rhodobacter sphaeroides cells adapting from respiratory to photosynthetic conditions: evidence for an anaerobic pathway for the formation of isocyclic ring E
Authors: Porra R.J. , Schäfer W. , Katheder Ingrid , Scheer H.
Published: April 2003

Using mass spectrometry, we have demonstrated 18O‐labelling of both the 131‐oxo and 3‐acetyl groups of newly‐formed bacteriochlorophyll a synthesized by Rhodobacter sphaeroides cells during adaptation from respiratory to photosynthetic conditions in the presence of H2
18O. This derivation of the 131‐oxo group of bacteriochlorophyll a from water provides a stark constrast with that of chlorophylls in higher plants where ring E formation is an aerobic process in which the 131‐oxo group arises from molecular oxygen via an oxygenase activity. The formation of the 3‐acetyl group of bacteriochlorophyll a, however, is consistent with the enzymic hydration of the 3‐vinyl group of a derivative of chlorophyll a.
Interaction of glyceraldehyde‐3‐phosphate dehydrogenase with SH‐containing compounds: evidence for the binding of l‐cysteine and for the dependence of the binding on the functional state of the enzyme
Authors: Schmalhausen Elena V. , Muronetz Vladimir I. , Nagradova Natalia K.
Published: April 2003

Incorporation of l‐[35S]cysteine into rabbit muscle glyceraldehyde‐3‐phosphate dehydrogenase was detected following incubation of the enzyme in a mixture containing glyceraldehyde‐3‐phosphate, NAD+ and the labeled cysteine. Insignificant binding occurred in the absence of either the substrate or NAD+, suggesting that formation of an acylated enzyme form was a prerequisite for the binding. Stoichiometry of the binding depended on the number of functioning active centers; up to 4 moles of l[35S]cysteine bound per mole tetramer with fresh enzyme preparations. The l‐[35S]cysteine incorporation depended on pH and was maximal when a group having pKa of 8.5 is protonated. To clarify the relevance of this finding to the effect of SH‐containing compounds previously shown to decrease the rate of 3‐phosphoglyceroyl‐enzyme hydrolysis [Kuzminskaya et al., FEBS Lett. 336 (1993) 208–210], the pH‐dependence of the effect of glutathione on the hydrolysis rate was determined and found to be close to the pH‐dependence of l‐[35S]cysteine binding.
Mechanism of Lac repressor switch‐off: orientation of the Lac repressor DNA‐binding domain is reversed upon inducer binding
Authors: Kamashev Dimitrii E. , Esipova Natalia G. , Ebralidse Konstantin K. , Mirzabekov A.D.
Published: April 2003

Lac repressor's DNA‐binding domains contain helix‐turn‐helix motif which, though similar to those of phage λ Cro protein, are oriented differently with respect to DNA: in the specific complexes with Lac operator, N termini of the repressor's subunits are facing inwards. We demonstrate that, in the presence of an inducer, the repressor's N termini cross‐link to the operator's outermost nucleotides. We suggest that the inducer fixes the repressor's DNA‐binding domains in the Cro‐type configuration and thus garbles its recognition surface. Since the Cro‐type configuration is perfectly suitable for binding the DNA, this also explains how the switched‐off repressor retains its non‐specific DNA‐binding.
Analysis of a novel defective HTLV‐I provirus and detection of a new HTLV‐I‐induced cellular transcript
Authors: Kubota Satoshi , Furuta Rika A. , Siomi Haruhiko , Maki Masatoshi , Hatanaka Masakazu
Published: April 2003

HTLV‐I generally integrates at least one full‐length copy in adult T‐cell leukemia (ATL) cells. A group of patients without full‐length provirus have a unique conserved truncation of the provirus which retains env‐pX‐3′LTR. Tumor cells of a patient from this group were genetically analyzed. Analysis of the 5′ and 3′ cellular flanking region adjacent to the provirus suggest that the defective provirus was integrated immediately downstream of a promoter of an unknown cellular gene. The activity of the promoter was weak but was responsive to Tax‐like HTLV‐I LTR. The provirus may have utilized it as a substitute for the 5′LTR and thus 3′LTR may have become an alternative promoter for the cellular gene, which may give similar viral‐cellular interactions to that of general cases with full‐length proviruses. Surprisingly, the 3′ cellular flanking region which is thought to be controlled originally by the promoter is constitutively expressed specifically in an HTLV‐I producing ATL cell line HUT102G, in which the corresponding region is not modified by provirus. The detection of this HTLV‐I‐induced transcript provides a probe to find an HTLV‐I inducible unknown cellular gene that may be related to the pathogenesis of ATL.
Hucolin, a new corticosteroid‐binding protein from human plasma with structural similarities to ficolins, transforming growth factor‐β1‐binding proteins
Authors: Edgar Paul F.
Published: April 2003

In order to study the cortisol‐binding factors in blood, human plasma was applied to a 11β‐hydroxy‐3‐oxo‐4‐androstene‐17β‐carboxyaminoethylamine‐(HACA) 1,4‐butanediol diglycidyl ether‐Sepharose column. Elution of the column with cortisol buffer produced two protein peaks, the minor peak yielded a protein complex of molecular weight ∼200 kDa, subsequently termed hucolin. SDS‐PAGE analysis under reducing and non‐reducing conditions revealed hucolin was a disulphide‐linked complex of 35‐kDa and 75‐kDa subunits. Twenty‐five amino acid residues of the N‐terminus of the 35‐kDa subunit were determined and homology searches revealed an 88% sequence identity with the N‐terminal region of β‐ficolin, a transforming growth factor‐β1 (TGF‐β1)‐binding protein purified from porcine uterus.
Mapping the heparin‐binding domain of boar spermadhesins
Authors: Calvete Juan J. , Dostàlovà Zuzana , Sanz Libia , Adermann Knut , Thole Hubert H. , Töpfer-Petersen Edda
Published: April 2003

Boar spermadhesins are a group of seminal plasma, heparin‐binding proteins which appear to be involved in sperm capacitation and gamete interaction. Using a proteolytic protection assay we have identified regions of AQN‐1, AQN‐3, PSP‐I and AWN which remain attached to a heparin‐Sepharose column following in‐column digestion of bound spermadhesins with chymotrypsin and elastase. In addition, the complete amino acid sequence of spermadhesin AWN was synthesized as overlapping peptides, and their ability to bind to a heparin‐Sepharose column and to inhibit the interaction of soluble heparin with purified ELISA plate‐coated AWN was tested. Both approaches gave similar results and as a whole showed that different regions of AWN may converge in its tertiary structure to form a composite heparin‐binding site. The conformational heparin‐binding surface resides on the GFCC′C″ face of the proposed structural model for AWN and is in an opposite location to the carbohydrate‐binding region of the spermadhesin.
Cyclic aristeromycin diphosphate ribose: A potent and poorly hydrolysable Ca2+‐mobilising mimic of cyclic adenosine diphosphate ribose
Authors: Bailey Victoria C. , Fortt Simon M. , Summerhill Robin J. , Galione Antony , Potter Barry V.L.
Published: April 2003

Cyclic aristeromycin diphosphate ribose, a carbocyclic analogue of cyclic adenosine diphosphate ribose, was synthesised using a chemo‐enzymatic route involving activation of aristeromycin 5′‐phosphate by diphenyl phosphochloridate. The calcium‐releasing properties of this novel analogue were investigated in sea urchin egg homogenates. While cyclic aristeromycin diphosphate ribose has a calcium release profile similar to that of cyclic adenosine diphosphate ribose (EC
50 values are 80 nM and 30 nM, respectively), it is degraded significantly more slowly ( values are 170 min and 15 min, respectively) and may, therefore, be a useful tool to investigate the activities of cyclic adenosine diphosphate ribose.
The nuclear‐encoded 18 kDa (IP) AQDQ subunit of bovine heart complex I is phosphorylated by the mitochondrial cAMP‐dependent protein kinase
Authors: Papa Sergio , Sardanelli Anna Maria , Cocco Tiziana , Speranza Francesco , Scacco Salvatore C. , Technikova-Dobrova Zuzana
Published: April 2003

In bovine heart mitochondria a protein of M
r 18 kDa, phosphorylated by mtPKA, is associated to the NADH‐ubiquinone oxidoreductase in the inner membrane and is present in purified preparation of this complex. The 18 kDa phosphoprotein has now been isolated and sequenced. It is identified as the 18 kDa (IP) AQDQ subunit of complex I, a protein of 133 amino acids with a phosphorylation consensus site RVS at position 129–131.
Partial purification and characterization of a protein kinase that is activated by nuclear localization signal peptides
Authors: Kurihara Toshinao , Hori Masatsugu , Takeda Hiroshi , Inoue Michitoshi , Yoneda Yoshihiro
Published: April 2003

A nuclear localization signal (NLS) is required for the transport of karyophilic proteins from the cytoplasm to the nucleus. In this study, NLS was examined in terms of its effect on diverse cellular functions such as protein phosphorylation reactions. When synthetic peptides containing the NLS of SV40 T‐antigen were injected into the cytoplasm of Xenopus oocytes, and the oocytes incubated with [32P]phosphorus‐containing medium, a 32 kDa protein was found to be preferentially phosphorylated in an NLS‐dependent manner. The incubation of fractionated cytosolic extracts prepared from mouse Ehrlich ascites tumor cells with [γ‐32P]ATP in the presence of the NLS peptides, results in the stimulation of the phosphorylation of several proteins. Similar in vitro stimulation was observed by other functional NLS peptides such as those of polyoma virus T‐antigen and nucleoplasmin. Little or no stimulation, however, was detected for peptides of mutant type and reverse type NLS of SV40 T‐antigen, and the C‐terminal portion of lamin B. Using an in vitro assay, the phosphorylation activity was fractionated chromatographically and a fraction was obtained which contained a high level of activity. The fraction was found to contain three major proteins having molecular masses of 95, 70, and 43 kDa. The in vivo and in vitro results are consistent with the existence of a protein kinase, called NLS kinase, that is specifically activated by NLS peptides.
The SV40 T‐antigen induces premature apoptotic mammary gland involution during late pregnancy in transgenic mice
Authors: Tzeng Y.J. , Gottlob K. , Santarelli R. , Graessmann A.
Published: April 2003

Transgenic animals of the line 8 contain the WAP‐SV‐T transgene. Females of this line synthesise the SV40 T‐antigen in mammary gland epithelial cells during pregnancy and the lactation period. All females are ‘milk‐less’ and the offspring have to be nursed by foster mothers. The reason for this phenomenon is a premature apoptosis during late pregnancy. Nonetheless a significant number of mammary epithelial cells escape apoptosis and all transgenic females develop breast cancer after the first lactation period.
Association of Drosophila cysteine string proteins with membranes
Authors: van de Goor Jana , Kelly Regis B.
Published: April 2003

Cysteine string proteins are putative synaptic vesicle proteins that lack a transmembrane domain. Our analysis shows that Drosophila cysteine string proteins are extensively modified by hydroxylamine‐sensitive fatty acylation. This modification could be responsible for association of csp's with membranes. Extensive deacylation of Dcsp's by a 20 h incubation in 1 M hydroxylamine, pH 7.0, or methanolic KOH produces a protein of 6–7 kDa lower mass than untreated Dcsp's. Surprisingly, the hydroxylamine treatment does not cause release of Dcsp's from membranes. On the other hand, alkaline stripping of membranes isolated from Drosophila brain by 0.1 M sodium carbonate, pH 11.5, causes a significant release of Dcsp's from membranes into the cytosol. These results indicate that fatty acylation may not form the main anchor of Dcsp's in membranes. Taking advantage of the endocytotic block in the Drosophila mutant shibire
ts1, we analyzed the acylation states of Dcsp's in two stages during synaptic vesicle recycling and found no evidence for an acylation/ deacylation cycle of Dcsp's in the brain nerve terminals.
Functional regulation of reconstituted Na, K‐ATPase by protein kinase A phosphorylation
Authors: Cornelius Flemming , Logvinenko Ninel
Published: April 2003

Reconstituted Na+,K+‐ATPase from either pig kidney or shark rectal glands was phosphorylated by cAMP dependent protein kinase, PKA. The stoichiometry was ∼ 0.9 mole Pi/mole α‐subunit in the pig kidney enzyme and ∼ 0.2 mol Pi/mol α‐subunit in the shark enzyme. In shark Na+,K+‐ATPase PKA phosphorylation increased the maximum hydrolytic activity for cytoplasmic Na+ activation and extracellular K+ activation without affecting the apparent K
m values. In contrast, no significant functional effect after PKA phosphorylation was observed in pig kidney Na+,K+‐ATPase.
SNAP‐25 is differentially expressed by noradrenergic and adrenergic chromaffin cells
Authors: Kannan Rama , Grant Nancy J. , Aunis Dominique , Langley Keith
Published: April 2003

This study examines chromaffin cell expression of the synaptosomal‐associated protein SNAP‐25 in the adrenal medulla by immunoblotting, immunocytochemistry and PCR. Both mRNAs coding for the SNAP‐25 isoforms a and b were detected and SNAP‐25 was found to be present in all chromaffin cells in adult rat adrenal gland sections. It was essentially restricted to a zone close to the cytoplasmic face of the plasma membrane in the majority of cells, but located extensively throughout the cytoplasm in a chromaffin cell sub‐population, identified by double immunofluorescence labelling to have a noradrenergic phenotype. This differential SNAP‐25 expression may reflect different stages in the phenotypic development of the sympathoadrenal lineage and be related to an additional functional role in noradrenergic chromaffin cells not associated with secretion.
Inhibition of neutrophil elastase by the α1‐proteinase inhibitor‐immunoglobulin A complex
Authors: Adam Christophe , Bieth Joseph G.
Published: April 2003

Neutrophil elastase is thought to be involved in cartilage destruction occurring in rheumatoid arthritis despite the local presence of α
1‐proteinase inhibitor. Part of synovial fluid α
1‐proteinase inhibitor forms a mixed disulfide with immunoglobulin A, which has been postulated to lack inhibitory activity. We show here that the immunoglobulin‐inhibitor complex tightly inhibits neutrophil elastase and cathepsin G, bovine pancreatic trypsin and chymotrypsin, and porcine pancreatic elastase. Although the rate constant of inhibition of neutrophil elastase by immunoglobulin A‐bound α
1‐proteinase inhibitor (k
ass = 9.2 × 105 M−1 · s−1) is about 10‐fold lower than that measured with the free inhibitor, it is high enough to enable efficient inhibition of elastase in vivo.
Gene duplication and the evolution of photosynthetic reaction center proteins
Authors: Lockhart Peter J. , Steel Michael A. , Larkum Anthony W.D.
Published: April 2003

We investigate the evolutionary relationships between photosynthetic reaction center proteins (D1, D2, L and M) and demonstrate that the pattern of nucleotide substitution in these is more complicated than has been assumed in previous phylogenetic analyses. We show that there are serious violations of methodological assumptions in previous published studies. We conclude that there is equal support for hypotheses indicating (i) a single gene duplication of an ancestral reaction center protein followed by diversification and (ii) two independent gene duplications giving rise to proteins in oxygenic and anoxygenic systems.
Dependence on Mg2+ ions of the activities of dimeric hammerhead minizymes
Authors: Amontov Sergei , Nishikawa Satoshi , Taira Kazunari
Published: April 2003

A minizyme is a hammerhead ribozyme with short oligonucleotide linkers instead of stem/loop II. In a previous study we demonstrated that a minizyme with high‐level activity forms a dimeric structure with a common stem II [Amontov and Taira (1996) J. Am. Chem. Soc. 118, 1624–1628]. We now demonstrate that the stability of the dimeric structure is influenced by Mg2+ ions. We found that the dependence on Mg2+ ions of the activity of homodimeric minizyme (a dimer with two identical binding sites) has composite biphasic characteristics. When the concentration of Mg2+ ions reached a specific critical level, the dependence on the concentration of Mg2+ ions lost its tendency to reach a plateau. In the case of the heterodimeric minizyme (a dimer with two different binding sites), we investigated the kinetic behavior of two different forms of the dimer, namely, free dimer and the complex of the dimer with an uncleavable substrate. The kinetic behavior of the free heterodimer was very similar to that of the homodimeric minizyme. In contrast, in the presence of the uncleavable substrate at a concentration as high as that of the minizyme, the curve for the dependence on Mg2+ ions showed normal saturation kinetics. While, at low concentrations of Mg2+ ions, the activity of the heterodimers was much higher when the dimeric structure was stabilized by the presence of the uncleavable substrate, at high concentrations of Mg2+ ions, this difference in activity became less and less significant. Thus, high concentrations of Mg2+ ions were able to stabilize the dimeric minizymes in the absence of the uncleavable substrate.
Human high affinity, Na+‐dependent l‐glutamate/l‐aspartate transporter GLAST‐1 (EAAT‐1) : gene structure and localization to chromosome 5p11‐p12
Authors: Stoffel Wilhelm , Sasse Jürgen , Düker Maria , Müller Rolf , Hofmann Kay , Fink Thomas , Lichter Peter
Published: April 2003

The gene of the human l‐glutamate transporter hGLAST‐1 (EAAT‐1) has been isolated and characterized. The 1626 bp cDNA open reading frame (542 aa) is distributed over ten exons and at least 85 kb on chromosome 5p11‐p12. The gene is unrelated to any other previously described neurotransmitter transporter gene family, but its exon/intron structure corresponds largely to that of the Na+‐dependent neutral amino acid transporter ASCT‐1. GLAST‐1, ASCT‐1 and the glutamate transporters GLT‐1 and EAAC‐1 have strongly similar amino acid sequences. The l‐glutamate transporter gene structures might help to understand the correlation of l‐glutamate reuptake in neurodegenerative disorders.
RpoS‐dependent regulation of genes expressed at late stationary phase in Escherichia coli
Authors: Talukder Ali Azam , Yanai Satoshi , Nitta Takeshi , Kato Akio , Yamada Mamoru
Published: April 2003

We have identified 6 Escherichia coli genomic genes, including 4 new genes, responsive to the stationary phase. One of them was regulated positively by RpoS at the stationary phase, and the remaining 5 negatively at a late stationary phase, all of them responding to multiple environmental stresses. Nucleotide sequences as well as such multiple responses revealed that those genes may have more than one overlapping‐promoter recognized by different σ‐factors which regulate gene expressions during their cell growth.
Molecular structure and genetic mapping of the mouse gastrin gene
Authors: Friis-Hansen Lennart , Rourke Ian J. , Bundgaard Jens R. , Rehfeld Jens F. , Samuelson Linda C.
Published: April 2003

The mouse gastrin gene has three exons totalling 460 bp and a deduced preprogastrin of 101 amino acids. The sequence of murine gastrin‐34 is 94% identical to rat gastrin‐34 and 76% identical to human gastrin‐34. At Arg79, mouse progastrin has a unique cleavage site that might allow species‐specific synthesis of gastrin‐13. Northern analysis and RT‐PCR demonstrated that gastrin gene transcripts are abundant in mouse stomach and duodenum and present at low levels in brain, ovary and pancreas, similar to the pattern described for other mammals. The gastrin gene was mapped to the distal region of mouse chromosome 11.
Differential display PCR reveals increased expression of 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase by lithium
Authors: Wang Jun-Feng , Young L.Trevor
Published: April 2003

Differential display PCR was used to study the effects of lithium on gene expression. Four candidate genes were isolated and verified by Northern hybridization after 1 week treatment of C6 glioma cells with therapeutically relevant concentrations of LiCl (1 mM). Sequencing analysis revealed three previously unidentified cDNA fragments in addition to a sequence with 99% homology with the cDNA for 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase type II (CNPaseII). Since CNPaseII is important in myelinogenesis and possibly neuronal growth and repair, the present findings suggest that lithium treatment may regulate these processes.
The design of an alternative, covalently flavinylated 6‐hydroxy‐d‐nicotine oxidase by replacing the FAD‐binding histidine by cysteine and reconstitution of the holoenzyme with 8‐(methylsulfonyl)FAD
Authors: Stoltz Michaela , Henninger Hans-Peter , Brandsch Roderich
Published: April 2003

The cofactor of several flavoenzymes is autocatalytically bound to the polypeptide via a histidyl(N3)‐(8α)‐FAD linkage which makes the generation of apoenzyme difficult. We introduced an alternative covalent protein‐FAD bond at the active site of 6‐hydroxy‐n‐nicotine oxidase (6HDNO) by replacing the FAD‐binding histidine with cysteine. The resulting mutant enzyme was expressed with noncovalently attached cofactor. Incubation with 8‐(methylsulfonyl)FAD, and less efficiently with 8‐chloro‐FAD, resulted in the spontaneous replacement of the noncovalently bound FAD by the flavin derivative and the formation of an 8‐(N‐acetylcysteinyl)FAD linkage. The flavinylated 6HDNO.cys exhibited close to wild‐type activity levels. This strategy may be generally applicable to the attachment of artificially designed flavin derivatives to the active site of covalently flavinylated enzymes.
Granulocyte colony‐stimulating factor induces the binding of STAT1 and STAT3 to the IFNγ response region within the promoter of the FcγRI/CD64 gene in human neutrophils
Authors: Bovolenta Chiara , Gasperini Sara , Cassatella Marco A.
Published: April 2003

Granulocyte colony‐stimulating factor (G‐CSF) has been recently shown to induce the high‐affinity Fc receptor for IgG (FcγRI/CD64) in human polymorphonuclear neutrophils (PMN). To elucidate the molecular mechanisms whereby G‐CSF exerts this effect, we examined whether the cytokine induces the binding of transcription factors to the IFNγ response region (GRR), a well characterized regulatory element in the FCγRI promoter that is responsible for the transcriptional induction of this gene. Using electrophoretic mobility shift assays, we show that in human PMN, G‐CSF activates a GRR‐binding complex which contains members of the signal transducer and activator of transcription (STAT) family of proteins, namely STAT1 and STAT3. In keeping with this result, treatment of neutrophils with G‐CSF led to tyrosine phosphorylation of STAT3, as determined by immunoprecipitation followed by immunoblotting with antiphosphotyrosine antibodies. This is the first demonstration that in human neutrophils, the induction by G‐CSF of FcγRI gene expression may be mediated by the binding of STAT1 and STAT3 to the GRR sequence.
5S rRNA binding ribosomal proteins from Thermus thermophilus: identification and some structural properties
Authors: Gongadze G. , Kashparov I. , Lorenz S. , Schroeder W. , Erdmann V.A. , Liljas A. , Garber M.
Published: April 2003

An unusual acidic ribosomal protein from Thermus thermophilus, TL5, that binds to 5S rRNA specifically and strongly, has been investigated. The N‐terminal sequence of TL5 does not reveal any homology with known ribosomal proteins. Two large tryptic fragments of TL5 have been isolated and characterized. 5S rRNA protected TL5 and its unstable N‐terminal fragment against trypsin action. The 5S rRNA binding ability of TL5 is probably inherent in its N‐terminal part. The other 5S rRNA binding ribosomal protein from T. thermophilus, TL4, has been identified as a homolog of the ribosomal protein L5 from Escherichia coli.
The cloning, expression and crystallisation of a thermostable arginase
Authors: Bewley Maria C. , Lott J.Shaun , Baker Edward N. , Patchett Mark L.
Published: April 2003

The gene for the thermostable arginase from the thermophilic bacterium ‘Bacillus caldovelox’ has been cloned and sequenced. Expression of recombinant arginase at high levels has been achieved in E. coli using an inducible T7 RNA polymerase‐based system. A facile purification procedure incorporating a heat‐treatment step yielded 0.2 g of recombinant arginase per litre of induced culture. The kinetic properties of the purified recombinant protein are essentially identical to the native enzyme. The recombinant protein has been crystallised and one crystal form is isomorphous to crystals of the native protein.
Efficient expression, processing and secretion of a biologically active mammalian protein by Vibrio cholerae
Authors: Ghorpade Anuja , Garg Lalit C.
Published: April 2003

The use of Vibrio cholerae as a secretory expression system for the expression of a mammalian protein, namely human growth hormone, under the control of the heat labile enterotoxin chain B signal sequence is reported. The protein is efficiently expressed and processed. The mature protein is exported to the periplasm after which it is secreted to the extracellular milieu. The expressed and secreted hGH actively binds to its receptor as established by its receptor binding activity. The biological activity of the protein is demonstrated in vitro in a Nb2 proliferation assay.
Two bacteriorhodopsin M intermediates differing in accesibility of the Schiff base for azide
Authors: Radionov Alexey N. , Kaulen Andrey D.
Published: April 2003

Glutaraldehyde treatment leads to the inhibition (i) of the M intermediate decay in wild‐type bacteriorhodopsin (bR) and (ii) of the azide‐facilitated M decay in the D96N mutant bR. LuCl3 is shown to be a more potent inhibitor of both processes. Glycerol and sucrose are also inhibitors. None of these agents change the linearity of the azide concentration dependency of the M decay in the D96N mutant but they do shift this dependency to higher azide concentrations. It is concluded that the two M forms are in equilibrium. These M forms differ in the accessibility of the Schiff base for azide and, probably, also for water molecules. The above‐mentioned agents shift the equilibrium toward the less accessible M form. The data obtained are in line with the model of azide action as the penetrating proton donor and can hardly be realized within the framework of the model of Le Coutre et al. [(1995) Proc. Natl. Acad. Sci. USA 92, 4962–4966] which assumes that a bound anionic form of azide catalyzes proton transfer to the Schiff base.
Reconstitution of GTP‐γ‐S‐dependent phospholipase D activity with ARF, RhoA, and a soluble 36‐kDa protein
Authors: Shimooku Kiyoshi , Akisue Toshihiro , Jinnai Hitoshi , Hitomi Tomohiro , Ogino Chiaki , Yoshida Kimihisa , Nakamura Shun-ichi , Nishizuka Yasutomi
Published: April 2003

For activation of kidney membrane phospholipase D (PLD), cytosol is absolutely needed in addition to GTP‐γ‐S. The active component of cytosol consists of three protein factors: ADP‐ribosylation factor, RhoA, and a soluble 36‐kDa protein. Any combination of these two factors synergistically activates PLD to some extent, but the presence of the three factors causes full activation. The 36‐kDa protein is stable at 60°C but inactivated at 80°C for 10 min. Tissue distribution of the 36‐kDa protein roughly coincides with that of PLD, suggesting physiological relevance of the protein in the regulation of PLD.
Reverse transcription, amplification and sequencing of poliovirus RNA by Taq DNA polymerase
Authors: Grabko V.I. , Chistyakova L.G. , Lyapustin V.N. , Korobko V.G. , Miroshnikov A.I.
Published: April 2003

A model for virion RNA of the poliomyelitis virus, which does not pass the stage of DNA copies during biogenesis, demonstrates that Taq DNA polymerase is capable of synthesizing 960‐bp cDNA with the specific primer. When comparing the nucleotide sequence of the starting virion RNA and recombinant DNAs, isolated from several independent clones, copying and amplification of virion RNA appear accurate (one substitution per 960 bp). A comparison of Taq and Tth DNA polymerases in RT/PCR indicates that the sensitivity of Taq polymerase seems to be two orders of magnitude higher than that of Tth polymerase. The RNA detection level under the chosen conditions approached 104 RNA copies per test. The present investigation indicates the great versatility of Taq polymerase, which promoted the reverse transcription reaction of RNA, cDNA amplification, screening of recombinant clones as well as sequencing of recombinant DNA. Thus application of Taq polymerase is rather promising not only to detect nucleic acids in biological samples, but also for isolating and cloning individual genes, encoded on DNA and/or on RNA templates.
Selective control of cytosolic glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase mRNA stability by selenium supply
Authors: Bermano Giovanna , Arthur John R. , Hesketh John E.
Published: April 2003

Selenium depletion of H4 hepatoma cells reduced cytosolic glutathione peroxidase (cGSH‐Px) mRNA abundance but had no effect on phospholipid hydroperoxide glutathione peroxidase (PHGSH‐Px) mRNA abundance. Actinomycin D chase experiments showed that selenium depletion had no effect on the stability of PHGSH‐Px mRNA but decreased the stability of cGSH‐Px mRNA. In Se‐replete cells puromycin decreased the stability of both cGSH‐Px and PHGSH‐Px mRNAs. The results suggest that when selenium supply is limiting PHGSH‐Px mRNA translation is maintained more than that of cGSH‐Px mRNA, and thus more cGSH‐Px mRNA is released from polysomes and degraded.
Effect of analogues of diaminopimelic acid on the meso‐diaminopimelate‐adding enzyme from Escherichia coli
Authors: Auger Geneviève , van Heijenoort Jean , Vederas John C. , Blanot Didier
Published: April 2003

Several analogues of diaminopimelic acid (A2pm) were tested as substrates or inhibitors of the meso‐diaminopimelate‐adding enzyme from Escherichia coli. They included lanthionine derivatives, a phosphonic analogue, heterocyclic compounds, 3‐fluoro‐A2pm, 4‐methylene‐A2pm and N‐hydroxy‐A2pm. The best substrates were, in decreasing order of specific enzyme activity, (2S,3R,6S)‐3‐fluoro‐A2pm, meso‐lanthionine sulfoxide and N‐hydroxy‐A2pm (mixture of stereoisomers). In those cases where all the stereoisomers were available, the specificity could be described as meso ⪢ DD ≈ LL. N‐Hydroxy‐A2pm (mixture of stereoisomers) strongly inhibited the addition of radioactive meso‐A2pm to UDP‐N‐acetylmuramoyl‐dipeptide.
Selective uptake and degradation of c‐Fos and v‐Fos by rat liver lysosomes
Authors: Aniento Fernando , Papavassiliou Athanasios G. , Knecht Erwin , Roche Enrique
Published: April 2003

The transcription factor c‐Fos is a short‐lived protein and calpains and ubiquitin‐dependent systems have been proposed to be involved in its degradation. In this report, we consider a lysosomal degradation pathway for c‐Fos. Using a cell‐free assay, we have found that freshly isolated lysosomes can take up and degrade c‐Fos with high efficiency. v‐Fos, the oncogenic counterpart of c‐Fos, can also be taken up by lysosomes, yet the amount of incorporated protein is much lower. c‐Fos uptake is independent of its phosphorylation state but it appears to be regulated by dimerization with differentially phosphorylated forms of c‐Jun, while v‐Fos escapes this regulation. Moreover, we show that c‐Fos is immunologically detected in lysosomes isolated from the liver of rats treated with the protease inhibitor leupeptin. Altogether, these results suggest that lysosomes can also participate in the selective degradation of c‐Fos in rat liver.
Identification of an Arabidopsis thaliana cDNA encoding a HSP70‐related protein belonging to the HSP110/SSE1 subfamily
Authors: Storozhenko Sergei , De Pauw Pascal , Kushnir Sergei , Van Montagu Marc , Inzé Dirk
Published: April 2003

Heat‐shock protein 70 (HSP70)‐related proteins are classified in two main subfamilies: the DnaK subfamily and the HSP110/SSE1 subfamily. We have characterized the first plant member of the HSP110/SSE1 subfamily, HSP91. At least two, tightly linked genes encoding HSP91 are present per haploid Arabidopsis genome. HSP91 is constitutively expressed in non‐stressed Arabidopsis plants and is transiently induced by heat shock.
Site‐directed mutagenesis of the formate dehydrogenase active centre: role of the His332‐Gln313 pair in enzyme catalysis
Authors: Tishkov Vladimir I. , Matorin Andrey D. , Rojkova Alexandra M. , Fedorchuk Vladimir V. , Savitsky Pavel A. , Dementieva Larissa A. , Lamzin Victor S. , Mezentzev Alexander V. , Popov Vladimir O.
Published: April 2003

Gln313 and His332 residues in the active centre of NAD+‐dependent formate dehydrogenase (EC 1.2.1.2, FDH) from the bacterium Pseudomonas sp. 101 are conserved in all FDHs and are equivalent to the glutamate‐histidine pair in active sites of d‐specific 2‐hydroxyacid dehydrogenases. Two mutants of formate dehydrogenase from Pseudomonas sp. 101, Gln313Glu and His332Phe, have been obtained and characterised. The Gln313Glu mutation shifts the pK of the group controlling formate binding from less than 5.5 in wild‐type enzyme to 7.6 thus indicating that Gln313 is essential for the broad pH affinity profile towards substrate. His332Phe mutation leads to a complete loss of enzyme activity. The His332Phe mutant is still able to bind coenzyme but not substrate or analogues. The role of histidine in the active centre of FDH is discussed. The protonation state of His332 is not critical for catalysis but vital for substrate binding. A partial positive charge on the histidine imidazole, required for substrate binding, is provided via tight H‐bond to the Gln313 carboxamide.
A novel murine cathelin‐like protein expressed in bone marrow
Authors: Popsueva Anna E. , Zinovjeva Marina V. , Visser Jan W.M. , Zijlmans J.Mark J.M. , Fibbe Willem E. , Belyavsky Alexander V.
Published: April 2003

A novel cDNA encoding a putative secreted protein was isolated from murine bone marrow. The encoded protein named MCLP (urine athelin‐ike rotein) was found to be highly homologous to the pig cathelin, and to four neutrophil antimicrobial polypeptides: CAP 18, indolicidin, Bac 5 and FALL‐39. Secondary structure prediction studies identified a highly cationic region in the C‐terminal part of prepro‐MCLP with a tendency to adopt an amphipathic α‐helical conformation, as observed in many antimicrobial peptides. However, no antibacterial activity was observed with the synthetic peptide corresponding to this region of MCLP.