bluuebunny/crossref_metadata_2025 huggingface dataset:
A covalently bound catalytic intermediate in Escherichia coli asparaginase : Crystal structure of a Thr‐89‐Val mutant
Authors: Palm Gottfried J. , Lubkowski Jacek , Derst Christian , Schleper Stefan , Röhm Klaus-Heinrich , Wlodawer Alexander
Published: July 2002

Escherichia coli asparaginase II catalyzes the hydrolysis of l‐asparagine to l‐aspartate via a threonine‐bound acylenzyme intermediate. A nearly inactive mutant in which one of the active site threomines, Thr‐89, was replaced by valine was constructed, expressed, and crystallized. Its structure, solved at 2.2 Å resolution, shows high overall similarity to the wild‐type enzyme, but an aspartyl moiety is covalently bound to Thr‐12, resembling a reaction intermediate. Kinetic analysis confirms the deacylation deficiency, which is also explained on a structural basis. The previously identified oxyanion hole is described in more detail.
The mitochondrial oxoglutarate carrier protein contains a disulfide bridge between intramembranous cysteines 221 and 224
Authors: Bisaccia F. , Capobianco L. , Mazzeo M. , Palmieri F.
Published: July 2002

The oxoglutarate carrier (OGC) purified from bovine heart mitochondria was treated, both in its active and in its SDS‐denatured state, with the fluorescent N‐(1‐pyrenyl)maleimide and other SH reagents before and after reduction with dithioerythritol or β‐mercaptoethanol. The number of SH groups per OGC polypeptide chain was found to be about 1 for the oxidized carrier and 3 for the reduced carrier. The bovine oxoglutarate carrier contains three cysteines: Cys‐184, Cys‐221 and Cys‐224. Sequencing of BrCN cleavage products of oxoglutarate carrier showed that N‐(1‐pyrenyl)maleimide binds to only Cys‐184 of the oxidized protein and also to Cys‐221 and Cys‐224 after reduction of the protein. These results show the presence of a disulfide bridge between the latter two cysteines of the purified carrier. The oxidized and the reduced forms of the oxoglutarate carrier exhibited different V
max but virtually the same K
m values for oxoglutarate.
Expression of the gene for tear lipocalin/von Ebner's gland protein in human prostate
Authors: Holzfeind Paul , Merschak Petra , Rogatsch Hermann , Culig Zoran , Feichtinger Hans , Klocker Helmut , Redl Bernhard
Published: July 2002

Northern analysis of human multiple tissue blots containing poly A+ RNA from spleen, thymus, prostate, testis, ovary, small intestine, colon and peripheral blood leukocytes revealed that a prostate specific transcript hybridizes to a tear lipocalin/von Ebner's gland protein (TL/VEGP) gene probe. To characterize this transcript, the corresponding cDNA was amplified by reverse transcription (RT)‐PCR. Cloning and sequence analysis showed that it was identical to the tear lipocalin cDNA isolated from human lachrymal glands. Immunohistochemical analysis on thin layer sections of human prostate using a tear lipocalin specific antiserum confirmed the expression of this cDNA in prostate. Thus, our results clearly argue against a unique function of TL/VEGP in human tear fluid or saliva. The human cDNA was expressed in E. coli using the pQE system yielding a recombinant protein which shows biochemical properties identical to the native TL/VEGP.
Gene structure of human cholecystokinin (CCK) type‐A receptor: body fat content is related to CCK type‐A receptor gene promoter polymorphism
Authors: Funakoshi Akihiro , Miyasaka Kyoko , Matsumoto Hideo , Yamamori Shunji , Takiguchi Sohichi , Kataoka Kazuhiro , Takata Yutaka , Matsusue Kimihiko , Kono Akira , Shimokata Hiroshi
Published: July 2002

The transcriptional start site of the human cholecystokinin (CCK)‐A receptor gene was determined by the Capsite Hunting method. Two sequence changes were detected, a G to T change in nucleotide −128, and an A to G change in nucleotide −81. The homozygote (T/T, G/G) was detected in 25 of 1296 individuals (1.9%) in the cohort study. This polymorphism showed a significantly higher percent body fat and higher levels of serum insulin and leptin, compared with wild type and heterozygotes. Our study provided the possibility that polymorphism in the promoter region of the CCK‐A receptor gene may be one of genetic factors affecting fat deposition.
Multiple sites of in vivo phosphorylation in the MDM2 oncoprotein cluster within two important functional domains
Authors: Hay Trevor J , Meek David W
Published: July 2002

The MDM2 oncoprotein is a negative regulatory partner of the p53 tumour suppressor. MDM2 mediates ubiquitination of p53 and targets the protein to the cytoplasm for 26S proteosome‐dependent degradation. In this paper, we show that MDM2 is modified in cultured cells by multisite phosphorylation. Deletion analysis of MDM2 indicated that the sites of modification fall into two clusters which map respectively within the N‐terminal region encompassing the p53 binding domain and nuclear export sequence, and the central acidic domain that mediates p14ARF binding, p53 ubiquitination and cytoplasmic shuttling. The data are consistent with potential regulation of MDM2 function by multisite phosphorylation.
Characterization of the AfaD‐like family of invasins encoded by pathogenic Escherichia coli associated with intestinal and extra‐intestinal infections
Authors: Garcia Marie-Isabelle , Jouve Mabel , Nataro James P. , Gounon Pierre , Le Bouguénec Chantal
Published: July 2002

The afimbrial adhesive sheath, encoded by the afa‐3 gene cluster, is composed of two proteins with different roles in bacterium–HeLa cell interactions. AfaE is required for adhesion and AfaD for internalization. In this study, we found that the AfaD invasin was structurally and functionally conserved among human afa‐expressing strains, independently of AfaE subtype and clinical origin of the Escherichia coli isolate. The AggB protein from enteroaggregative E. coli was also found to be an AfaD‐related invasin. These data suggest that AfaD is the prototype of a family of invasins encoded by adhesion‐associated operons in pathogenic E. coli.
C2PA, a new protein expressed during mouse spermatogenesis
Authors: Linares José-Luis , Wendling Corinne , Tomasetto Catherine , Rio Marie-Christine
Published: July 2002

C2PA is a novel protein that contains a C2 membrane binding domain, a PDZ protein/protein interaction domain, and an ATP/GTP binding domain. C2PA is expressed during embryogenesis from 8.5 days post‐coitum (dpc) until birth. After birth, C2PA expression is mainly observed in the post‐natal and adult testis. During spermatogenesis, C2PA transcripts are specifically observed in the spermatocytes, whereas spermatogonia and spermatids are negative. Taken together, these results suggest that C2PA might be involved in cell signaling pathways occurring during spermatogenesis.
Genomic Exploration of the Hemiascomycetous Yeasts: 12.Kluyveromyces marxianusvar.marxianus
Authors: Llorente Bertrand , Malpertuy Alain , Blandin Gaëlle , Artiguenave François , Wincker Patrick , Dujon Bernard
Published: July 2002

As part of the comparative genomics project ‘GENOLEVURES’, we studied theKluyveromyces marxianusvar.marxianusstrain CBS712 using a partial random sequencing strategy. With a 0.2×genome equivalent coverage, we identified ca. 1300 novel genes encoding proteins, some containing spliceosomal introns with consensus splice sites identical to those ofSaccharomyces cerevisiae, 28 tRNA genes, the whole rDNA repeat, and retrotransposons of the Ty1/2 family ofS. cerevisiaewith diverged Long Terminal Repeats. Functional classification of theK. marxianusgenes, as well as the analysis of the paralogous gene families revealed few differences with respect toS. cerevisiae. Only 42K. marxianusidentified genes are without detectable homolog in the baker's yeast. However, we identified several genetic rearrangements between these two yeast species.
Genomic Exploration of the Hemiascomycetous Yeasts: 18. Comparative analysis of chromosome maps and synteny withSaccharomyces cerevisiae
Authors: Llorente Bertrand , Malpertuy Alain , Neuvéglise Cécile , de Montigny Jacky , Aigle Michel , Artiguenave François , Blandin Gaëlle , Bolotin-Fukuhara Monique , Bon Elisabeth , Brottier Philippe , Casaregola Serge , Durrens Pascal , Gaillardin Claude , Lépingle Andrée , Ozier-Kalogéropoulos Odile , Potier Serge , Saurin William , Tekaia Fredj , Toffano-Nioche Claire , Wésolowski-Louvel Micheline , Wincker Patrick , Weissenbach Jean , Souciet Jean-Luc , Dujon Bernard
Published: July 2002

We have analyzed the evolution of chromosome maps of Hemiascomycetes by comparing gene order and orientation of the 13 yeast species partially sequenced in this program with the genome map ofSaccharomyces cerevisiae. From the analysis of nearly 8000 situations in which two distinct genes having homologs inS. cerevisiaecould be identified on the sequenced inserts of another yeast species, we have quantified the loss of synteny, the frequency of single gene deletion and the occurrence of gene inversion. Traces of ancestral duplications in the genome ofS. cerevisiaecould be identified from the comparison with the other species that do not entirely coincide with those identified from the comparison ofS. cerevisiaewith itself. From such duplications and from the correlation observed between gene inversion and loss of synteny, a model is proposed for the molecular evolution of Hemiascomycetes. This model, which can possibly be extended to other eukaryotes, is based on the reiteration of events of duplication of chromosome segments, creating transient merodiploids that are subsequently resolved by single gene deletion events.
Proteolytic cleavage of epidermal growth factor receptor by caspases
Authors: Bae Sun Sik , Choi Jang Hyun , Oh Yong Seok , Perry David K. , Ryu Sung Ho , Suh Pann-Ghill
Published: July 2002

Apoptotic proteases cleave and inactivate survival signaling molecules such as Akt/PKB, phospholipase C (PLC)‐γ1, and Bcl‐2. We have found that treatment of A431 cells with tumor necrosis factor‐α in the presence of cycloheximide resulted in the cleavage of epidermal growth factor receptor (EGFR) as well as the activation of caspase‐3. Among various caspases, caspase‐1, caspase‐3 and caspase‐7 were most potent in the cleavage of EGFR in vitro. Proteolytic cleavage of EGFR was inhibited by both YVAD‐cmk and DEVD‐fmk in vitro. We also investigated the effect of caspase‐dependent cleavage of EGFR upon the mediation of signals to downstream signaling molecules such as PLC‐γ1. Cleavage of EGFR by caspase‐3 significantly impaired the tyrosine phosphorylation of PLC‐γ1 in vitro. Given these results, we suggest that apoptotic protease specifically cleaves and inactivates EGFR, which plays crucial roles in anti‐apoptotic signaling, to abrogate the activation of EGFR‐dependent downstream survival signaling molecules.
A redistribution of actin and myosin IIA accompanies Ca2+‐dependent insulin secretion
Authors: Wilson Justine R. , Ludowyke Russell I. , Biden Trevor J.
Published: July 2002

The study addressed the functional link between remodelling of the actomyosin cytoskeleton in pancreatic β‐cells and the regulation of insulin secretion. Confocal microscopy revealed that myosin heavy chain (MHC) IIA co‐localized very well with filamentous (F)‐actin in RINm5F cells but MHCIIB did not. Subcellular localization of MHCIIB was not altered by stimulation with 30 mM KCl (which evokes Ca2+‐dependent insulin secretion). In contrast MHCIIA redistributed in a manner similar to F‐actin, especially towards the apical surface, but also away from peripheral regions towards cell contact points on the basal surface. Finally, Ca2+‐dependent insulin secretion was inhibited by stabilization of actin filaments with jasplakinolide. The results support a role for the MHCIIA/actin cytoskeleton in regulating insulin secretion.
Decreased antioxidant defense during replicative aging of the yeast Saccharomyces cerevisiae studied using the ‘baby machine’ method
Authors: Grzelak Agnieszka , Skierski Janusz , Bartosz Grzegorz
Published: July 2002

Replicatively senescent cells of Saccharomyces cerevisiae were obtained using the ‘baby machine’ method by immobilizing cells on CovaLink™ NH2 plates and allowing them to divide while exchanging medium and removing daughter cells. Centrifugation in a Percoll density gradient was employed for further purification of replicatively old yeast cells. Comparison of senescent cells showing more than 20 bud scars with cells from early stationary culture demonstrated a significant reduction of total and reduced glutathione and decrease of superoxide dismutase activity during replicative aging of yeast cells.
The Ras GDP/GTP cycle is regulated by oxidizing agents at the level of Ras regulators and effectors
Authors: Accorsi Katia , Giglione Carmela , Vanoni Marco , Parmeggiani Andrea
Published: July 2002

Reactive oxygen species (ROS) have been found to play important roles in regulating cellular functions. Their action in vivo has been related to specific effects on signal transduction pathways, such as Ras pathway. In order to characterize which elements of Ras pathway are affected by ROS, we have analyzed the action of different oxidizing agents on the ability of GTPase activating protein GAP and nucleotide exchange factor GEF to enhance the intrinsic activities of Ras. The action of these agents on the binding between H‐Ras and its effector c‐Raf‐1 was also investigated. No effects were observed on the intrinsic activities of H‐Ras or Ras2p. On the other hand, reversible inhibitions of GEF and GAP actions on Ras were found, whose extent was dependent on the agent used. As tested with the scintillation proximity assay, these agents also inhibited the binding of c‐Raf‐1 to H‐Ras. Our data reveal new potential targets for the action of ROS on Ras pathway and suggest that they can influence the Ras activation state indirectly via regulators and effectors.
Functional site of bukatoxin, an α‐type sodium channel neurotoxin from the Chinese scorpion (Buthus martensi Karsch) venom: probable role of the 52PDKVP56 loop
Authors: Srinivasan K.N. , Nirthanan S. , Sasaki T. , Sato K. , Cheng B. , Gwee M.C.E. , Kini R.M. , Gopalakrishnakone P.
Published: July 2002

α‐Toxins from scorpion venoms prolong the action potential of excitable cells by blocking sodium channel inactivation. We have purified bukatoxin, an α‐toxin from scorpion (Buthus martensi Karsch) venom, to homogeneity. Bukatoxin produced marked relaxant responses in the carbachol‐precontracted rat anococcygeus muscle (ACM), which were mediated through the L‐arginine–nitric oxide synthase–nitric oxide pathway, consequent to a neuronal release of nitric oxide. Based on the presence of proline residues in the flanking segments of protein–protein interaction sites, we predicted the site between 52PP56 to be the potential interaction site of bukatoxin. A homology model of bukatoxin indicated the presence of this site on the surface. Buka11, a synthetic peptide designed based on this predicted site, produced a concentration‐dependent nitric oxide‐mediated relaxant response in ACM. Using alanine‐substituted peptides, we have shown the importance 53DKV55 flanked by proline residues in the functional site of bukatoxin.
Binding of tenascin‐X to decorin
Authors: Elefteriou Florent , Exposito Jean-Yves , Garrone Robert , Lethias Claire
Published: July 2002

Tenascin‐X (TN‐X) is an extracellular matrix protein whose absence results in an alteration of the mechanical properties of connective tissue. To understand the mechanisms of integration of TN‐X in the extracellular matrix, overlay blot assays were performed on skin extracts. A 100 kDa molecule interacting with TN‐X was identified by this method and this interaction was abolished when the extract was digested by chondroitinase. By solid‐phase assays, we showed that dermatan sulfate chains of decorin bind to the heparin‐binding site included within the fibronectin‐type III domains 10 and 11 of TN‐X. We thus postulate that the association of TN‐X with collagen fibrils is mediated by decorin and contributes to the integrity of the extracellular network.
N‐terminal EF‐hand‐like domain is required for phosphoinositide‐specific phospholipase C activity in Arabidopsis thaliana
Authors: Otterhag Lotta , Sommarin Marianne , Pical Christophe
Published: July 2002

Phosphoinositide‐specific phospholipase C's (PI‐PLCs) are ubiquitous in eukaryotes, from plants to animals, and catalyze the hydrolysis of phosphatidylinositol 4,5‐bisphosphate into the two second messengers inositol 1,4,5‐trisphosphate and diacylglycerol. In animals, four distinct subfamilies of PI‐PLCs have been identified, and the three‐dimensional structure of one rat isozyme, PLC‐δ1, determined. Plants appear to contain only one gene family encoding PI‐PLCs. The catalytic properties of plant PI‐PLCs are very similar to those of animal enzymes. However, very little is known about the regulation of plant PI‐PLCs. All plant PI‐PLCs comprise three domains, X, Y and C2, which are also conserved in isoforms from animals and yeast. We here show that one PI‐PLC isozyme from Arabidopsis thaliana, AtPLC2, is predominantly localized in the plasma membrane, and that the conserved N‐terminal domain may represent an EF‐hand domain that is required for catalytic activity but not for lipid binding.
Conservation of amino acids in multiple alignments: aspartic acid has unexpected conservation
Authors: Fiser András , Simon István , J. Barton Geoffrey
Published: July 2002

Analysis of the relationship between surface accessibility and amino acid conservation in multiple sequence alignments of homologous proteins confirms expected trends for hydrophobic amino acids, but reveals an unexpected difference between the conservation of Asp, Glu and Gln. Even when not in an active site, Asp is more highly conserved than Glu. There is a clear preference for conserved and buried Asp to be present in coil, but there is no tendency for Asp to conserve φ/ψ in the ++ region of the Ramachandran map. Glu does not show any preference to be conserved in a particular secondary structure. Analysis of recently derived substitution matrices (e.g. BLOSUM) confirms that Glu tends to substitute more frequently with other amino acids than does Asp. Analysis of relative accessibility versus relative conservation for individual amino acid positions in alignments shows a negative correlation for all amino acid types. With the exception of Arg, Lys, Gly, Glu, Asp and Tyr, a relative conservation of > 2 suggests the amino acid will have a relative accessibility of < 50%. Observation of conserved Cys, Gly or Asp in a reliable multiple aligmnent suggests a position important for the structure of the protein. Furthermore, the Asp is likely to be involved in polar interactions through its side chain oxygen atoms. In contrast, Gln is the least conserved amino acid overall.
Regulatable production of mature insulin from a hepatocyte cell line: insulin production is up‐regulated by cAMP and glucocorticoids, and down‐regulated by insulin
Authors: Lu Danhong , Hoshino Hideki , Takeuchi Toshiyuki
Published: July 2002

We engineered a hepatoma cell line that produces an up‐regulation of insulin in response to cAMP, dexamethasone, and retinoic acid, and a down‐regulation in response to insulin. We devised a regulatory secretion system by placing proinsulin DNA under the regulatable promoter for phosphoenolpyruvate carboxykinase (PEPCK). To assess the ability to regulate insulin secretion, we used the rat hepatoma cell line, H4IIE. The H4IIE cells secreted immunoreactive insulin (IRI) constantly at a level of 1–3 fmol/106 cells/h. IRI increased approximately two‐fold upon stimulation with 0.5 mM cAMP and five‐fold with the addition of the cAMP‐dependent phosphodiesterase inhibitor IBMX, as compared to baseline IRI secretion. IRI increased 18‐fold by 1–500 nM dexamethasone together with cAMP and IBMX. Addition of exogenous insulin to the culture medium significantly decreased insulin mRNA expression on Northern blot.
Lack of vacuolar proton ATPase association with the cytoskeleton in osteoclasts of osteosclerotic (oc/oc) mice
Authors: Nakamura Ichiro , Takahashi Naoyuki , Udagawa Nobuyuki , Moriyama Yoshinori , Kurokawa Takahide , Jimi Eijiro , Sasaki Takahisa , Suda Tatsuo
Published: July 2002

We examined the pathogenetic mechanism underlying the lack of bone resorption in osteosclerotic oc/oc mice. An immunoelectron microscopic analysis revealed that in the osteoclasts of these mice, no ruffled borders formed, and that vacuolar H+‐ATPase (V‐ATPase) was present throughout the cytoplasm but not on the apical membranes. The activity of V‐ATPase in oc/oc mice was similar to that in normal mice. In normal spleen cell‐derived osteoclast‐like cells (OCLs), immunoreactivity for V‐ATPase was detected in association with Triton X‐100‐insoluble cellular structure, but not in oc/oc spleen cell‐derived OCLs. Moreover, in renal proximal convoluted tubules of oc/oc mice, the basal striation did not form. These results suggest that osteosclerosis in oc/oc mice is possibly due to the dissociation of V‐ATPase and cytoskeleton in osteoclasts.
Expression and activity of a recombinant chimeric protein composed of pokeweed antiviral protein and of human interleukin‐2
Authors: Dore Jean-Michel , Gras Evelyne , Wijdenes John
Published: July 2002

The pokeweed antiviral protein (PAP) has already been used to chemically construct immunotoxins. Here we tested the recombinant approach for the production of PAP‐containing cytotoxic fusion‐proteins. A cDNA encoding a mutated PAP (PAP9), which is expressed at high levels in bacteria, was fused to human interleukin‐2 (IL‐2) cDNA. The resulting PAP9–IL‐2 protein was as active as the free PAP9 in inhibiting an eukaryotic cell‐free translation system. Only the chimeric protein desaminated the 28S rRNA and inhibited translation of the CTLL‐2 cell line which expresses the IL‐2 receptor. These results show that PAP is a suitable toxin for the production of recombinant immunotoxins.
Engineering thermolysin‐like proteases whose stability is largely independent of calcium
Authors: Veltman Oene R , Vriend Gert , van den Burg Bertus , Hardy Florence , Venema Gerard , Eijsink Vincent G.H
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Distribution of sequence‐dependent curvature in genomic DNA sequences
Authors: Gabrielian Andrei , Vlahovicek Kristian , Pongor Sándor
Published: July 2002

The distribution of inherent, sequence‐dependent curvature was calculated for a number of prokaryotic (M. genitalium, H. influenzae, M. jannaschii), viral (adenovirus 2, equine herpes virus 1), phage (M13, lambda), eukaryotic (S. cerevisiae) and mitochondrial genomes as well as E. coli and human genomic fragments. The genomic averages are in the range of 6–8°/helical turn and only about 20% of DNA is curved less than 3°/helical turn. The prokaryotes and phages appear to have a consistently higher frequency of curved DNA in their genomes than the other genomes tested. Long, highly curved segments, similar to artificially designed curved DNA, are apparently absent from the genomes. Short, curved segments, differing in G+C content may provide environmentally modulated conformational signals for gene regulation. A WWW‐server was constructed for the prediction of curved sites from DNA sequences 〈http://icgeb.trieste.it/dna/curve_it.html/〉.
Replacement of Pro109 by His in TlpA, a thioredoxin‐like protein from Bradyrhizobium japonicum, alters its redox properties but not its in vivo functions
Authors: Rossmann Reinhild , Stern Dorothee , Loferer Hannes , Jacobi Alexander , Glockshuber Rudi , Hennecke Hauke
Published: July 2002

TlpA, the membrane‐anchored, thioredoxin‐like protein from Bradyrhizobium japonicum, is essential for cytochrome aa
3 biogenesis. The periplasmic domain of TlpA was previously shown to have protein thiol:disulfide oxidoreductase activity and reducing properties similar to those of cytoplasmic thioredoxins. Here, we replaced the proline‐109 in its active‐site sequence C107V108P109C110 by a histidine residue. The resulting active‐site motif (CVHC) resembles that of oxidizing thiol:disulfide oxidoreductases such as protein disulfide isomerase (PDI) and DsbA. Indeed, the TlpA variant P109H was by 66 mV more oxidizing than the wild‐type protein. Nevertheless, the altered protein was even more efficient in catalyzing the reduction of insulin disulfides by dithiothreitol than the wild‐type due to a faster recycling of its catalytically active, reduced form. Cells of B. japonicum expressing only the mutated tlpA gene had the same phenotypes as wild‐type cells, suggesting that the change in the redox potential of TlpA was not critical for its in vivo function.
Molecular cloning of a diacylglycerol kinase isozyme predominantly expressed in rat retina
Authors: Kohyama-Koganeya Ayako , Watanabe Masahiko , Hotta Yoshiki
Published: July 2002

We have cloned and characterized a new diacylglycerol kinase (DGK) isozyme which is expressed in the retina and the brain of rat. The cDNA contains an open reading frame of 567 amino acid residues with a predicted protein of 64 kDa and shows very high homology to human DGKϵ. The new DGK isozyme contains two distinctive zinc‐finger structures and a putative catalytic domain. This DGK expressed predominantly in the inner and outer nuclear layers of retina. This expression pattern is different from those of the previously cloned DGKs including the human DGKϵ, suggesting that this DGK isozyme has potential importance in visual functions as was the case in Drosophila retinal cells.
Purification, amino acid sequence and immunological characterization of Ole e 6, a cysteine‐enriched allergen from olive tree pollen
Authors: Batanero Eva , Ledesma Amalia , Villalba Mayte , Rodrı́guez Rosalı́a
Published: July 2002

The Ole e 6 allergen from olive tree pollen has been isolated by combining gel permeation and reverse‐phase chromatographies. It is a single and highly acidic (pI 4.2) polypeptide chain protein. Its NH2‐terminal amino acid sequence has been determined by Edman degradation. Total RNA from the olive tree pollen was isolated, and a specific cDNA was amplified by the polymerase chain reaction using a degenerate oligonucleotide primer designed according to the NH2‐terminal sequence of the protein. The nucleotide sequencing of the cDNA rendered an open reading frame encoding a 50 amino acid polypeptide chain, in which two sets of the sequential motif Cys‐X3‐Cys‐X3‐Cys are present. No sequence similarity has been found between this protein and other previously described polypeptides.
Interaction of bacteriophage T4 AsiA protein with Escherichia coliσ70 and its variant
Authors: Pahari Susanta , Chatterji Dipankar
Published: July 2002

Bacteriophage T4 produces a small protein AsiA, which inhibits transcription from σ
70‐dependent promoters in E. coli by tightly binding to σ
70 and is therefore termed as anti‐sigma factor. We observed that there was no inhibition of single round transcription at lac UV5 promoter when AsiA was added to preformed open complex between RNA polymerase and template DNA. However, transcription was found to proceed normally at ‘extended −10’ promoters in the presence of AsiA. It appears therefore that AsiA binds σ
70 at its 4.2‐subdomain or in its close vicinity. Further experiments on immunoprecipitation of σ
70 and a mutant σ
70‐V576G with AsiA seem to corroborate such conclusion.
Bcl‐2 antagonizes apoptotic cell death induced by two new ceramide analogues
Authors: Wieder Thomas , Geilen Christoph C , Kolter Thomas , Sadeghlar Farsaneh , Sandhoff Konrad , Brossmer Reinhard , Ihrig Petra , Perry David , Orfanos Constantin E , Hannun Yusuf A
Published: July 2002

Ceramides which arise in part from the breakdown of sphingomyelin comprise a class of antiproliferative lipids and have been implicated in the regulation of programmed cell death better known as apoptosis. In the present study, two new synthetic ceramide analogues, N‐thioacetylsphingosine and FS‐5, were used in Molt 4 cells to induce cell death. Besides their cytotoxic effects at concentrations ≥14 μM the data obtained clearly show that both analogues induced apoptosis at concentrations below this critical concentration as assessed by trypan blue exclusion and cleavage of the death substrate poly‐(ADP‐ribose) polymerase (PARP). Additional experiments in bcl‐2‐transfected Molt 4 cells revealed that the apoptotic but not the lytic effects of the analogues were antagonized by the apoptosis inhibitor Bcl‐2. Furthermore, neither N‐thio‐acetylsphingosine nor FS‐5 induced PARP cleavage in bcl‐2‐transfected Molt 4 cells indicating that the induction of apoptotic cell death by cell permeable ceramides is not due to unspecific disturbance of the cell membrane.
Annexin I targets S100C to early endosomes
Authors: Seemann Joachim , Weber Klaus , Gerke Volker
Published: July 2002

Immunofluorescence and subcellular fractionation localize annexin I and the EF hand protein S100C to the same membranous structures which in part correspond to transferrin receptor‐positive endosomes. The association of S100C with endosomal membranes is strictly dependent on annexin I binding since a D91stop–S100C mutant protein, in which the residues essential for annexin I binding have been removed, fails to co‐localize with membraneous structures. This indicates that annexin I and S100C form a complex in vivo and that the endosomal localization of this complex is mediated through an interaction of annexin I with the endosomal membrane.
Selection of down‐regulated sequences along the monocytic differentiation of leukemic HL60 cells
Authors: Herblot S , Vekris A , Rouzaut A , Najeme F , de Miguel C , Bezian J.H , Bonnet J
Published: July 2002

In order to dissect the molecular mechanisms of monocytic differentiation we have developed a subtractive hybridisation method based on a simplified `representational difference analysis'. We have selected 16 sequences and confirmed their down‐regulation along the TPA‐induced monocytic differentiation of HL60 cells. Among these sequences we have identified the α‐tubulin, the TaxREB protein and two ribosomal protein sequences which had not been previously described as differentially expressed. These results add to our knowledge about the molecules implicated along the monocytic differentiation and growth arrest of leukemic cells and provide a first step in the study of their respective roles.
Identification of novel homologues of mouse importin α, the α subunit of the nuclear pore‐targeting complex, and their tissue‐specific expression
Authors: Tsuji Lyuji , Takumi Toru , Imamoto Naoko , Yoneda Yoshihiro
Published: July 2002

Transport of karyophilic proteins into the nucleus is mediated by nuclear localization signals (NLSs) via a multistep process. The karyophiles are recognized by the importin α subunit in the cytoplasm to form a stable complex, termed the nuclear pore‐targeting complex (PTAC). To date, three different mammalian α subunits (mSRP1/NPI‐1, PTAC58/mPendulin/Rch1 and Qip1) have been identified. In this study, we report the identification of three additional mouse genes homologous to the known α subunits using RT‐PCR methodology and show that the mouse α subunits can be classified into at least three subfamilies, α‐P, α‐Q and α‐S families, each composed of closely related members (more than 80% amino acid sequence identity). These three subfamilies, however, have ∼50% amino acid identity to one another. Northern blot analysis showed that all were differentially expressed in various mouse tissues. These results suggest that the function of these proteins may be controlled in a tissue‐specific manner and that their combinatorial expression may play a role in differentiation and organogenesis.
Chemotherapeutic activity of synthetic antimicrobial peptides: correlation between chemotherapeutic activity and neutrophil‐activating activity
Authors: Nakajima Yuki , Alvarez-Bravo Juan , Cho Jang-hyun , Homma Ko-ichi , Kanegasaki Shiro , Natori Shunji
Published: July 2002

The chemotherapeutic activity of three synthetic antibacterial peptides was investigated. KLKLLLLLKLK‐NH2 and its d‐enantiomer showed significant chemotherapeutic activity in MRSA‐infected mice, whereas KLKLLLKLK‐NH2, which showed the highest antibacterial activity among them in vitro, was found to have almost no ability to prevent MRSA infection. These results suggest that the antibacterial activity of peptides assessed in vitro does not necessarily correlate with their chemotherapeutic activity. We found that KLKLLLLLKLK‐NH2 and its d‐enantiomer, but not KLKLLLKLK‐NH2, have the ability to activate human neutrophils to produce superoxide, suggesting that the prevention of MRSA infection by these peptides is not simply due to their direct bactericidal activity but to augmentation of the systemic defense mechanism mediated by neutrophils.
Epitope mapping by screening of phage display libraries of a monoclonal antibody directed against the receptor binding domain of human α2‐macroglobulin
Authors: Birkenmeier Gerd , Osman Awad A , Kopperschläger Gerhard , Thomas Mothes
Published: July 2002

The human proteinase inhibitor, α2‐macroglobulin (α2‐M), inhibits a large number of proteinases. α2‐M‐proteinase complexes are rapidly cleared from the circulation by binding to a cellular receptor (α2‐M‐R/LRP) via the receptor binding domain (RBD) which is made up of a 20 kDa C‐terminal stretch of the 180 kDa monomer of the inhibitor. A monoclonal antibody (mab α‐1) has been described which reacts with the receptor‐recognizable form of the inhibitor, the so called transformed α2‐M (α2‐Mt). By screening of a phage display library an epitope in the RBD of the inhibitor was identified that reacts with mab α‐1. Out of 25 phage clones a heptapeptide sequence (S‐x1‐x2‐D‐x3‐x4‐K) was obtained containing identical amino acids in three positions. A consensus peptide (S‐R‐S‐D‐P‐P‐K) was synthesized and found to displace α2‐Mt from binding to mab α‐1 and to receptor. The specificity of competition was demonstrated by a reversed peptide and a control antibody. By structural comparison it was found that the consensus heptapeptide mimics a discontinuous conformationally constrained epitope present in the RBD of the inhibitor. This is the first report describing the detection of discontinuous epitopes by phage display using a short linear peptide.
Populating the equilibrium molten globule state of apomyoglobin under conditions suitable for structural characterization by NMR
Authors: Eliezer David , Jennings Patricia A. , Dyson H.Jane , Wright Peter E.
Published: July 2002

Conditions have been determined under which the equilibrium molten globule state of apomyoglobin is stable and remains monomeric for periods of time sufficient for the application of three‐dimensional heteronuclear NMR experiments. The quality of initial two‐dimensional NMR spectra suggests that sequence‐specific assignments can be made for a majority of the protein resonances under these conditions. A pH titration of the protein followed using two‐dimensional 1H‐15N correlation experiments indicates that the equilibrium intermediate undergoes fast exchange on the chemical shift time scale with the unfolded state and intermediate time scale exchange with the native state, and suggests a strategy to assist with backbone resonance assignments. The conditions and techniques described may be applicable to the characterization of other equilibrium folding intermediates.
A stable intermediate product of the archaeal zinc‐containing 7Fe ferredoxin from Sulfolobus sp. strain 7 by artificial oxidative conversion
Authors: Iwasaki Toshio , Oshima Tairo
Published: July 2002

Irreversible conversion of the purified zinc‐containing 7Fe ferredoxin from the thermoacidophilic archaeon Sulfolobus sp. strain 7 was found to occur under aerobic conditions at pH 5.0 and at 4°C. This process accompanied a substantial increase of the electron paramagnetic resonance signal attributed to a [3Fe‐4S]1+ cluster, and the converted form containing ∼6 Fe/Zn (mol/mol) had a net charge different from that of the native 7Fe ferredoxin. These data provide evidence for the formation of a stable intermediate product of the archaeal ferredoxin having two [3Fe‐4S] clusters and a zinc center by artificial oxidative degradation. This further explains the discrepancy that a zinc center and two [3Fe‐4S] clusters (but not a zinc center and one [3Fe‐4S] cluster plus one [4Fe‐4S] cluster) are observed in the crystal structure at pH 5.0.
Construction, expression and characterization of a soluble form of human endothelin‐converting‐enzyme‐1
Authors: Korth Petra , Egidy Giorgia , Parnot Charles , LeMoullec Jean-Michel , Corvol Pierre , Pinet Florence
Published: July 2002

Endothelin‐converting‐enzyme‐1 (ECE‐1) belongs to the family of zinc metallopeptidases and is responsible for generating endothelin (ET) peptides from their inactive precursors the big endothelins (bigET). The enzyme is a type II integral membrane protein consisting of a short amino‐terminal cytosolic domain of 56 amino acids, a single transmembrane domain and a large putative extracellular domain containing the catalytic site. Recombinant and native ECE‐1 are expressed as a dimer. We have constructed a soluble form of ECE, named sECE*, by fusing the cleavable signal peptide of pro‐opiomelanocortin in frame to the complete extracellular domain of human ECE‐1. Stable expression of this construct in CHO cells resulted in the secretion of a fully active enzyme. In contrast to membrane‐bound ECE, sECE* was expressed as a monomer, highly glycosylated, as assessed by gel filtration and Western blot. However, recombinant sECE* converted bigET‐1 with similar specific activity as ECE‐1a. This activity was completely inhibited by phosphoramidon, but not by thiorphan and captopril. sECE* was active in a broad range of pH, showing an optimum of 6.6–6.8 for bigET‐1. Thus, the extracellular domain alone is sufficient for conferring full ECE‐1 activity, inhibitors recognition and substrate specificity.
The C‐terminus of yeast plasma membrane H+‐ATPase is essential for the regulation of this enzyme by heat shock protein Hsp30, but not for stress activation
Authors: Braley R , Piper P.W
Published: July 2002

Several stresses cause additional activation to the glucose‐stimulated plasma membrane H+‐ATPase activity of yeast, but it is not clear how this is achieved. We recently reported that cells lacking the integral plasma membrane heat shock protein Hsp30 display enhanced increases in plasma membrane H+‐ATPase activity with either heat shock or weak organic acid stress (Piper, P.W., Ortiz‐Calderon, C., Holyoak, C., Coote, P. and Cole, M. (1997) Cell Stress and Chaperones 2, 12–24), indicating that the stress induction of Hsp30 acts to reduce stress stimulation of the H+‐ATPase. In this study it is shown that Hsp30 acts to reduce the V
max of H+‐ATPase in heat shocked cells. Its action is lost with deletion of the C‐terminal 11 amino acids of the H+‐ATPase, a deletion that does not abolish the stress stimulation of this enzyme. Mutation of the Thr‐912 residue within the C‐terminal domain of H+‐ATPase, a potential site of phosphorylation by the Ca2+‐calmodulin‐dependent protein kinase, also abolishes any effect of Hsp30. Hsp30 may therefore be acting on a Thr‐912 phosphorylated form of the H+‐ATPase.
Determination of fast proton exchange rates of biomolecules by NMR using water selective diffusion experiments
Authors: Böckmann Anja , Guittet Eric
Published: July 2002

We present a new water selective pulse sequence allowing rapid determination of exchange rates of labile protons on the millisecond time scale. Using diffusion measurements, exchange rates of resolved protons can be determined without prior knowledge of relaxation parameters in a short overnight experiment. The use of a sensitive, highly selective and easy to implement water excitation scheme allows for its straightforward application to a wide range of biomolecules. The results obtained for the imino proton exchange rates of a 16 bp DNA are in strong agreement with values obtained by the classical approach of two‐dimensional exchange spectroscopy.
Cortisol increases transfection efficiency of cells
Authors: Bernasconi Alessio G.F , Rebuffat Alexandre G , Lovati Emanuela , Frey Brigitte M , Frey Felix J , Galli Ivo
Published: July 2002

DNA uptake can be facilitated by addition of physiological amounts of 11β‐hydroxy glucocorticosteroids (such as cortisol) during transfection. In the presence of cortisol, but not of the inactive 11‐keto glucocorticoid cortisone, twice as many cells uptake and express the reporter gene. The effect is specific and dose‐dependent; the amounts of glucocorticosteroids needed to enhance transfection efficiency are in the nanomolar range, which corresponds to the dissociation constant of glucocorticoids for the glucocorticoid receptor in vitro. This effect can be abolished by an excess of the glucocorticoid antagonist RU486. We infer that the activated cytoplasmic glucocorticoid receptors enhance nuclear translocation of the incoming transfected DNA.
Pyrene excimer fluorescence as a proximity probe for investigation of residual structure in the unfolded state of human carbonic anhydrase II
Authors: Hammarström Per , Kalman Björn , Jonsson Bengt-Harald , Carlsson Uno
Published: July 2002

The excimer fluorescence from two pyrenyl moieties attached to cysteines in human carbonic anhydrase II has been monitored to characterize residual structure retained under strong denaturing conditions. A position in β‐strand 3, N67C, together with the single naturally occurring cysteine 206 in β‐strand 7, were used as attachment sites. The eximer formation by the pyrenyls, requiring proximity of the probes, revealed an unfolding transition at a GuHCl concentration significantly higher than that required to induce unfolding of the molten globule state as monitored by CD. These results indicate that the excimer transition monitors the unfolding of a residual compact structure that spans β‐strands 3–7. This region constitutes the central and the most hydrophobic part of the molecule, emphasizing the importance of hydrophobic interaction in maintaining residual structure under strong unfolding conditions.
Subcellular localization of vanillyl‐alcohol oxidase in Penicillium simplicissimum
Authors: Fraaije Marco W , Sjollema Klaas A , Veenhuis Marten , van Berkel Willem J.H
Published: July 2002

Growth of Penicillium simplicissimum on anisyl alcohol, veratryl alcohol or 4‐(methoxymethyl)phenol, is associated with the synthesis of relatively large amounts of the hydrogen peroxide producing flavoprotein vanillyl‐alcohol oxidase (VAO). Immunocytochemistry revealed that the enzyme has a dual location namely in peroxisomes and in the cytosol. The C‐terminus of the primary structure of VAO displays a WKL‐COOH sequence which might function as a peroxisomal targeting signal type 1 (PTS1). As VAO activity results in production of hydrogen peroxide also the subcellular location of a recently characterized co‐inducible catalase‐peroxidase was studied. As VAO, this hydroperoxidase is also distributed throughout the cytosol and peroxisomes.
MK‐801 blocks monoamine transporters expressed in HEK cells
Authors: Nishimura Mitsuhiro , Sato Kohji , Okada Tomoya , Schloss Patrick , Shimada Shoichi , Tohyama Masaya
Published: July 2002

(+)‐MK‐801 is known to be a specific non‐competitive antagonist of N‐methyl‐d‐aspartate (NMDA) receptors. However, besides having an anticonvulsant effect, this compound possesses a central sympathomimetic effect and an anxiolytic‐like action, raising the possibility that (+)‐MK‐801 might affect monoamine uptake systems. To elucidate this possibility, we investigated the effects of (+)‐MK‐801 on monoamine transporters expressed in HEK cells. (+)‐MK‐801 significantly inhibited the uptake of all three monoamine transporters in a dose‐dependent manner and the inhibitions were competitive with respect to monoamines. The K
i values of (+)‐MK‐801 on the norepinephrine, dopamine and serotonin transporters were 3.2 μM, 40 μM and 43 μM, respectively. In addition, (−)‐MK‐801, a less potent antagonist of NMDA receptors, also inhibited monoamine transporters with a similar potency as that of (+)‐MK‐801. These results clearly indicate that MK‐801, a non‐competitive antagonist of NMDA receptors, competitively inhibits monoamine transporters without stereoselectivity.
Updated catalogue of homologues to human disease‐related proteins in the yeast genome1
Authors: Andrade Miguel A , Sander Chris , Valencia Alfonso
Published: July 2002

The recent availability of the full Saccharomyces cerevisiae genome offers a perfect opportunity for revising the number of homologues to human disease‐related proteins. We carried out automatic analysis of the complete S. cerevisiae genome and of the set of human disease‐related proteins as identified in the SwissProt sequence data base. We identified 285 yeast proteins similar to 155 human disease‐related proteins, including 239 possible cases of human‐yeast direct functional equivalence (orthology). Of these, 40 cases are suggested as new, previously undiscovered relationships. Four of them are particularly interesting, since the yeast sequence is the most phylogenetically distant member of the protein family, including proteins related to diseases such as phenylketonuria, lupus erythematosus, Norum and fish eye disease and Wiskott‐Aldrich syndrome.
Rapid and discrete isolation of oxygen‐evolving His‐tagged photosystem II core complex from Chlamydomonas reinhardtii by Ni2+ affinity column chromatography
Authors: Sugiura Miwa , Inoue Yorinao , Minagawa Jun
Published: July 2002

We have developed a simple and rapid procedure to isolate an oxygen‐evolving photosystem II (PS II) core complex from Chlamydomonas reinhardtii. A His‐tag made of six consecutive histidine residues was genetically attached at the carboxy terminus of D2 protein to create a metal binding site on the PS II supramolecular complex. The recombinant cells producing the His‐tagged variant of D2 protein grew photoautotrophically as well as the wild‐type cells. Characterization of the oxygen evolution and the thermoluminescence properties revealed that the His‐tagging did not affect the functional integrity of the PS II reaction center. A PS II core complex was isolated from the detergent‐solubilized thylakoids of the recombinant cells in 4 h by a single one‐step Ni2+ affinity column chromatography. This preparation consists of D1, D2, CP43, CP47, 33 kDa, and a few low molecular weight proteins, and retains a high rate of oxygen‐evolving activity (=1000 μmol/mg Chl/h).
Examination of temperature‐induced ‘gel‐sol’ transformation of α‐actinin/cross‐linked actin networks by static light scattering
Authors: Goldmann Wolfgang H. , Guttenberg Zeno
Published: July 2002

We studied the gel‐sol transformation of F‐actin/α‐actinin solutions. Cross‐linking of actin filaments by α‐actinin shows a temperature‐dependent increase in light scatter signal, (I)T. Higher F‐actin/α‐actinin molar ratios, r
Aα as well as increases in F‐actin concentration, [A], and reduction of actin filament lengths, r
AG, augment the maximal light intensity, I and shift the gel‐sol transition point, T
g to higher temperatures. This behavior is interpreted in terms of the model developed by Tempel, M., Isenberg, G. and Sackmann, E. (1996) (Physical Review E 54, 1802–1810) based on the percolation theory. Using the temperature‐dependent binding model of this theory allows instant prediction of the equilibrium constant, K for F‐actin/α‐actinin solutions at temperatures T<T
g.