bluuebunny/crossref_metadata_2025 huggingface dataset:
The prokaryotic thermophilic TF1‐ATPase is functionally compatible with the eukaryotic CFo‐part of the chloroplast ATP‐synthase
Authors: Galmiche Jean Michel , Pezennec Stephane , Zhao Rongbao , Girault Guy , Baeuerlein Edmund
Published: July 2002

The ATP synthase from chloroplasts, CFo · F1, was reconstituted into liposomes, from which most of CF1 was removed by a short treatment with guanidinium chloride. ATP‐dependent proton uptake was restored with these CFo‐liposomes even better by the addition of the bacterial TF1‐ than of the related CF1‐part. This proton uptake was prevented by tentoxin, a specific inhibitor of the CF1‐ATPase, in these CFo · F1‐liposomes, but not in the hybrid CFo · TF1‐liposomes. Venturicidin, a specific inhibitor of proton flow through CFo, was able to block it in both the hybrid CFo· TF1‐liposomes and reconstituted CFo· F1‐liposomes. These results indicate that the bacterial TF1‐part binds to the eukaryotic CFo‐part of four subunits forming a functional CFo · TF1‐ATPase.
Inhibition of epidermal growth factor‐dependent protein tyrosine phosphorylation by phorbol myristate acetate is mediated by protein tyrosine phosphatase activity
Authors: Errasfa Mourad , Stern Arnold
Published: July 2002

Incubation of HER 14 cells with phorbol myristate acetate (PMA) decreases epidermal growth factor (EGF)‐dependent protein tyrosine phosphorylation, except for a 40‐kDa MAP kinase II‐like protein, whose tyrosine phosphorylation is further enhanced. The inhibitory effect of PMA on EGF‐dependent protein tyrosine phosphorylation is reversed if cells are pre‐incubated with a combination of Na3VO4 and NaF, two known inhibitors of protein tyrosine phosphatase activity. Protein tyrosine phosphatase activity of cell homogenate was measured on immunopurified EGF receptor, and was found to be enhanced in PMA‐treated cells. These data suggest that the inhibitory effect of PMA on EGF‐dependent protein tyrosine phosphorylation in HER14 cells may be mediated by protein tyrosine phosphatase activity.
A monoclonal antibody that interferes with the post‐aggregation adhesion of Dictyostelium discoideum cells
Authors: Keller Thomas , Eitle Eveline , Balding Kirsten , Corrick Catherine , Parish Roger W.
Published: July 2002

A monoclonal antibody that interferes with the EDTA‐resistant adhesion of Dictyostelium discoideum slug cells recognised a carbohydrate epitope on four major antigens (95, 90, 35 and 30 kDa) in slug cells. The 35 and 30 kDa antigens were specific for stalks and spores, respectively. The 30 kDa antigen was identified as the cell surface glycoprotein, PsA. Cyclic AMP, acting via cell surface receptors, induced only the 90 kDa slug cell antigen. Slug cell adhesion proteins may be involved in cell‐sorting and the glycosylation of the 95 and 90 kDa antigens appeared to be abnormal in a mutant defective in cell‐sorting. Previously, a 150 kDa glycoprotein has been strongly implicated in slug cell adhesion and the present work suggests that additional glycoprotein(s) are involved.
A porcine gut polypeptide identical to the pancreatic hormone PP (pancreatic polypeptide)
Authors: Chen Zheng-wang , Bergman Tomas , Östenson Claes-Göran , Höög Anders , Näslund Jan , Norberg Åke , Carlquist Mats , Efendic Suad , Mutt Viktor , Jörnvall Hans
Published: July 2002

A peptide hormone has been isolated from porcine intestine. Its primary structure was found to consist of 36 amino acid residues in a sequence identical to that of the porcine pancreatic polypeptide, previously not isolated from intestines or a tissue other than pancreas. The gut polypeptide significantly suppresses glucose‐induced insulin secretion in vitro. Using an immunohistochemical technique, we also identified cells in the porcine gastrointestinal tract that were immunoreactive with pancreatic polypeptide. The immunoreactivity disappeared after absorption with the isolated gut polypeptide or synthetic human pancreatic polypeptide.
Differential response of NF‐κB1 p105 and NF‐κB2 p100 to HTLV‐I encoded tax
Authors: Watanabe Mitsuo , Muramatsu Masa-aki , Tsuboi Akio , Arai Ken-ichi
Published: July 2002

In a previous study we found that HTLV‐I encoded Tax transactivator, which binds to NF‐κ‐B1 p105, suppresses p105‐mediated Iκ‐B activity, thereby allowing entry of NF‐κB p65 (Re1A) and p50 into the nucleus. In the present report, we compared the effect of Tax on NF‐κB2 p100, which also binds to Tax, with that of p105 in transfected COS7 cells. While p 105 is processed to the DNA binding form, p50, processing of p100 was much less efficient both in the presence and absence of Tax. Both p105 and p100 showed IfκB activity in sequestering NF‐κB p65 into the cytoplasm. However, only p105‐mediated IκB activity, and not that of p100, was inhibited by Tax. Chimeric molecules between p100 and p105 suggested that inefficient processing of p100 can be attributed to the Rel homologous domain, rather than to the ankyrin repeat domain. p100, but not p105, potently suppressed Tax‐ and p65‐induced transactivation of a GM‐CSF promoter in Jurkat cells. Taken together, these results suggest that p105 and p100 may have distinct effects on Tax‐induced transactivation events.
Phosphorylation and accumulation of tau without any concomitant increase in tubulin levels in Chinese Hamster Ovary cells stably transfected with human tau441
Authors: Haque Niloufar , Denman Robert B , Merz George , Grundke-Iqbal Inge , Iqbal Khalid
Published: July 2002

Eucaryotic expression vectors bearing a 1.4 kb cDNA encoding the 4 repeat isoform of human tau, tau441, in either the sense or anto‐sense orientation with respect to a cytomegalovirus (CMV) promoter were constructed. The resulting constructs were used to transiently express tau in Chinese Hamster Ovary cells and to generate non‐neuronal stable cell lines. Immunocytochemical studies of these cells show that tau is expressed in the sense but not the anti‐sense or vector containing lines. Some of the cells expressing tau showed fine elongated processes which were stained by tau antibodies. The general tau immunostaining pattern appeared diffuse and punctuate. The expressed tau was seen both unbound and bound to microtubules. In some cells labeling with antibodies that specifically recognize hyperphosphorylation of tau was observed. The size of this population increased with increasing numbers of cell passages. However, no increase in steady‐state tubulin level was observed following tau441 expression. These studies show that tau can accumulate in the cells without a concomitant increase in tubulin.
Reactivity of histidyl residues in d‐amino acid oxidase from Rhodotorula gracilis
Authors: Gadda Giovanni , Beretta Giovanni L. , Pilone Mirella S.
Published: July 2002

Incubation of d‐amino acid oxidase from the yeast Rhodotorula gracilis with excess dansyl chloride at pH 6.6 and 18°C caused an irreversible inactivation of d‐amino acid oxidase. Benzoate, a competitive inhibitor of the enzyme, completely protected the enzyme from inactivation. The dansylated‐enzyme, isolated by gel‐filtration, was in part still active while the substrate specificity was altered substantially. It was completely reduced by d‐alanine in anaerobiosic conditions and did stabilize the red anion semiquinone upon photochemical reduction with EDTA. The results provide evidence for the presence of essential histidyl residue(s) in the active center of the yeast enzyme.
Agonist regulation of the expression of the delta opioid receptor in NG108‐15 cells
Authors: Kim Dong Sun , Chin Hemin , Klee Werner A.
Published: July 2002

Exposure of neuronal cells to the chronic presence of opiates leads to a complex series of biochemical events which reflect the changes that result in tolerance and dependence in animals. To achieve a better understanding of the molecular mechanisms underlying these processes, we have examined the effect of agonist efficacy on the regulation of the δ‐opioid receptor mRNA in NG108‐15 cells. Incubation with various opiates decreased receptor numbers in the order of their efficacy. Northern blot analysis showed that there are 4 size classes of mRNA coding for the δ‐opioid receptor in NG108‐15 cells even though only one known protein species is found. Moreover, the amount of each transcript is coordinately decreased by long‐term etorphine treatment, but not necessarily to the same extent. The etorphine‐induced decrease in receptor mRNA was found to be slow in onset, whereas a much more rapid loss of receptor number was observed. This disparity suggests that the down‐regulation induced by etorphine can occur both at the levels of receptor protein modification and receptor gene expression, and that the mechanisms of the two processes may be different.
Identification of a cryptic protein kinase CK2 phosphorylation site in human complement protease C1r, and its use to probe intramolecular interaction
Authors: Pelloux Sophie , Thielens Nicole M. , Hudry-Clergeon Gilbert , Pétillot Yves , Filhol Odile , Arlaud Gérard J.
Published: July 2002

Treatment of human Cr by CK2 resulted in the incorporation of [32P]phosphate into the N‐terminal α region of its non‐catalytic A chain. Fragmentation of 32P‐labelled Cr followed by N‐terminal sequence and mass spectrometry analyses allowed identification of Ser189 as the phosphorylation site. Accessibility of Ser189 was low in intact C1r, due in part to the presence of one of the oligosaccharides borne by the α region, further reduced in the presence of calcium, and abolished when C1r was incorporated into the C1s‐C1r‐C1r‐C1s treatment or the C1 complex. In contrast, phosphorylation was enhanced in the isolated α fragment and insensitive to calcium. Taken together, these data provide support for the occurrence of a Ca2+‐dependent interaction between the α region and the remainder of the C1r molecule.
The Sulfolobus solfataricus P2 genome project
Authors: Charlebois Robert L. , Gaasterland Terry , Ragan Mark A. , Ford Doolittle W. , Sensen Christoph W.
Published: July 2002

Over 800 kbp of the 3‐Mbp genome of Sulfolobus solfataricus have been sequenced to date. Our approach is to sequence subclones of mapped cosmids, followed by sequencing directly on cosmid templates with custom primers. Using a prototype automated system for genome‐scale analysis, known as MAGPIE, along with other tools, we have discovered one open reading frame of at least 100 amino acids per kbp of sequence, and have been able to associate 50% of these with known genes through database searches. An examination of completely sequenced cosmids suggests a clustering of genes by function in the S. solfataricus genome.
Mammalian microsomal and soluble Ras‐processing peptidase activities are distinct
Authors: Hitz Anna M. , Georgopapadakou Nafsika H.
Published: July 2002

Microsomal and soluble peptidases from bovine liver and pig brain hydrolyze the farnesylated, Ras‐based CAAX peptide [3H]Ac‐fCVIM‐OH. However, they differ in their sensitivity to substrate‐based inhibitors, sulfhydryl and chelating agents, pH and ionic strength optima, and stability. The microsomal activity was exquisitely sensitive to the substrate‐based inhibitor Boc‐fC[CH2]VIM‐OH, moderately sensitive to the sulfhydryl agent pCMB, but insensitive to NEM and the metal‐chelating agent o‐phenanthroline. The soluble activity was insensitive to Boc‐fC[CH2]VIM‐OH, but very sensitive to pCMB, NEM and o‐phenanthroline, suggesting it to be the previously reported (Biochem. Biophys. Res. Commun. 198, 787–794 (1994)) zinc metallopeptidase. The microsomal activity is most likely to be a cysteine peptidase involved in the post‐translational processing of Ras proteins.
Testing the eukaryotic promoters for efficient expression of exogenous genes in chondrocytes and synoviocytes
Authors: Su Suming , Gedamu Lashitew , Dehnade Faramaze , Zafarullah Muhammad
Published: July 2002

To identify suitable promoters for expressing exogenous genes in arthritic joints, the constitutive, simian virus 40 (SV 40) and IL‐1 or metal inducible, human stromelysin and metallothionein (MT) gene promoters were tested for their activity in chondrocytes and synovial fibroblasts. Transient transfection with plasmids containing the reporter chloramphenicol acetyltransferase (CAT) gene attached to these promoters showed that SV40, stromelysin and MT promoters drove CAT expression with different strengths in primary bovine chondrocytes. The MTI‐F and MT‐IG gene promoters were also functional in human chondrocytes. The SV40, IL‐1 inducible stromelysin‐1 and MT‐IG driven CAT activity was also detectable in human synoviocytes. Therefore, chondrocytes and synoviocytes have the trans‐acting factors necessary for transcription from the respective promoters which may be conserved in bovine and human cells. These promoters could be useful for expressing potentially therapeutic anti‐inflammatory and anti‐erosive genes in arthritic joints.
Ser644 is important for catalytic activity but is not involved in cAMP‐dependent phosphorylation of yeast 6‐phosphofructo‐2‐kinase
Authors: Keßler Renate , Eschrich Klaus
Published: July 2002

To identify the target amino acid for the cAMP‐dependent phosphorylation of yeast 6‐phosphofructo‐2‐kinase Ser644 was mutated to Ala. The plasmid‐encoded wild‐type and mutant enzymes were overexpressed in E. coli TG2 cells and in the yeast strain DFY658. Like the wild‐type enzyme, the Ser644 → Ala mutant was phosphorylated in vivo after addition of glucose to yeast cells and in vitro by the catalytic subunit of protein kinase A. The specific activity of the mutant enzyme was 6‐fold lower than that of the wild‐type yeast 6‐phosphofructo‐2‐kinase, but both enzymes were activated in response to the addition of glucose to yeast cells.
The influence of sub‐optimal protein nutrition on insulin hypersecretion evoked by high‐energy/high‐fat feeding in rats
Authors: Holness Mark J.
Published: July 2002

Low (8%) protein feeding during pregnancy impairs the functional development of the fetal endocrine pancreas. Continued low‐protein feeding post‐natally decreases pancreatic insulin content and secretion, whereas transfer to standard diet evoked β‐cell recuperation. Glucose‐stimulated insulin secretion (GSIS) and insulin action were examined in vivo at 28 days after transfer from 8% protein diet to a high‐energy/high‐fat/standard (20%)‐protein diet (HEF diet). HEF feeding dramatically enhanced GSIS after intravenous glucose in control rats, but not in rats previously maintained on the low‐protein diet. However, glucose disappearance after intravenous glucose, and glucose production and whole‐body glucose disposal during euglycaemic‐hyperinsulinaemic clamps were unaffected by prior protein malnutrition. In conclusion, impaired insulin secretion after protein malnutrition is exacerbated by high‐energy/high‐fat feeding, but this response is not linked to enhanced whole‐body insulin resistance.
Normal human endometrial cells in culture: Characterization and immortalization of epithelial and stromal cells by SV 40 large T antigen
Authors: Merviel Philippe , Degeorges Armelle , Salat‐Baroux Jacques , Calvo Fabien
Published: July 2002

Summary— Thirty endometrial biopsies were cultured in order to separate stromal and epithelial cells. Using epidermal growth factor (EGF), cortisol, cholera toxin, insulin with 5% horse serum for epithelial cells or a medium with 20% fetal calf serum for stromal cells, we could specifically enrich endometrial culture in epithelial or stromal cells and culture them for 1 or 2 months. These cultures retained the phenotypic characteristics of epithelial (cytokeratins, mucin HMFG 1) and stromal (vimentin, smooth muscle actin, desmin) lineage by immunostaining analysis. Epithelial and stromal cultures from one individual were respectively immortalized by the SV 40 large T antigen. The immortalized cell lines kept the phenotype of the normal cells from which they derived.
Key amino acids of vasopressin V1a receptor responsible for the species difference in the affinity of OPC‐21268
Authors: Shinoura Hitomi , Take Hitoshi , Hirasawa Akira , Inoue Kazuhide , Ohno Yasuo , Hashimoto Keitaro , Tsujimoto Gozoh
Published: July 2002

A non‐peptide, vasopressin V1a receptor‐selective antagonist, OPC‐21268, exhibited a markedly higher affinity for the rat V1a receptor (K
i=380 nM) than for the human V1a receptor (K
i=140 μM). To delineate the region responsible for the high affinity binding of OPC‐21268 for the rat V1a receptor, we have constructed a series of chimeric human and rat V1a receptors, and examined the chimeric and point‐mutated receptors by competitive radioligand binding analysis. The results showed that the transmembrane domain (TMD) VI–VII of the vasopressin V1a receptor, in particular the amino acid residue Ala‐342 in TMD VII, is the major component conferring the rat‐selective binding of OPC‐21268 to the V1a receptor.
Spin label EPR structural studies of the N‐terminus of α‐spectrin
Authors: Cherry L , Menhart N , Fung L.W.-M
Published: July 2002

Spectrin, a vital component in human erythrocyte, is composed of α‐ and β‐subunits, which associate to form (αβ)2 tetramers. The tetramerization site is believed to involve the α‐spectrin N‐terminus and the β‐spectrin C‐terminus. Abnormal interactions in this region may lead to blood disorders. It has been proposed that both termini consist of partial structural domains and that tetramerization involves the association of these partial domains. We have studied the N‐terminal region of a model peptide for α‐spectrin by making a series of double spin‐labeled peptides and studying their dipolar interaction by electron paramagnetic resonance methods. Our results indicate that residues 21–42 of the N‐terminus region exhibit an α‐helical conformation, even in the absence of β‐spectrin.
The species specificity of growth hormone requires the cooperative interaction of two motifs
Authors: Peterson Francis C , Brooks Charles L
Published: July 2002

Primate growth hormones (GH) activate both primate and non‐primate somatotrophic receptors (GH receptors), but non‐primate GHs do not activate primate GH receptors. Previous studies argued the interaction of Asp171 of human GH and Arg43 of the receptor produced an attractive ionic interaction. In non‐primate GHs, His170 replaces the homologous Asp171, producing a repulsive interaction with Arg43 of the primate receptor which was believed to reduce the attraction of non‐primate GH for the human GH receptor, thus providing species specificity. In this report, H170D bovine GH had activity and affinity for human GH receptors approaching those of human GH. In contrast, replacing Asp171 of human GH with His did not significantly reduce somatotrophic activity, indicating that species specificity is not wholly explained by this residue's interaction with Arg43 of the receptor. Deletion of either Phe44 (a residue present only in primate GHs) or residues 32–46 (20‐kDa form of human GH) each only marginally reduced somatotrophic activities. But the combination of the D171H mutation with either ΔPhe44 or Δ32–46 in human GH reduced binding and activity in a greater than additive fashion, indicated a functional interaction between these distant structural features. In bovine GH addition of phenylalanine at position 44 increased the somatotrophic activity and receptor affinity in cells containing the human GH receptor. The combination of the H170D mutation and the addition of phenylalanine at position 44 created a bovine GH with activity indistinguishable from wild‐type human GH. Based on evidence from both bovine and human GHs, the cooperative interaction of these two distant motifs determined the species specificity and indicated that structural plasticity was a critical feature necessary for the species specificity of somatotrophic activity.
Impaired stimulation of intestinal glucose absorption by portal insulin via hepatoenteral nerves in chronically ethanol‐intoxicated rats
Authors: Kucera Tomas , Jungermann Kurt , Stümpel Frank
Published: July 2002

In the isolated, jointly perfused small intestine and liver of rats insulin, infused into the portal vein, induced an increase in intestinal glucose absorption via hepatoenteral cholinergic nerves. The possible loss of function of these nerves due to ethanol‐induced neuropathy was investigated with 6 weeks ethanol‐fed rats. Portal insulin or arterial carbachol failed to increase intestinal glucose absorption but cAMP still did so. The intact stimulatory effect of cAMP indicated an undisturbed capacity of the enterocytes. The loss of action of portal insulin and of arterial carbachol can be explained by the impairment of the hepatoenteral nerves in line with an ethanol‐induced neuropathy.
Blue light regeneration of bacteriorhodopsin bleached by continuous light
Authors: Dancsházy Zsolt , Tokaji Zsolt
Published: July 2002

Photobleaching of bacteriorhodopsin (BR) by continuous light has recently been demonstrated. This bleaching consists of at least two subsequent product states. One of them is absorbing maximally in the blue spectral region. Our present study shows that upon illumination of the bleached sample with blue light a back photoprocess appears, resulting in regeneration of the original BR state. From a technical point of view, the observed phenomenon is similar to the reverting effect of blue light on the photocycle. An important difference is that the photobleached state of BR is much more stable than any of the photocycle intermediates, and may provide an advantage for several technical applications.
Analyses of the genomic methylation status of the human cyclin A1 promoter by a novel real‐time PCR‐based methodology
Authors: Müller-Tidow Carsten , Bornemann Christina , Diederichs Sven , Westermann Annette , Klümpen Silvia , Zuo Peijun , Wang Wenbing , Berdel Wolfgang E , Serve Hubert
Published: July 2002

The role of CpG methylation in the regulation of tissue‐specific gene expression is highly controversial. Cyclin A1 is a tissue‐specifically expressed gene that is strongly methylated in non‐expressing tumor cell lines. We have established a novel real‐time PCR method to quantitate genomic CpG methylation of the cyclin A1 promoter. Genomic DNA samples from different human organs were treated with bisulfite and amplified with methylation‐specific primers and with primers amplifying methylated as well as non‐methylated DNA. PCR product quantitation was obtained by using a fluorogenic probe labeled with FAM and TAMRA. These analyses demonstrated that the human cyclin A1 promoter was methylated in kidney, colon, spleen, testis, and small intestine, but not in brain, liver, pancreas, or heart. Expression of cyclin A1 was predominantly found in testis. Low level expression of cyclin A1 was present in spleen, prostate, leukocytes, colon, and thymus. Taken together, our data provide evidence that CpG methylation patterns of the human cyclin A1 promoter in human organs do not generally correlate with cyclin A1 gene expression in vivo.
p‐Hydroxyphenylacetaldehyde, the major product of tyrosine oxidation by the activated myeloperoxidase system can act as an antioxidant in LDL
Authors: Exner M , Alt E , Hermann M , Hofbauer R , Kapiotis S , Quehenberger P , Speiser W , Minar E , Gmeiner B
Published: July 2002

The oxidative modification of low density lipoprotein (LDL) may play a significant role in atherogenesis. HOCl generated by the myeloperoxidase/H2O2/Cl− system of activated neutrophils may be operative in vivo making LDL atherogenic. Tyrosine has been found to be oxidized by HOCl to p‐hydroxyphenylacetaldehyde (p‐HA) capable of modifying phospholipid amino groups in LDL. As an amphiphatic phenolic compound, p‐HA may have the potential to act as an antioxidant in the lipid phase of LDL. The present results show that (a) tyrosine exerts a protective effect on LDL modification by HOCl, (b) p‐HA could act as antioxidant associated with the lipoprotein preventing cell‐ and transition metal ion‐mediated LDL oxidation and (c) p‐HA was able to scavenge free radicals.
Inhibition of gap junctional intercellular communication in rat liver epithelial cells with transforming RNA
Authors: Hayashi Tomonori , Trosko James E. , Hamada Katsutomo
Published: July 2002

Previous studies indicated that transforming RNA, derived from the 3′ half of the U5 small nuclear RNA first stem structure, suppressed the secretory protein translation in vitro. Gap junctions facilitate homeostatic control of cell growth and differentiation and their dysfunction has been correlated with carcinogenesis. Here, we reported that transforming RNA directly suppressed the gap junction protein, connexin 43, translation and thereby inhibited functional gap junction function in rat epithelial cells. Together with previous data, this implies that altered expression of transforming RNA itself is a potential mechanism in inhibiting gap junction function during carcinogenesis.
Bidirectional activity and orientation‐dependent specificity of the rat aldolase C promoter in transgenic mice
Authors: Phan Dinh Tuy Françoise , Porteu Arlette , Kahn Axel , Skala Henriette
Published: July 2002

We previously reported that the rat aldolase C 115 bp promoter is sufficient to ensure the brain specific expression of the chloramphenicol acetyltransferase reporter gene in transgenic mice. We identify in a further reduced 84 bp promoter several putative binding sites for the transcriptional factors Sp1, USF, AP1, and AP2. Deletion or mutation of these partially overlapping binding sites results in inactivation of the cognate transgenes. Moreover, we show that the 115 bp sequence is able to direct bidirectional transcription in vivo but, surprisingly, transcriptional activity in the opposite direction is no more brain specific.
Estramustine phosphate but not estramustine inhibits the interaction of microtubule associated protein 2 (MAP2) with actin filaments
Authors: Pedrotti Barbara , Islam Khalid
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Calpains are involved in phosphatidylinositol 3′,4′‐bisphosphate synthesis dependent on the αIIbβ3 integrin engagement in thrombin‐stimulated platelets
Authors: Montsarrat Nathalie , Racaud-Sultan Claire , Mauco Gérard , Plantavid Monique , Payrastre Bernard , Breton-Douillon Monique , Chap Hugues
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Molecular mobility of nitroxyl‐labelled taxol during tubulin assembly
Authors: Nicholov R , Kingston D.G.I , Chordia M.C , DiCosmo F
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Ligands regulate GroEL thermostability
Authors: Surin A.K , Kotova N.V , Kashparov I.A , Marchenkov V.V , Marchenkova S.Yu , Semisotnov G.V
Published: July 2002

Escherichia coli heat‐shock proteins GroEL and GroES stimulate (in an ATP‐dependent manner) the folding of various proteins. In this study scanning microcalorimetry was applied to investigate GroEL thermostability in the presence of its ligands. Mg2+ and K+ ions stabilize while ADP destabilizes the GroEL molecule against the action of temperature. Furthermore, ADP essentially increases the number of binding sites for the hydrophobic probe (ANS) and the number of GroEL SH‐groups accessible to Ellman's reagent as well as the accessibility of the protein to the action of trypsin. The interaction of GroEL with GroES in the presence of Mg2+‐ADP eliminates the destabilizing effect of ADP on the GroEL molecule against the action of temperature and Ellman's reagent but does not change its hydrophobicity and accessibility to trypsin.
Blood group MN‐dependent difference in degree of galactosylation of O‐glycans of glycophorin A is restricted to the GalNAc residues located on amino acid residues 2–4 of the glycophorin polypeptide chain1
Authors: Krotkiewski Hubert , Duk Maria , Syper Danuta , Lis Halina , Sharon Nathan , Lisowska Elwira
Published: July 2002

Glycophorin A (GPA) of human erythrocytes contains a minor number of unsubstituted GalNAc residues (Tn receptors) which are recognized by Moluccella laevis lectin (MLL). The lectin reacts better with blood group N‐ than M‐type of GPA which suggests a higher number of Tn receptors in GPA‐N than in GPA‐M. To find out whether this difference is restricted to a defined domain of GPA, the N‐terminal tryptic glycopeptides of GPA‐M and GPA‐N (a.a. residues 1–39) and their fragments obtained by degradation with CNBr (a.a. residues 1–8 and 9–39) were analyzed. The untreated and desialylated glycopeptides were tested as inhibitors of MLL in ELISA, and the content of GalNAc‐ol was determined in the products of β‐elimination of the asialoglycopeptides by gas‐liquid chromatography/mass spectrometry. The asialoglycopeptides 1–39 and 1–8 derived from GPA‐N showed about 2 and 4 times higher content of non‐galactosylated GalNAc residues, respectively, and higher reactivity with MLL than their counterparts derived from GPA‐M, while asialoglycopeptides 9–39 of GPA‐M and GPA‐N did not show such differences. These results demonstrate that higher expression of non‐galactosylated GalNAc in GPA‐N than in GPA‐M is confined to GalNAc residues located in the amino‐terminal portion of GPA polypeptide chain, between the blood group M‐ and N‐specific amino acid residues 1 and 5.
Spin trapping agent phenyl N‐tert‐butylnitrone protects against the onset of drug‐induced insulin‐dependent diabetes mellitus
Authors: Tabatabaie Tahereh , Kotake Yashige , Wallis Gemma , Jacob Jane M , Floyd Robert A
Published: July 2002

Insulin‐dependent diabetes mellitus is an autoimmune disease believed to be caused by an inflammatory process in the pancreas leading to selective destruction of the β‐cells. Cytokines and nitric oxide (NO) have been shown to be involved in this destruction. Phenyl N‐tert‐butylnitrone (PBN) has demonstrated protective effects against several pathological conditions including ischemia‐reperfusion injury and endotoxin‐induced shock. We report here that PBN co‐administration can prevent the onset of the STZ‐induced diabetes in mice. PBN co‐treatment inhibited the streptozotocin (STZ)‐induced hyperglycemia, the elevation in the level of glycated hemoglobin and weight loss in the treated mice. Histological observations indicated destruction of β‐cells in the STZ‐treated animals and its prevention by PBN co‐treatment. EPR spin trapping experiments in the pancreas indicated the in vivo formation of NO in STZ‐treated animals and its attenuation by PBN treatment.
A native electrostatic environment near QB is not sufficient to ensure rapid proton delivery in photosynthetic reaction centers
Authors: Valerio-Lepiniec Marie , Delcroix Jean-Dominique , Schiffer Marianne , Hanson Deborah K , Sebban Pierre
Published: July 2002

Flash‐induced absorption spectroscopy has been used to characterize Rhodobacter capsulatus reaction centers mutated in the secondary quinone acceptor site (QB). We compared the wild‐type, the L212Glu‐L213Asp→Ala‐Ala photosynthetically incompetent double mutant (DM), and two photocompetent revertants, the DM+L217Arg→Cys and the DM+M5Asn→Asp strains. The electrostatic environment for QB
− is different in the two revertant strains. Only the L217Arg→Cys mutation nearly restores the native electrostatic environment of QB
−. However, the level of recovery of the reaction center function, measured by the rates of second electron transfer and cytochrome c turnover, is quite incomplete in both strains. This shows that a wild‐type‐like electrostatic environment of QB
− cannot ensure on its own, rapid and efficient proton transfer to QB
−.
Functional interference between contacting amino acids of homeodomains
Authors: Pellizzari Lucia , Tell Gianluca , Fabbro Dora , Pucillo Carlo , Damante Giuseppe
Published: July 2002

In a protein, the function of an amino acid at some position depends on the amino acids at other positions. Here we demonstrate a functional interference between base‐contacting amino acids (at positions 50 and 54) of homeodomains. When, in the context of Antennapedia or Goosecoid homeodomains, Lys50 is paired to Tyr54 or Ala54 and Gln50 is paired to Met54, the resulting proteins efficiently discriminate among different DNA sequences. In contrast, in the presence of the pair Lys50‐Met54, both homeodomains show a reduced capability to discriminate among different DNA sequences. Sequence selection experiments performed in the context of the Goosecoid homeodomain suggest that the presence of Met54 precludes the base‐discriminating function of Lys50. These results may explain why the pair Lys50‐Met54 is never found in natural homeodomains.
Biogenesis of Candida albicans Can1 permease expressed in Saccharomyces cerevisiae
Authors: Matı̀jèková Alena , Sychrová Hana
Published: July 2002

The Candida albicans CAN1 gene, encoding a high‐affinity permease for arginine, lysine and histidine, was tagged at its C‐terminus with a c‐myc epitope and introduced into strains of Saccharomyces cerevisiae lacking basic amino acid permeases. The expression levels of Ca‐Can1p were influenced by the available nitrogen source, being almost negligible when cells were grown in the presence of ammonia. Ca‐Can1p was shown to follow the secretory pathway in S. cerevisiae. Ca‐Can1p activity was not detected in a secretion‐defective sec1‐1 mutant grown at a non‐permissive temperature. Shr3p, an ER protein that participates in the biogenesis of amino acid permeases was also required for the functional expression of Ca‐Can1p. The shr3 mutation does not affect the affinity for substrate but does decrease the number of Can1p molecules reaching the plasma membrane.
Involvement of soluble proteinous factors in auxin‐induced modulation of P‐type ATPase in rice (Oryza sativa L.) seedlings
Authors: Kim Donghern , Kim Yong-Sam , Jung Jin
Published: July 2002

The marked difference in auxin sensitivity between plant roots and shoots was studied in terms of auxin‐induced stimulation of membrane P‐type ATPase. The results suggest the existence in rice seedlings of, at least, two isoforms of soluble proteinous factors (SPF), SPFI and SPFII, which are involved in the stimulatory action of auxin on the enzyme. It also is indicated that SPFI which mediates the auxin effect in low hormone concentration range (10−10–10−7 M IAA) is the dominant isoform in roots, whereas SPFII which does in high hormone concentration range (10−7–10−4 M IAA) is that in shoots.
RNA polymerase I from S. cerevisiae depends on an additional factor to release terminated transcripts from the template
Authors: Tschochne Herbert , Milkereit Philipp
Published: July 2002

Terminated transcripts were generated at the ends of linearized DNA templates and at DNA‐bound lac repressor by in vitro transcription with highly enriched or purified yeast RNA polymerase I (pol I). The release of the synthesized transcripts from the DNA was analyzed using immobilized DNA as template for the transcription reaction. An additional activity distinguishable from pol I was necessary to remove the terminated RNA from the template. Efficiency of transcript release could be improved if a thymidine‐rich DNA fragment was located upstream of the transcriptional arrest caused by the DNA‐bound lac repressor. The release activity interacted with different forms of polymerases, pol I able to initiate on the ribosomal gene promoter and pol I only active in non‐specific transcription.
Role of the distal hinge region of C1‐inhibitor in the regulation of C1s activity
Authors: He Shiping , Yang J.-C , Tsang S , Sim R.B , Whaley K
Published: July 2002

A synthetic peptide corresponding to residues 448–459 of C1‐inhibitor (C1‐inh) binds to C1s, is a non‐competitive inhibitor of C1s activity and prevents formation of an SDS‐stable C1s–C1‐inh complex. Substitutions of residues Q452, Q453 or F455 in this peptide resulted in loss of C1s binding and inhibitory activity of the peptide. NMR analysis of the peptide showed an area of well‐defined structure from E450 to F455. The side chains of Q452, Q453 and Q455 were exposed to the solvent and therefore available for C1s binding. The defined structure in the peptide is compatible with our computer model of the serpin domain of C1‐inh.
Nuclear levels of NF‐κB correlate with syncytium‐forming capacity of 8e51 cells, expressing a defective HIV virus
Authors: Guerrini Luisa , De Santis Claudio , Robbioni Piera , Beretta Alberto , Blasi Francesco
Published: July 2002

The double NF‐κB site identified in the LTR of the human immunodeficiency virus‐1 (HIV‐1) has been demonstrated to be necessary for efficient viral transcription. In this report we present the characterisation of NF‐κB subunits engaged in complexes binding to the HIV‐1 NF‐κB site in human 8e51 T‐cells, that harbour a defective HIV‐1. At least four different specific NF‐κB complexes are present in the nucleus of these cells. With the use of specific antibodies we have determined the composition of each complex using electrophoretic mobility shift assays. The results show the presence of several NF‐κB family members, with the transactivating RelA being engaged in multiple complexes. The importance of NF‐κB complexes in viral functions has been established comparing the level of NF‐κB DNA‐binding complexes with syncytia‐forming activity of 8e51 cells. In fact, 8e51 cells that had almost lost their syncytia‐forming capacity were found to contain at least 10 times less active NF‐κB DNA‐binding complex than the actively fusing cells. The correlation is specific as the level of at least three other transcription factors did not change.
mRNP3 and mRNP4 are phosphorylatable by casein kinase II in Xenopus oocytes, but phosphorylation does not modify RNA‐binding affinity
Authors: Deschamps Stéphane , Jacquemin-Sablon Hélène , Triqueneaux Gérard , Mulner-Lorillon Odile , Potier Michel , Le Caer Jean-Pierre , Dautry François , le Maire Marc
Published: July 2002

mRNP3 and mRNP4 (also called FRGY2) are two mRNA‐binding proteins which are major constituents of the maternal RNA storage particles of Xenopus laevis oocytes. The phosphorylation of mRNP3–4 has been implicated in the regulation of mRNA masking. In this study, we have investigated their phosphorylation by casein kinase II and its consequence on their affinity for RNA. Comparison of the phosphopeptide map of mRNP3–4 phosphorylated in vivo with that obtained after phosphorylation in vitro by purified Xenopus laevis casein kinase II strongly suggests that casein kinase II is responsible for the in vivo phosphorylation of mRNP3–4 in oocytes. The phosphorylation occurs on a serine residue in a central domain of the proteins. The affinity of mRNP3–4 for RNA substrates remained unchanged after the treatment with casein kinase II or calf intestine phosphatase in vitro. This suggests that phosphorylation of these proteins does not regulate their interaction with RNA but rather controls their interactions with other proteins.
The D13C variant of Bacillus schlegelii 7Fe ferredoxin is an 8Fe ferredoxin as revealed by 1H‐NMR spectroscopy
Authors: Aono Shigetoshi , Bentrop Detlef , Bertini Ivano , Luchinat Claudio , Macinai Riccardo
Published: July 2002

The N‐terminal cluster binding motif Cys8XXXXXXXCys16....Cys49 of Bacillus schlegelii 7Fe ferredoxin, which provides the ligands to the [Fe3S4]+ cluster, was modified by the mutation Asp13→Cys. The mutant D13C is expressed in Escherichia coli as an 8Fe ferredoxin, with NMR properties similar to those of clostridial‐type ferredoxins. The full assignment of the hyperfine shifted resonances indicates that Cys13 serves as ligand to the new fourth iron atom in the N‐terminal cluster despite the atypical binding sequence CysXXXXCysXXCys....Cys. The Cα–Cβ–S–Fe dihedral angles of all cysteine ligands to the two [Fe4S4]2+ clusters of the D13C variant are similar to those observed in other 8Fe and 4Fe ferredoxins.
Expression of PEP carboxylase from Escherichia coli complements the phenotypic effects of pyruvate carboxylase mutations in Saccharomyces cerevisiae
Authors: Flores Carmen-Lisset , Gancedo Carlos
Published: July 2002

We investigated the effects of the expression of the Escherichia coli ppc gene encoding PEP carboxylase in Saccharomyces cerevisiae mutants devoid of pyruvate carboxylase. Functional expression of the ppc gene restored the ability of the yeast mutants to grow in glucose‐ammonium medium. Growth yield in this medium was the same in the transformed yeast than in the wild type although the growth rate of the transformed yeast was slower. Growth in pyruvate was slowed down in the transformed strain, likely due to a futile cycle produced by the simultaneous action of PEP carboxykinase and PEP carboxylase.
Translocation of NLS–BSA conjugates into nuclei of permeabilized mammalian cells can be supported by protoplast extract
Authors: Broder Yehoshua C , Stanhill Ariel , Zakai Nehama , Friedler Assaf , Gilon Chaim , Loyter Abraham
Published: July 2002

An heterologous experimental system, which allows the study of the yet unknown cytosolic factors involved in nuclear import of nuclear localization signal (NLS)‐containing proteins in plants, has been established. The ability of plant cell extract to substitute mammalian cytosol and to promote translocation of NLS‐containing proteins into nuclei of permeabilized HeLa cells was demonstrated. The results described in the present work show that nuclear import of fluorescently labeled BSA conjugates bearing the NLS sequence of SV40 large T antigen could be supported by petunia cell cytoplasmic extract. This heterologous system shows the characteristic features of the homologous mammalian system, namely, is ATP dependent and is inhibited by WGA, GTPγS as well as by non‐fluorescent NLS–BSA conjugates. The system described here offers an experimental method to study and characterise cytosolic factors which are required for nuclear import in plants.
Purification, characterization and cloning of a novel variant of the superantigen Yersinia pseudotuberculosis‐derived mitogen
Authors: Ramamurthy T. , Yoshino Ken-ichi , Abe Jun , Ikeda Norikazu , Takeda Tae
Published: July 2002

The novel superantigen designated as Yersinia pseudotuberculosis‐derived mitogen typeB (YPMb) was purified from the cell lysate of Y. pseudotuberculosis O:6 strain (R‐104). Proliferative response of human peripheral blood mononuclear cells to the purified YPMb was detectable at a concentration of as low as 1 pg/ml, that is comparable to the previously documented YPM (YPMa). The Vβ repertoire specificity (3, 9, 13.1 and 13.2) of YPMb was also the same as that of YPMa. A gene (ypmB) encoding YPMb was cloned and its nucleotide sequence was determined. The open reading frame (ORF 453 bp) of the ypmB encodes a protein with 150 amino acid residues, though the precursor protein of YPMa consists of 151 amino acid residues. There is a nucleotide sequence homology of 88.9% between ypmB and ypmA. The low homology in the downstream of the structural gene between ypmB and ypmA and the difference in the GC content in the ORF of ypmB and ypmA from that of the base usage of Y. pseudotuberculosis suggest that the ypm gene originates from another organism. The alignment of the amino acid sequences of mature proteins of YPMb and YPMa revealed that there is 83% homology (108 amino acid residues are identical). Between YPMa and YPMb, the central region is less homologous than the N‐ and C‐terminal regions. Based on the functional similarity of two superantigen molecules, the less homology suggested that the central region is less important for the function of both YPM molecules.
Identification and tissue expression of a splice variant for the growth arrest‐specific gene gas6
Authors: Marcandalli Paolo , Gostissa Monica , Varnum Brian , Goruppi Sandro , Schneider Claudio
Published: July 2002

The growth arrest‐specific gene gas6 encodes a secreted protein (Gas6) which is a member of the vitamin K‐dependent protein family and was identified as a ligand for the Ax1 tyrosine kinase receptor family. Gas6 shares significant similarity with protein S and a similar domain organisation: an extensively γ‐carboxylated amino‐terminal, four epidermal growth factor‐like motifs and a large carboxy‐terminal region, known as the D domain. Here we report on the isolation of a splice variant (gas6SV) characterised by an in‐frame 129 bp insertion between the fourth EGF domain and the D domain. The gene gas6 was previously mapped on chromosome 13. The genomic organisation of gas6 has been investigated demonstrating the presence of alternative splicing consensus sites. Expression of gas6SV has been investigated in various human tissues and found to have a similar distribution pattern as gas6, with the exception of the spleen where gas6SV seems to be the predominant form.
Bactericidal activity of human lysozymes carrying various lengths of polyproline chain at the C‐terminus
Authors: Ito Yoshihiro , Kwon Oh Hyeong , Ueda Mitsuyoshi , Tanaka Atsuo , Imanishi Yukio
Published: July 2002

The amphiphilic polypeptide polyproline having different chain lengths was connected to the C‐terminus of human lysozyme by the recombinant DNA technique. The hydrophobicity of human lysozyme increased with increasing length of the polyproline chain. Although the bactericidal activity of wild‐type lysozyme is limited to Gram‐positive bacteria and the hydrolytic activity of the mutant lysozyme decreased with increasing chain length of polyproline, the mutant lysozymes showed bactericidal activity to Gram‐negative bacteria and the activity increased with increasing hydrophobicity of the mutant enzyme. Experiments with Escherichia coli phospholipid liposomes revealed that the mutant human lysozymes dissipated the valinomycin‐induced transmembrane electrochemical potential, and the dissipation increased with increasing hydrophobicity. The increased hydrophobicity of the mutant enzyme may induce interaction of lysozyme with the outer membrane and subsequent penetration into the inner membrane of E. coli, resulting in an increase of bactericidal activity.
Induction by peroxisome proliferators and triiodothyronine of serine:pyruvate/alanine:glyoxylate aminotransferase of rat liver
Authors: Oda Toshiaki , Funai Tsuneyoshi , Ichiyama Arata
Published: July 2002

In rat liver, a single serine:pyruvate/alanine:glyoxylate aminotransferase (SPT or SPT/AGT) gene is transcribed from two transcription initiation sites. Transcription from the upstream site generates the mRNA encoding the precursor for mitochondrial SPT (pSPTm) and is markedly enhanced by the administration of glucagon or cAMP. In this report we show the increase in the downstream transcript, the peroxisomal SPT (SPTp) mRNA, caused by peroxisome proliferators and triiodothyronine (T3). In the case of T3, the pSPTm mRNA was also increased 72 h after a single administration of the hormone in addition to an earlier increase in SPTp mRNA.
Roles of acidic residues in the hydrophilic loop regions of metal‐tetracycline/H+ antiporter Tet(K) of Staphylococcus aureus
Authors: Fujihira Erika , Kimura Tomomi , Yamaguchi Akihito
Published: July 2002

Three transmembrane glutamic acid residues play essential roles in the metal‐tetracycline/H+ antiporter Tet(K) of Staphylococcus aureus [Fujihira et al., FEBS Lett. 391 (1996) 243–246]. In the putative hydrophilic loop region of the Tet(K) and Tet(L) proteins, six acidic residues are conserved. Asp74, Asp200, Asp318 and Glu381 are located on the putative cytoplasmic side, and Asp39 and Glu345 on the putative periplasmic side. These residues were replaced by a neutral amino acid residue or a charge‐conserved one. In contrast to the transmembrane glutamic acid residues, the replacement of the two glutamic acid residues (Glu345 and Glu381) did not affect the tetracycline resistance level. Out of the other four aspartic acid residues, the only essential residue is Asp318, any replacement of which resulted in complete loss of the tetracycline resistance and transport activity. Asp318 is located in cytoplasmic loop 10–11 in the putative 14‐transmembrane‐segment topology of Tet(K). In the case of the tetracycline exporters of Gram‐negative bacteria, the only essential acidic residue in the cytoplasmic loop region is located in loop 2–3 [Yamaguchi et al., Biochemistry 31 (1992) 8344–8348]. It may be a general role for tetracycline efflux proteins that three transmembrane and one cytoplasmic acidic residues are mandatory for the tetracycline transport function.