bluuebunny/crossref_metadata_2025 huggingface dataset:
Growth hormone induces the tyrosine phosphorylation and nuclear accumulation of components of the ISGF3 transcription factor complex
Authors: Kilgour Elaine , Anderson Neil G.
Published: July 2002

Growth hormone induced the accumulation of the stat91 and p84 subunits of the transcription factor complex ISGF3 in nuclear fractions of 3T3‐F442A cells. Nuclear levels of p84 and stat91 peaked 30–60 min after addition of growth hormone. Growth hormone also induced the tyrosine phosphorylation of two proteins, with similar sizes to stat91 and p84, in both nuclear and cytosolic fractions. The time course of growth hormone‐induced tyrosine phosphorylation of these proteins paralleled the nuclear accumulation of stat91 and p84. Immunoprecipitation with stat91‐specific antibodies confirmed that growth hormone induced the tyrosine phosphorylation of stat91 and an associated protein of M
r, ≈ 120 kDa. These findings suggest a mechanism for the modulation of specific gene transcription by growth hormone.
Localization of an arginine‐specific mono‐ADP‐ribosyltransferase in skeletal muscle sarcolemma and transverse tubules
Authors: Klebl Bert M. , Matsushita Shigeki , Pette Dirk
Published: July 2002

The precise localization of a membrane‐bound, arginine‐specific mono‐ADP‐ribosyltransferase (mADP‐RT) was assessed in rabbit skeletal muscle by studying membrane fractions isolated by successive sucrose density gradient centrifugations. mADP‐RT activity was 10‐fold enriched in sarcolemmal and T‐tubular membranes. The catalytic activity, determined in preparations with mainly right‐side‐out vesicles, was found to be on the cytoplasmic face. As revealed by SDS‐PAGE and autoradiography endogenous mADP‐RT activity labeled several proteins in the range between 15 kDa and 250 kDa. T‐tubules contained the highest number of [32P]ADP‐ribose‐labeled proteins.
Modulation of cytosolic RNase activity by endogenous RNase inhibitor in rat vaginal epithelial cells on estradiol administration
Authors: Rao K.Sridhar , Sirdeshmukh Ravi , Gupta P.D.
Published: July 2002

The cytosolic, alkaline RNase in rat vaginal epithelial cells (VEC) from normal, immature rats was found to be present largely in the free, active form unlike in many other mammalian tissues where it is known to be present in a latent form as a complex with RNase inhibitor (RNasin). Estradiol (E2) administration induced expression of RNasin activity in the VEC from such animals and caused virtually total inhibition of cytosolic RNase activity in these cells by 12 h after the hormone injection. These changes may have metabolic implications in relation to other biochemical events stimulated by estradiol in rat VEC.
Modulation of endothelin receptor expression in human vascular smooth muscle cells by interleukin‐1β
Authors: Newman P. , Kakkar V.V. , Kanse S.M.
Published: July 2002

Endothelin may play a role in atherosclerosis as it causes smooth muscle cell proliferation and its levels are elevated in patients with atherosclerosis. We report that interleukin‐1β is a potent inducer of endothelin receptor expression in cultured human vascular smooth muscle cells. The effect is dose‐ and time‐dependent and is due to an increase in receptor number. Endothelin receptor mRNA levels are also elevated. Interleukin‐1β is a major regulator of endothelin release so, together with its effects on endothelin receptors, it may be responsible for a generalized activation of the endothelin system in diseased vessels.
Opposite regulation of bilirubin and 4‐nitrophenol UDP‐glucuronosyltransferase mRNA levels by 3,3′,5 triiodo‐l‐thyronine in rat liver
Authors: Masmoudi Taoufik , Planells Richard , Mounié Jacques , Artur Yves , Magdalou Jacques , Goudonnet Hervé
Published: July 2002

The effects of 3,3′,5 triiodo‐l‐thyronine (L‐T3) on the constitutive levels of hepatic mRNA encoding two UDP‐glucuronosyltransferase (UGT) isoforms implicated in the glucuronidation of planar phenolic substrates (UGTI∗06) and bilirubin (UGT1∗0) were investigated in rat liver. The amount of UGT mRNA was quantitated by reverse transcription and amplification methods (RT‐PCR). Treatment with L‐T3 significantly increased UGT1∗06 and decreased UGT1∗0 mRNA levels by 41% and 54%, respectively. The opposite situation was observed in thyroidectomised animals. A good relationship observed between UGT activity toward 4‐nitrophenol and bilirubin and mRNA levels emphasizes the key role played by the thyroid hormone L‐T3 on UGT expression.
An essential tyrosine residue of Aspergillus polygalacturonase
Authors: Stratilová Eva , Dzúrová Mária , Markovič Oskar , Jörnvall Hans
Published: July 2002

Based on strict conservation of a tyrosine residue in 24 polygalacturonases, tyrosine modification was assessed in two different forms of the Aspergillus enzyme. The second subform was unknown in structure but submitted to sequence analysis and was found also to have the conserved tyrosine residue. Results of chemical modifications are consistent in showing inactivation of the proteins with all tyrosine‐reactive agents tested, acetic anhydride, N‐acetyl imidazole, and tetranitromethane. Furthermore, after acetylation, regeneration of enzyme activity was possible with hydroxylamine. Spectrophotometric pH titration showed that one accessible tyrosine residue is ionized at pH 9.3–9.5, whereas the remaining, masked residues are all ionized at pH 10.5. It is concluded that one tyrosine residue is catalytically important, in agreement with the inactivation and reactivation data, that this residue is accessible, and that it is likely to correspond to the strictly conserved residue observed in all forms.
Crystallization and preliminary crystallographic analysis of the signal recognition particle SRPΦ14‐9 fusion protein
Authors: Birse Darcy E.A. , Doublié Sylvie , Kapp Ulrike , Strub Katharina , Cusack Stephen , Åberg Anders
Published: July 2002

The SRPΦ14‐9 fusion protein, which can functionally replace the SRP9/14 heterodimer in the mammalian signal recognition particle (SRP), has been crystallized using the vapor diffusion method. Four different crystal forms were grown. SRPΦ14‐9 form IV crystals belong to the space group P4122/P4322 with cell parameters . A complete data set to 2.8 Å resolution with an R
sym on intensities of 7.0% was collected on a single flashfrozen crystal.
Down‐regulation of plant V‐type H+‐ATPase genes after light‐induced inhibition of growth
Authors: Viereck Ruth , Kirsch Matthias , Löw Rainer , Rausch Thomas
Published: July 2002

Cell extension growth in the mesocotyl tip of dark‐grown Zea mays L. seedlings is dependent on vacuole enlargement and massive flux of ER and Golgi vesicles. Water flow into the expanding vacuole is driven by ion accumulation, which in turn is energized by the vacuolar H+‐ATPase (V‐ATPase). The V‐ATPase energizes the secondary ion transport into the expanding vacuole. As light exposure leads to a strong inhibition of extension growth, the effect of light on transcript levels for subunits A and c of the V‐ATPase was analyzed. Partial homologous cDNAs for subunit A and two isoforms of subunit c were cloned by RT‐PCR. In dark‐grown seedlings transcript levels for both subunits were much higher in the growing mesocotyl tip than in the fully differentiated mesocotyl tissue. Only in the tip region did light exposure lead to a strong and coordinate down‐regulation of both mRNAs whereas in the differentiated mesocotyl only a slight decrease was observed. The results indicate that expression of the ‘housekeeping’ V‐type H+‐ATPase is strongly regulated in response to growth rate.
Reversible zinc exchange between metallothionein and the estrogen receptor zinc finger
Authors: Cano-Gauci Danielle F. , Sarkar Bibudhendra
Published: July 2002

We report here the first demonstration that reversible metal exchange occurs between metallothionein (MT) and full‐length estrogen receptor (ER). Specific binding of ER to estrogen response element is inhibited in the presence of 40 μM thionein and restored by 120 μM zinc. Moreover, ER in metal‐depleted nuclear extracts exhibits reduced DNA binding which can be restored by 140 μM native MT. Hence, thionein inhibits DNA binding by abstracting zinc from functional ER while native MT is capable of restoring binding to metal‐depleted extracts by donating metal to ER. This indicates MT may be an important physiological regulator of intracellular zinc and/or other metals.
Enhancement of catalytic activities of serine proteases by tripeptides compounds
Authors: Hiwasa Takaki , Ogawa Sachiko , Kobayashi Hisashi , Ike Yoshimasa
Published: July 2002

The tripeptide compounds, Glu‐Arg‐Pro‐amide (ERPm), d‐Pro‐Thr‐Trp‐amide (dPTWm) and thioproline‐Thr‐Trp (tPTW), were obtained by screening of synthetic peptides for growth‐inhibitory activity toward cultured transformed cells. The effects of these peptide compounds on proteases were investigated and the results showed that these compounds enhanced the amidolytic activity of serine proteases despite the fact that each reaction was carried out under optimal conditions. ERPm stimulated the activities of trypsin, chymotrypsin, thrombin, plasmin urokinase and elastase. dPTWm also showed similar effects except that toward chymotrypsin. tPTW elevated the activity only of trypsin, chymotrypsin and thrombin. Stimulation of trypsin activity by these compounds was also confirmed by using casein as a substrate. None of these compounds affected the amidolytic activities of metalloproteinases (MMP‐1 and MMP‐9), cysteine proteinases (m‐ and μ‐calpains, cathepsin B and papain) or an exopeptidase (leucine aminopeptidase). The activation was at least partly due to the stabilization of the catalytic activity of proteases as well as prevention of autolysis.
Determination of binding site of anti‐tumour necrosis factor‐α monoclonal antibody using hybrid and mutant proteins
Authors: Shingarova Ludmila N. , Sagaidak Lubov N. , Berkova Nadia , Korobko Vyacheslav G.
Published: July 2002

In order to map the immunogenic epitope for the monoclonal antibody E7H2 on the human tumour necrosis factor (hTNF‐α) molecule, a number of chimeric proteins were developed by in‐frame joining segments of the human genes encoding TNF‐α and lymphotoxin (TNF‐β) as well as by coupling appropriate coding regions for human and mouse TNF‐α. High level expression of these chimeric genes was achieved in Escherichia coli by placing the coding sequences under control of either E. coli trp‐promoter or a tandem of bacteriophage T7 constitutive promoters A2 and A3. As revealed by Western blot analysis with monoclonal antibody E7H2 directed against human TNF‐α, the region involved in the binding of this antibody includes sequence ValGluLeuArg in the N‐terminal part of the TNF‐α molecule.
Calnexin : its molecular cloning and expression in the liver of the frog, Rana rugosa
Authors: Yamamoto Satoshi , Nakamura Masahisa
Published: July 2002

A 2.2‐kb cDNA clone encoding the endoplasmic reticulum (ER) resident protein, calnexin (CLX), was isolated from the frog Rana rugosa liver cDNA library and sequenced. The sequence encoded 622 amino acids and was 77% similar to mammalian CLXs and 56% to mouse CLX‐t. In the phylogenetic tree, mouse CLX‐t was clearly diverged from other CLXs including frog. The amino acid sequence of CLX showed regions similar to those in frog calreticulin (CLT), although CLX, but not CLT, contained a histidine‐rich region at the NH2‐terminus. CLX gene expression was observed only in the liver among various tissues examined. Additionally, its expression was strong in the liver in 2‐month post‐metamorphosis frogs, but very weak in adults. The results suggest that the CLX gene is expressed in a tissue‐ and stage‐dependent manner in the frog, R. rugosa, and that CLX is widely distributed in eukaryotic organisms.
Ligand dependence of cytochrome P450c17 protection against proteolytic inactivation: structural, methodological and functional implications
Authors: Nikolaus Kühn-Velten W. , Löhr Jörg B.
Published: July 2002

Rate constants for the subtilisin‐catalyzed proteolytic inactivation of cytochrome P450c17 (CYP17), the endoplasmic reticulum membrane‐bound limiting enzyme of gonadal androgen synthesis, have been determined in the absence and presence of various CYP17 ligands and correlated with fractional enzyme saturation (Y). Extrapolation to Y = 1 reveals 15.1‐, 4.0‐ and 7.4‐fold enzyme stabilization with progesterone (substrate‐type ligand), testosterone (product‐type ligand) and ketoconazole (imidazole‐type inhibitory ligand), respectively. Structural features of ligand accommodation can therefore be monitored by the susceptibility of target enzymes to proteolysis. It is further proposed that specific protection of a membrane protein by ligand binding during proteolytic digestion may assist in the purification of that protein. Evidence is finally presented that the gonadotropin‐induced rapid CYP17 down‐regulation is not promoted by an elevation of steroid hormone levels.
Effects of phorbol esters on insulin‐induced activation of phosphatidylinositol 3‐kinase, glucose transport, and glycogen synthase in rat adipocytes
Authors: Standaert M.L. , Bandyopadhyay G. , Galloway L. , Farese R.V.
Published: July 2002

In rat adipocytes, phorbol ester‐induced activation of PKC did not inhibit insulin signalling through IRS‐1‐dependent phosphatidylinositol (PI) 3‐kinase activation. Moreover, phorbol esters alone provoked an increase in membrane PI 3‐kinase activity. These findings may be relevant to the failure of phorbol esters to inhibit insulin effects on glucose transport and glycogen synthesis in rat adipocytes.
Fructose‐1,6‐bisphosphatase. Primary structure of the rabbit liver enzyme. ‘Intermediate’ variability of an oligomeric protein
Authors: Kaiser Rudolf , Olsson Heléne , Erman Mary , Weeks Charles M. , Hjelmqvist Lars , Ghosh Debashis , Jörnvall Hans
Published: July 2002

The primary structure of rabbit liver fructose‐1,6‐bisphosphatase was determined by peptide analysis of digests with different proteases. The results establish the primary structure, complete data bank entries, and show that this enzyme variant is indeed homologous with other liver fructose‐1,6‐bisphosphatases. Residue differences with the enzymes from other mammals are 9–15%, with those from plants and yeasts about 50%, and with those from characterized prokaryotes up to 70%, showing an enzyme variability intermediate between those of ‘variable’ and ‘constant’ oligomeric dehydrogenases. Structural relationships, conformations and catalytic mechanisms are consistent within the family of fructose‐1,6‐bisphosphatases, and the rabbit protein is a typical rather than an aberrant form of the enzyme.
αIIbβ3‐integrin mediated adhesion of human platelets to a fibrinogen matrix triggers phospholipase C activation and phosphatidylinositol 3′,4′‐bisphosphate accumulation
Authors: Gironcel Daisy , Racaud-Sultan Claire , Payrastre Bernard , Horicot Martine , Borchert Gudrun , Kieffer Nelly , Breton Monique , Chap Hugues
Published: July 2002

This study focused on the variations in phosphoinositide metabolism depending upon αIIbβ3‐integrin/fibrinogen interaction without previous activation of platelet agonist receptors. We found that adhesion of resting human platelets to immobilized fibrinogen stimulates phosphatidic acid production and a concomitant decrease in phosphatidylinositol 4′,5′‐bisphosphate. These results, and the absence of a transphosphatidylation reaction, argue in favor of the activation of a phospholipase C. Moreover, we observed the accumulation of phosphatidylinositol 3′,4′‐bisphosphate in adherent platelets as a consequence of the activation of a phosphatidylinositol 3‐kinase. This effect was inhibited by ADP scavengers. Our results demonstrate that in adherent platelets, whereas phosphatidylinositol 3‐kinase activation is controlled by both αIIbβ‐integrin engagement and released ADP, phospholipase C stimulation is triggered only by αIIbβ‐integrin/fibrinogen interaction.
DNA initiates polymorphic structural transitions in lecithin
Authors: Tarahovsky Y.S. , Khusainova R.S. , Gorelov A.V. , Nicolaeva T.I. , Deev A.A. , Dawson A.K. , Ivanitsky G.R.
Published: July 2002

The inverted micellar phase, obtained by treating lecithin and Ca2+‐DNA complex with chloroform, was used as an intermediate step in the preparation of DNA‐Ca2+‐lecithin complex. DSC analysis demonstrated the involvement of a large fraction of lipid in the interaction with DNA. Freeze‐fracture electron microscopy revealed (i) rod‐like structures on the hydrophobic fracture surface of membranes and (ii) regular bundles of fibrils with a repeat distance of about 6 nm, which were located free in solution. Similar regular bundles of fibrils were also revealed by staining the samples with uranyl acetate. According to the suggested model, the observed structures are hexagonally packed inverted lipid tubes, with DNA located in their central cores. The possible biological relevance of the capability of Ca2+‐DNA to initiate polymorphic phase transitions of lecithin is discussed.
Compromised inhibition of human lung lavage cell elastases
Authors: Smith S.F. , Roberts N.A. , Guz A. , Tetley T.D.
Published: July 2002

Increased elastinolytic activity has been correlated with the degree of lung damage occurring in a variety of lung diseases including cystic fibrosis; serine proteinase inhibitors are currently on trial for the treatment of some lung disorders. However, human lung lavage cells also secrete metallo‐dependent elastases. Here we show, for the first time, that whilst these are readily inhibited by EDTA, inhibition of serine elastases using serpins (serine proteinase inhibitors) is not always possible. This may reflect inactivation of serpins by uninhibited metalloproteinases and oxidants in a low protein milieu. Thus, the therapeutic inhibition of excessive elastinolytic activity may require a combination of inhibitors to work efficiently.
The use of the rat LAP LCR and promoter for the high‐level constitutive expression of K+ channel cDNAs in a rat liver cell line
Authors: Shelton P.A. , Dart C.
Published: July 2002

Locus control regions (LCRs) are cis‐acting elements that confer position‐independent and copy‐number‐dependent expression upon associated genes in transgenic mice. Here we show the second example of the use of an LCR (the rat LAP LCR) in a stable expression vector system, used here in conjunction with the rat liver (NRLM) cell line. Non‐transfected NRLM cells are electrically silent and highly suitable for patch clamp electrophysiology. We report reliable constitutive expression from two different K+ channel cDNAs; the voltage‐gated rat clone Kv3.4 and the inward rectifier mouse clone Kir2.1. We further show that constitutive expression levels are stable for at least 8 weeks from initial recording.
Phosphatidylcholine transfer protein from bovine liver contains highly unsaturated phosphatidylcholine species
Authors: Geijtenbeek Teunis B.H. , Westerman Jan , Heerma Wigger , Wirtz Karel W.A.
Published: July 2002

The phosphatidylcholine transfer protein (PC‐TP) from bovine liver contains one molecule of non‐covalently bound PC. In order to gain more insight into the physiological function of PC‐TP, PC was extracted from bovine liver PC‐TP and its molecular species composition identified by fast atom bombardment mass spectrometry. The prevailing molecular species were C18:0/C18:1‐, C18:0/C18:2‐, C18:OIC20:4‐, C18:0120:5‐ and C18:OIC22:5‐PC accounting for 85% of the PC species present. This molecular species composition is not representative for what is present in bovine liver where these species account for 43% of the total PC content [Montfoort et al. (1971) Biochim. Biophys. Acta 231, 335–342]. Another striking observation is that PC species carrying a palmitoyl chain at the sn‐1 position are nearly absent, despite these species being abundantly present in bovine liver. This study suggests that PC‐TP could play a role in the metabolism of highly unsaturated, stearoyl‐containing PC species.
Identification of residues in the putative 5th helical region of human interleukin‐6, important for activation of the IL‐6 signal transducer, gp130
Authors: Brakenhoff Just P.J , Klaasse Bos Hanny , Grötzinger Joachim , Rose-John Stefan , Aarden Lucien A
Published: July 2002

We have previously shown that L58 in the putative 5th helical region of human interleukin‐6 (IL‐6) is important for activation of the IL‐6 signal transducer gp130 [de Hon et al. (1995) FEBS Lett. 369, 187–191]. To further explore the importance of individual residues in this region for gp130 activation we have now combined Ala substitutions of residues E52, S53, S54, K55, E56, L58 and E60 with other substitutions in IL‐6, known to affect gp130 activation (Q160E and T163P). The combination mutant protein with L58A completely lost the capacity to induce the proliferation of XG‐1 myeloma cells, and could effectively antagonize wild type IL‐6 activity on these cells. Moreover, the data suggest that besides L58, S54 particularly, but also E52, S53, K55 and E56 contribute to gp130 activation.
Identification of the antigenic determinants of factors 8, 9, and 34 of genus Candida
Authors: Kobayashi Hidemitsu , Oyamada Hiroko , Suzuki Akifumi , Shibata Nobuyuki , Suzuki Shigeo , Okawa Yoshio
Published: July 2002

We investigated the antigenic determinants of factors 8, 9, and 34 of the genus Candida among pathogenic yeasts by enzyme‐linked immunosorbent assay (ELISA) using mannans of Saccharomyces cerevisiae wild type and mutant types, mnn 1‐mnn 4 and mnn 2. Results of ELISA including antisera against the antigenic factors of genus Candida (Candida Check, Iatron; FAbs) indicated that these three types of mannan distinctly react with FAbs 34, 8 and 9, respectively. To identify the recognition sites of these FAbs, we compared the ability of various oligosaccharides to inhibit the binding of the mannans to FAbs. The results indicated that FAb 34 preferentially recognizes linear side chains containing a non‐reducing terminal α‐1,3‐linked mannose residue, Manα1 → 3Manα1 → (2Manα1 →)
n
2Man (n ≥ 0), and that one of the recognition sites of FAb 9 is linear α‐1,6‐linked oligomannosyl series, Manα1 → (6Manα1 →)
n
6‐Man (n ≥ 2). On the other hand, the recognition site of FAb 8 apparently consisted of two α‐1,2‐linked oligomannosyl side chains and an α‐1,6‐linked mannose residue that originated from the mannan backbone, Manα1 → 2Manα1 → 2(Manα1 → 2Manα1 → 6)Man.
In Xenopus oocytes the human C3a and C5a receptors elicit a promiscuous response to the anaphylatoxins
Authors: Ames Robert S , Nuthulaganti Paru , Kumar Chandrika
Published: July 2002

The Xenopus laevis oocyte has been widely utilized for cloning and functional expression of G‐protein coupled receptors (GPCR). This system was used for the functional expression and characterization of the recently identified human C3a receptor. Complementary RNA from the human C3a receptor was transcribed in vitro and microinjected into Xenopus oocytes for functional characterization. A positive response to a synthetic C3a peptide agonist and to C3a, but not to platelet activating factor or fMetLeuPhe was detected. In addition, a response of approximately one third the amplitude obtained with C3a was obtained with rC5a. Conversely, oocytes co‐injected with the C5a receptor and total RNA isolated from U937 cells responded to C5a as well as to C3a and the C3a synthetic peptide. A functional response with the anaphylatoxin C3a receptor in oocytes was dependent on co‐injection of a pertussis toxin sensitive complementary human factor which could be supplied by co‐injection of total RNA isolated from U937 cells. Oocytes expressing the anaphylatoxin C3a and C5a receptors responded to both agonists, in each case the response to the cognate ligand was substantially more robust than the response elicited by the other anaphylatoxin.
Structure of a methionine‐rich segment of Escherichia coli Ffh protein
Authors: Oh Doo-Byoung , Yi Gwan-Su , Chi Seung-Wook , Kim Hyoungman
Published: July 2002

The methionine‐rich segments of the Ffh protein of Escherichia coli and its eukaryotic counterpart SRP54 are thought to bind signal sequences of secretory proteins. The structure of a chemically synthesized 25‐residue‐long peptide corresponding to one of the proposed methionine‐rich amphiphilic helices of Ffh was determined in water and in aqueous trifluroethanol (TFE) solution using CD and NMR. An appreciable α‐helix conformation exists even in water and this peptide assumes a stable α‐helix along most of its length in aqueous TFE solution. It is clear that this segment of Ffh protein has a very strong propensity to form α‐helical structure.
Novel DNA‐binding protein from Drosophila embryos identified by binding site selection
Authors: Todorović Vesna , Kojić Snežana , Vujanac Miloš , Savić Ana , Stefanović Dragana
Published: July 2002

The DNA sequence C/AGAGCGC/AGA, related to binding sites for GAF and Zeste transcription factors, was selected from a pool of degenerate PCR fragments for binding to the cytoplasmic protein of Drosophila preblastoderm embryos. Identical DNA binding activity was also detected in embryonic nuclei. Based on several criteria, such as size, intracellular distribution, sensitivity to ATP and protein kinase inhibitor 6‐DMAP, kinetics during development and lack of cross‐reaction with rabbit anti‐GAF serum, protein recognizing selected sequence was shown to differ from either Zeste or GAF.
Effect of early cortical lesion on the acute model of epilepsy
Authors: Ćulić M. , Šaponjić J. , Janković B. , Peković S. , Stojilković M. , Udović S. , Rakić Lj.
Published: July 2002

The experiments were performed in order to investigate the sparing of function following early postnatal cortical lesion in the acute rat model of epilepsy. Sensomotor cortex was unilaterally removed at 9 and 10 days of postnatal age in lesioned animals, while control animals were only sham operated (at the same early stage of life) or non‐operated (before implantation of the electrodes). Seizure activity was recorded by means of electroencephalograms at adult stage of life induced by parenteral administration of penicillin (1 000 000 I.U./kg, i.p.). Our results showed that when the cortical lesion was performed in infancy (on the contrary to the lesion performed in adulthood) there was no prolongation of seizure activity in an acute model of epilepsy.
Increased phosphorylation of neurofilament subunits in PC12 cells and rat dorsal root ganglion neurons treated with N‐Acetyl‐Leu‐Leu‐norleucinal
Authors: Jayaraman D. , Giasson B.I. , Mushynski W.E.
Published: July 2002

Treatment of PC12 cells or dorsal root ganglion neurons with the protease inhibitor, N‐Acetyl‐Leu‐Leu‐norleucinal, stimulated phosphorylation of the mid‐sized and heavy neurofilament subunits and caused the heavy subunit in the perikarya of dorsal root ganglion neurons to become hyperphosphorylated. The closely related inhibitor, N‐Acetyl‐Leu‐Leu‐methioninal, did not produce a similar effect. Okadaic acid increased the phosphorylation state of the heavy neurofilament subunit in PC12 cells in a fashion similar to N‐Acetyl‐Leu‐Leu‐norleucinal and the effect of both compounds together was greater than for either one alone. There was no increase in cyclin‐dependent kinase 5‐immunoprecitable histone H1 kinase activity in PC12 cells treated with N‐Acetyl‐Leu‐Leu‐norleucinal despite the presence of enzyme protein.

The present study demonstrates that a protease inhibitor can induce the hyperphosphorylation of neurofilament subunits to a level normally seen only in axons. This suggests that perturbations in intracellular proteolysis may lead to the accumulation of phosphorylated neurofilament epitopes in neuronal perikarya in certain pathological states. The results also show that the carboxy‐terminal tail domains of the two largest neurofilament subunits are phosphorylated even when cyclin dependent kinase 5 is inactive, indicating that other neuronal kinases are involved in the phosphorylation of Lys‐Ser‐Pro repeats.
Clinical and socio-demographic correlates of coping strategies in relatives of schizophrenic patients
Authors: Magliano L , Veltro F , Guarneri M , Marasco C
Published: July 2002

The study aimed to describe coping strategies in a sample of key-relatives of schizophrenic patients, and to explore their relationships with the following variables: patients' and key-relatives' socio-demographic characteristics; patients' and key-relatives' clinical status; and key-relatives' subjective burden. Significant correlations were found between the scores on coercion items and those on Present State Examination (PSE-9) factors “disorganized syndrome” and “psychomotor poverty syndrome”. A long duration of illness was found to be associated with relatives' strategies characterized by positive communication and patient's social involvement. Coercion, avoidance and resignation coping strategies were significantly associated with the occurrence of anxiety and depressive symptoms in patients' key-relatives.
Subcellular localization and processing of the lytic transglycosylase of the conjugative plasmid R1
Authors: Bayer Michaela , Bischof Karin , Noiges Rainer , Koraimann Günther
Published: July 2002

Protein P19 encoded by the conjugative resistance plasmid R1, is essential for efficient conjugative DNA transfer and infection by the pilus‐specific RNA phage R17. Based on sequence homologies P19 belongs to a family of lysozyme‐like virulence factors which are found in type III and type IV secretion systems. In this report we describe the processing and subcellular localization of P19. Pulse‐chase experiments were used to demonstrate the processing of P19 by the signal peptidase I of Escherichia coli. Translocation of P19 across the inner membrane was shown by gene 19‐phoA fusions. Cell fractionation studies of P19 expressing cells showed the presence of P19 in the membrane compartment. P19 was solubilized with the detergent Sarkosyl indicating an inner membrane localization. Using sucrose density gradient centrifugation to separate inner and outer membranes, P19 was found in both membrane fractions. Taken together, our data suggest that mature P19 is a periplasmic protein which may be attached to the proposed membrane‐spanning DNA transport complex.
Involvement of asparagine 118 in the nucleotide specificity of the catalytic subunit of protein kinase CK2
Authors: Jacob Germaine , Neckelman Guy , Jimenez Monica , Allende Catherine C , Allende Jorge E
Published: July 2002

Protein kinase CK2 is a heteromeric enzyme with catalytic (α) and regulatory (β) subunits which form an α2β2 holoenzyme and utilizes both ATP and GTP as nucleotide substrate. Site‐directed mutagenesis of CK2α subunit was used to study this capacity to use GTP. Deletion of asparagine 118 (αΔN118) or the mutant αN118E gives a 5–6‐fold increase in apparent K
m for GTP with little effect on the affinity for ATP. Mutants αN118A and αD120N did not alter significantly the K
m for either nucleotide. CK2αΔN118 has an apparent K
i for inosine 5′ triphosphate 5‐fold higher than wild‐type and is very heat labile. These studies complement recent crystallographic data indicating a role for CK2α asparagine 118 in binding the guanine base.
Presence of CuZn superoxide dismutase in human serum lipoproteins
Authors: Mondola P. , Bifulco M. , Serù R. , Annella T. , Ciriolo M.R. , Santillo M.
Published: July 2002

It has previously been demonstrated that CuZn‐superoxide dismutase (SOD) is secreted by several human cell lines. This suggests that the circulating enzyme derives from both hemolysis and peripheral tissues as a result of cellular secretion. In the present report, we evaluated the presence of CuZn‐SOD in human serum lipoproteins by both enzyme‐linked immunosorbent assay and Western blot analysis of immunoprecipitated lipoprotein samples. The distribution of CuZn‐SOD activity among the different lipoprotein fractions was also determined by the xanthine/xanthine oxidase method. The results demonstrated that CuZn‐SOD is noticeably present in serum lipoproteins and mainly in low and high density lipoproteins (LDL and HDL). Moreover, experiments performed by incubating CuZn‐SOD with a lipid emulsion and subsequent separation of the lipid fraction by ultracentrifugation showed that this enzyme associates in a saturable manner with lipids. The CuZn‐SOD bound to LDL and HDL could exert a physiological protective role against oxidative damage of these lipoprotein classes that carry out a crucial role in the cholesterol transport.
Amino acids and peptides. LVII. Synthetic peptide with a sequence of ribonuclease from Sulfolobus solfataricus, SSR(1–62), does not function as an RNase
Authors: Joshi Shima , Tsuda Yuko , Shintomi Noriyuki , Kondo Hiroyuki , Nishiyama Yasuhiro , Iwama Masanori , Ohgi Kazuko , Irie Masachika , Okada Yoshio
Published: July 2002

The 62 residue peptide, SSR(1–62), whose sequence corresponds to that of ribonuclease (RNase) from Sulfolobus solfataricus, and its related peptides, SSR(1–22) and SSR(10–62), were chemically synthesized and their RNase activity and DNA‐binding activity were examined. The RNase activity assay using yeast RNA or tRNAfMet as substrate showed that the synthetic peptide SSR(1–62) did not hydrolyze yeast RNA or tRNAfMet. These data were not consistent with previous reports that both the native peptide isolated from S. solfataricus [Fusi et al. (1993) Eur. J. Biochem. 211, 305–311] and the recombinant peptide expressed in Escherichia coli [Fusi et al. (1995) Gene 154, 99–103] were able to hydrolyze tRNAfMet. However, the synthetic SSR(1–62) exhibited DNA‐binding activity. In the presence of synthetic SSR(1–62), the cleavage of DNA (plasmid pUCRh2‐4) by restriction endonuclease (EcoRI) was not observed, suggesting that synthetic SSR(1–62) bound to DNA protected DNA from its enzymatic digestion. Neither SSR(1–22) nor SSR(10–62) prevented DNA from being cleaved by a restriction enzyme. These findings strongly suggest the importance of not only the N‐terminal region of SSR(1–62) but also the C‐terminal region for DNA‐binding. Circular dichroism spectroscopy of synthetic SSR(1–62) indicated a β‐sheet conformation, in contrast with synthetic SSR(1–22), which exhibited an unordered conformation.
Odorant and pheromone binding by aphrodisin, a hamster aphrodisiac protein
Authors: Briand Loı̈c , Huet Jean-Claude , Perez Valérie , Lenoir Guillaume , Nespoulous Claude , Boucher Yves , Trotier Didier , Pernollet Jean-Claude
Published: July 2002

Aphrodisin is a soluble glycoprotein of hamster vaginal discharges, which stimulates male copulatory behavior. Natural aphrodisin was purified and its post‐translational modifications characterized by MALDI‐MS peptide mapping. To evaluate its ability to bind small volatile ligands, the aphrodisiac protein was expressed in the yeast Pichia pastoris as two major isoforms differing in their glycosylation degree, but close in conformation to the natural protein. Dimeric recombinant aphrodisins were equally able to efficiently bind odors (2‐isobutyl‐3‐methoxypyrazine and methyl thiobutyrate) and a pheromone (dimethyl disulfide), suggesting that they could act as pheromone carriers instead of, or in addition to, direct vomeronasal neuron receptor activators.
pH‐dependent reversible inhibition of violaxanthin de‐epoxidase by pepstatin related to protonation‐induced structural change of the enzyme
Authors: Kawano Mitsuko , Kuwabara Tomohiko
Published: July 2002

The redox enzyme violaxanthin de‐epoxidase (VDE) was found to be sensitive to pepstatin, a specific inhibitor of aspartic protease. The inhibition was similar to that of aspartic protease in that it was reversible and accompanied by the protonation of the enzyme. Of the two peaks of VDE appearing on anion exchange chromatography, VDE‐I predominated at pH 7.2. On lowering the pH of the chromatography, VDE‐I decreased and VDE‐II increased. Furthermore, re‐chromatography of either peak yielded both peaks. These results suggest that VDE‐I and VDE‐II are interconvertible depending on pH, and thus, they represent the de‐protonated and protonated forms of the enzyme, respectively. Presumably the protonation‐induced structural change of the enzyme is responsible for the interaction with pepstatin, and also with substrate.
EMI, a novel cysteine‐rich domain of EMILINs and other extracellular proteins, interacts with the gC1q domains and participates in multimerization
Authors: Doliana Roberto , Bot Simonetta , Bonaldo Paolo , Colombatti Alfonso
Published: July 2002

The N‐terminal cysteine‐rich domain (EMI domain) of EMILIN‐1 is a new protein domain that is shared with two proteins (multimerin and EMILIN‐2) and with four additional database entries. The EMI domains are always located at the N‐terminus, have a common gene organization, and belong to proteins that are forming or are compatible with multimer formation. The potential role of the EMI domain in the assembly of EMILIN‐1 was investigated by the two‐hybrid system. No reporter gene activity was detected when EMI‐1 was co‐transformed with the C‐terminal gC1q‐1 domain excluding a head‐to‐tail multimerization; conversely, a strong interaction was detected when the EMI‐1 domain was co‐transformed with the gC1q‐2 domain of EMILIN‐2.
Tc1, from Tityus cambridgei, is the first member of a new subfamily of scorpion toxin that blocks K+‐channels
Authors: Batista Cesar V.F. , Gómez-Lagunas Froylan , Lucas Sylvia , Possani Lourival D.
Published: July 2002

A new peptide, Tc1, containing only 23 amino acids closely packed by three disulfide bridges was isolated from the Amazonian scorpion Tityus cambridgei. It blocks reversibly the Shaker B K+‐channels with a K
d of 65 nM and displaces binding of noxiustoxin to mouse brain synaptosome membranes. It is the shortest known peptide from scorpion venom that recognizes K+‐channels and constitutes a new structural subfamily of toxin, classified as alphaKTx 13.1.
Intrinsic structural differences between ‘tight couples’ and Kaltschmidt–Wittmann ribosomes evidenced by dielectric spectroscopy and scanning microcalorimetry
Authors: Bonincontro A , Nierhaus K.H , Onori G , Risuleo G
Published: July 2002

Measurements of dielectric spectroscopy (DS) and microcalorimetry (differential scanning calorimetry (DSC)) of Escherichia coli 70S, 50S and 30S were performed on particles prepared according either to the ‘classical’ twice NH4Cl‐washed ribosomes, also known as loose couples (LC), or to the ‘tight couples’ preparative protocol (TC). Results show that 70S particles prepared according to the two different protocols exhibit different structural properties. Two subsequent relaxation processes occur in both samples as measured by DS. However, in LC ribosomes the first one is shifted towards a lower frequency with a higher dielectric increment. This is suggestive of a more extensive exposure of RNA to the solvent and of an overall more relaxed structure. The smaller LC subunit exhibits only one relaxation while the TC 30S shows two dielectric dispersions as well as 70S. No substantial differences were evidenced in either 50S species. Two typical melting peaks were observed by DSC both in LC and TC 70S as well as in 50S. Thermograms obtained from the TC 30S show a single well structured peak while LC particles produce a large unstructured curve. On the basis of these results we conclude that TC 70S particles are more compact than LC ribosomes and that in the former ones the rRNA is less exposed to the solvent phase. Furthermore 30S particles obtained from TC show a more stable structure with respect to LC 30S. We conclude that the 30S subunit gives a major contribution to the compact character of the whole TC 70S. These differences might be related to the intrinsic and well documented functional difference between the two ribosome species.
The ascorbate‐driven reduction of extracellular ascorbate free radical by the erythrocyte is an electrogenic process
Authors: VanDuijn Martijn M , Van der Zee Jolanda , Van den Broek Peter J.A
Published: July 2002

Erythrocytes can reduce extracellular ascorbate free radicals by a plasma membrane redox system using intracellular ascorbate as an electron donor. In order to test whether the redox system has electrogenic properties, we studied the effect of ascorbate free radical reduction on the membrane potential of the cells using the fluorescent dye 3,3′‐dipropylthiadicarbocyanine iodide. It was found that the erythrocyte membrane depolarized when ascorbate free radicals were reduced. Also, the activity of the redox system proved to be susceptible to changes in the membrane potential. Hyperpolarized cells could reduce ascorbate free radical at a higher rate than depolarized cells. These results show that the ascorbate‐driven reduction of extracellular ascorbate free radicals is an electrogenic process, indicating that vectorial electron transport is involved in the reduction of extracellular ascorbate free radical.
Effect of divalent cations on the ATPase activity of Escherichia coli SecA
Authors: Kim Joon-Sik , Ahn Taeho , Ko Junsang , Park Chankyu , Kim Hyoungman
Published: July 2002

It was found that Ca2+ stimulates the intrinsic SecA ATPase activity in the absence as well as in the presence of liposome. On the other hand, Mg2+, the general cofactor for ATPase, did not affect the intrinsic SecA ATPase but reduced the portion of ATPase activity enhanced by Ca2+. The enhancement of SecA ATPase activity correlated well with the increase in 8‐anilino‐1‐naphthalene‐sulfonic acid binding of SecA, suggesting that increased exposure of hydrophobic residues stimulates the enzyme activity.
The Rev/Rex homolog HERV‐K cORF multimerizes via a C‐terminal domain
Authors: Boese Annette , Galli Uwe , Geyer Matthias , Sauter Marlies , Mueller-Lantzsch Nikolaus
Published: July 2002

Expression of human endogenous retrovirus K (HERV‐K) is associated with germ‐cell neoplasia. HERV‐K encodes a protein of the Rev/Rex family, cORF, that supports cellular transformation and binds the promyelocytic leukemia zinc finger (PLZF) protein implicated in spermatogenesis. Rev/Rex function invariably depends on multimerization. Here we show that cORF likewise self‐associates to form higher‐order oligomers. Amino acids (aa) 47–87 in cORF are sufficient, aa 75–87 essential for self‐association. Consistently, this domain is predicted to form a hydrophobic α‐helix that may represent an oligomerization interface. The existence of a dimerization‐competent cORF mutant lacking PLZF‐binding activity (cORF47–87) suggests a way of dominant negative inhibition of the proposed tumor susceptibility factor cORF.
A novel heterodimeric antimicrobial peptide from the tree‐frog Phyllomedusa distincta
Authors: Batista Cesar V.F , Scaloni Andrea , Rigden Daniel J , Silva Lindomar R , Rodrigues Romero Adela , Dukor Rina , Sebben Antonio , Talamo Fabio , Bloch Carlos
Published: July 2002

We present here the purification and the analysis of the structural and functional properties of distinctin, a 5.4 kDa heterodimeric peptide with antimicrobial activity from the tree‐frog Phyllomedusa distincta. This peptide was isolated from the crude extract of skin granular glands by different chromatographic steps. Its minimal inhibitory concentration was determined against pathogenic Escherichia coli, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa strains. Amino acid sequencing and mass spectrometric investigations demonstrated that distinctin is constituted of two different polypeptide chains connected by an intermolecular disulphide bridge. Circular dichroism and Fourier‐transformed infrared spectroscopy studies showed that this molecule adopts, in water, a structure containing a significant percentage of anti‐parallel β‐sheet. A conformational variation was observed under experimental conditions mimicking a membrane‐like environment. Database searches did not show sequence similarities with any known antimicrobial peptides. In the light of these results, we can consider distinctin as the first example of a new class of antimicrobial heterodimeric peptides from frog skin.
A novel dual‐specificity protein kinase targeted to the chloroplast in tobacco1
Authors: Cho Hye Sun , Yoon Gyeong Mee , Lee Sang Sook , Kim Young A. , Hwang Inhwan , Choi Doil , Pai Hyun-Sook
Published: July 2002

The NtDSK1 cDNA encoding a novel chloroplast‐targeted protein kinase was identified in Nicotiana tabacum. It contains the kinase domain at the C‐terminus and a putative regulatory domain at the N‐terminus. The recombinant NtDSK1 underwent autophosphorylation of serine, threonine, and tyrosine residues, indicating that NtDSK1 encodes a functional dual‐specificity protein kinase. The NtDSK1–green fluorescent protein fusion protein was targeted to chloroplasts. Furthermore, the NtDSK1 protein was immunodetected in chloroplast fractions isolated from tobacco seedlings. The NtDSK1 mRNA expression was developmentally regulated in different tissues, including anthers and germinating seeds, and strongly stimulated by gibberellin. The mRNA was rapidly light responsive during seedling growth. NtDSK1 may play a role in a light‐regulated signaling process in tobacco.
B cell receptor signaling involves physical and functional association of FAK with Lyn and IgM
Authors: Mlinaric-Rascan Irena , Yamamoto Tadashi
Published: July 2002

B cell receptor (BCR) stimulation induces phosphorylation of a number of proteins, leading to functional activation of B lymphocytes. Focal adhesion kinase (FAK) is a non‐receptor protein tyrosine kinase, involved in a variety of signaling pathways. In this study, we show that FAK is tyrosine‐phosphorylated and activated following BCR stimulation. We also demonstrate constitutive association of FAK with the Src‐family kinase Lyn and with components of the BCR. Association of Lyn with FAK which was not correlated with BCR‐induced activation of both kinases, appeared to be mediated via the binding of Lyn to the COOH‐terminal part of the FAK molecule. Our results indicate that FAK is a component of the BCR complex and that it participates in BCR signaling.
Deacylation of the transmembrane domains of Sindbis virus envelope glycoproteins E1 and E2 does not affect low‐pH‐induced viral membrane fusion activity
Authors: Smit Jolanda M. , Bittman Robert , Wilschut Jan
Published: July 2002

The envelope glycoproteins E1 and E2 of Sindbis virus are palmitoylated at cysteine residues within their transmembrane domains (E1 at position 430, and E2 at positions 388 and 390). Here, we investigated the in vitro membrane fusion activity of Sindbis virus variants (derived from the Toto 1101 infectious clone), in which the E1 C430 and/or E2 C388/390 residues had been substituted for alanines. Both the E1 and E2 mutant viruses, as well as a triple mutant virus, fused with liposomes in a strictly low‐pH‐dependent manner, the fusion characteristics being indistinguishable from those of the parent Toto 1101 virus. These results demonstrate that acylation of the transmembrane domain of Sindbis virus E1 and E2 is not required for expression of viral membrane fusion activity.
Does myoglobin protect Trypanosoma cruzi from the antiparasitic effects of nitric oxide?1
Authors: Ascenzi Paolo , Salvati Luca , Brunori Maurizio
Published: July 2002

The hemoflagellate protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, a progressive fatal cardiomyopathy widespread in South and Central America. Here, we postulate that the preferential colonization of cardiomyocytes by T. cruzi may reflect the role of myoglobin (Mb) as a nitric oxide (NO) scavenger, protecting the parasite from the trypanocidal effects of NO. The proposal of this novel function of Mb is based on knowledge that ferrous oxygenated Mb reacts rapidly and irreversibly with NO yielding nitrate and ferric oxidized Mb, which is reduced back to the physiologically active form by an intracellular reductase. The postulated protective role of Mb on the viability of T. cruzi is reminiscent of that postulated for hemoglobin in protecting intraerythrocytic Plasmodia from the parasiticidal effect of NO.