bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of cytoplasmic factors that bind to the 3' AU‐rich region of human Interferon β mRNA during early development of Xenopus laevis
Authors: Grafi Gideon , Galili Gad
Published: July 2002

Certain endogenous Xenopus mRNAs, carrying a destabilizing 3' AU‐rich sequence, are unusually very stable in oocytes and become unstable only after fertilization. In addition, heterologous short lived mRNA, containing 3' AU‐rich sequences, appear to be very stable when injected into Xenopus oocytes. In the present study, a human interferon β (hu‐IFNβ) mRNA, carrying the destabilizing 3' AU‐rich element, was used as a probe to identify Xenopus proteins that specifically bind to the 3' AU‐rich element as well as to study their relative levels during early embryonic development. While three major proteins that specifically bind to the 3' AU‐rich element were detected in human SV80 cells, that naturally express hu‐IFNβ (proteins termed AU‐F1, F2 and F3), only two proteins, migrating similarly to the SV80 AU‐F1 and AU‐F3, were detected in cytoplasmic extracts from Xenopus oocytes or eggs. Following fertilization, the intensity of the Xenopus AU‐F1 and AU‐F3 proteins increased considerably and a new protein, corresponding to SV80 AU‐F2, was also detected. Cyclohexamide applied either at the morula or at the early blastula stages reduced the intensity of the AU‐binding factors, while actinomycin D did not, indicating that the levels of these factors during these stages are regulated posttranscriptionally. In contrast, application of each of these metabolic inhibitors at the late blastula stage increased the intensity of the AU‐binding proteins. The possible function of these AU‐binding factors in regulating the expression and half life of AU‐rich mRNAs is discussed.
Amino acid sequence of alkaliphilic serine protease from silkworm, Bombyx mori, larval digestive juice
Authors: Sasaki Takuji , Hishida Tomoaki , Ichikawa Katsuomi , Asari Shin-ichiro
Published: July 2002

Alkaliphilic protease, P‐IIc, from silkworm, Bombyx mori, larval midgut digestive juice consists of 232 amino acids. It has a catalytic triad, Asp‐His‐Ser, invariably found in a serine protease. A shift of optimal pH value towards the alkaline side diminished at μ=1.0. This suggests the existence of an electrostatic interaction that affects the proteolytic activity. The higher Arg content may be responsible for this phenomenon. Two cysteine residues probably exist unpaired in a novel position among serine proteases.
A comparative picosecond‐resolved fluorescence study of tryptophan residues in iron‐sulfur proteins
Authors: Dorovska-Taran Victoria , van Hoek Arie , Link Thomas A. , Visser Antonie J.W.G. , Hagen Wilfred R.
Published: July 2002

The fluorescence intensity and anisotropy decays of the intrinsic tryptophan emission from six Fe/S proteins (ranging from the very simplest ones to enzyme complexes containing one, two or more Trp residues) were measured. All proteins were examined in the reduced and the oxidized state. In either redox state each protein exhibits ultrarapid tryptophan fluorescence decay on the picosecond timescale contributing up to 93% of the total decay. Correlation times in the range of 1 ns or less were found for all six iron‐sulfur proteins reflecting internal Trp motion. In addition, some proteins exhibit longer correlation times reflecting segmental motion and overall protein tumbling. The ultrarapid fluorescence decay in iron‐sulfur proteins indicates efficient radiationless energy transfer between distant tryptophan residues and iron‐sulfur clusters. Such an energy transfer mechanism can be accounted for by referring to the three‐dimensional structures of rubredoxin and ferredoxin in calculating the transfer efficiency of the single tryptophan—iron‐sulfur couple.
Absorption flattening in the optical spectra of liposome‐entrapped substances
Authors: Wittung Pernilla , Kajanus Johan , Kubista Mikael , Malmström Bo G.
Published: July 2002

Measuring optical spectra of chromophore molecules entrapped in liposomes can lead to considerable distortion because of absorption flattening. This phenomenon is analyzed theoretically, and it is shown that the deviation from the Beer—Lambert law becomes larger as the molar absorptivity of the chromophore increases and as the number of entrapped molecules becomes larger. The theoretical equations are tested experimentally with intermediate‐sized phospholipid vesicles containing entrapped cytochrome c. It is shown that considerable absorption flattening is observed with vesicles containing about 50 chromophore molecules. The equation given can be used to correct the spectrum.
The cis‐element CE‐LPH1 of the rat intestinal lactase gene promoter interacts in vitro with several nuclear factors present in endodermal tissues
Authors: Boukamel Rita , Freund Jean-Noël
Published: July 2002

We have shown by electrophoretic mobility shift assays that the nucleotide sequence CE‐LPH1, centred at position −49 with respect to the transcription start site of the rat gene encoding intestinal lactase‐phlorizin hydrolase, interacts in vitro with nuclear proteins present in the jejunum of suckling animals. Proteins binding to this element were also found in organs of endodermal origin that do not (or no longer) express lactase‐phlorizin hydrolase, i.e. the colon, lung and the liver, but not in the brain. However, a DNA—protein interaction was hardly detected with nuclear extracts prepared from adult tissues, although typical factors binding to the Sp1 binding site were detected at the adult stage as in the sucklings. Southwestern blotting experiments conducted with nuclear extracts prepared from the tissues of suckling rats indicated that CE‐LPH1 interacts with several factors in the jejunum, colon, lung and the liver. Some of these DNA‐binding proteins are specifically expressed in the jejunum or in the liver, whereas others seem to he shared with the colon and the lung. Hence, the cis‐element CE‐LPH1 located in close vicinity to the pseudo‐TATA‐box of the intestinal lactase‐phlorizin hydrolase gene promoter interacts in vitro with a family of nuclear proteins which may represent markers of the endodermal lineage predominantly expressed prior to weaning.
The existence of a growth‐specific DNA binding factor for the promoter region of mouse ST2 gene
Authors: Tominaga Shin-ichi , Kato-Yamazaki Masako , Yanagisawa Ken , Kawakami Kiyoshi , Tetsuka Tsunao
Published: July 2002

A comparison of the 5′‐flanking regions of human and mouse ST2 genes revealed the presence of two highly conserved DNA sequences. The promoter activity assay with a luciferase gene as a reporter showed that the deletion of the upstream conserved region diminished the transcriptional activity in growing BALB/c‐3T3 cells. By electrophoretic mobility‐shift analysis, the presence of a factor that binds to the positive regulatory region of the mouse ST2 gene was found in growing but not in quiescent BALB/c‐3T3 cells. These results suggest the functional importance of this conserved region and the requirement of a binding factor for the expression of the ST2 gene.
Identification of yeast MAS17 encoding the functional counterpart of the mitochondrial receptor complex protein MOM22 of Neurospora crassa
Authors: Masato Nakai  , Toshiya Endo
Published: July 2002

MOM22 of Neurospora crassa was suggested to be required for the transfer of mitochondrial precursor proteins from the receptors to the protein translocation machinery. We isolated a yeast mutant the viability of which depended on the expression of the introduced N. crassa MOM22 gene. The mutant cells showed defects in protein import into mitochondria when the cells were depleted of MOM22. By screening for suppressor genes for the mutant, we could identify the yeast gene MAS17 encoding the functional counterpart of MOM22. The MAS17 gene was found to be essential for the viability of yeast cells.
Synthesis of holo Paracoccus denitrificans cytochrome c550 requires targeting to the periplasm whereas that of holo Hydrogenobacter thermophilus cytochrome c552 does not
Authors: Sambongi Yoshihiro , Ferguson Stuart J.
Published: July 2002

Expression from a plasmid of the complete gene, including the codons for the N‐terminal periplasmic targeting signal, for cytochrome c
550 of Paracoccus denitrificans led to the formation of the holo protein in the periplasms of both P. denitrificans and Escherichia coli. Expression of the gene from which the region coding for the targeting signal had been specifically deleted resulted in formation of apo‐protein in the cytoplasms of both organisms. These findings are consistent with haem attachment occurring in the periplasm. In contrast, the formation of holo cytochrome c
552 from Hydrogenobacter thermophilus following expression of the gene lacking the periplasmic targeting sequence in either P. denitrificans or E. coli is attributed to spontaneous cytoplasmic attachment of haem to the thermostable protein.
Cytokines activate the nuclear factor κB (NF‐κB) and induce nitric oxide production in human pancreatic islets
Authors: Flodströma Malin , Welsh Nils , Eizirik Décio L.
Published: July 2002

We studied the ability of cytokines to activate the nuclear transcription factor NF‐κB in human pancreatic islets and the putative role of NF‐κB for cytokine‐induced NO production. Brief exposure (20 min) of human islets of Langerhans to a combination of interleukin‐lβ + interferon‐γ + tumor necrosis factor‐a induced a 2.6‐fold increase in nuclear NF‐κB activity in gel shift analysis. This increase was prevented by the NF‐κB inhibitor, pyrrolidine dithiocarbamate (PDTC), which also counteracted NO production by human islets exposed for 14 h to the cytokine combination. High concentrations of interleukin‐lβ alone (150 and 250 U/ml) increased NF‐κB nuclear binding but failed to induce NO formation in human islets. The present data are the first to demonstrate that cytokines activate NF‐κB in primary adult human pancreatic islets and suggest that activation of NF‐KB may be a necessary but not sufficient signal for cytokine‐induced iNOS expression in human islets of Langerhans.
Suppressed mixed lymphocyte reaction in couples with intrauterine growth retardation pregnancies
Authors: Tsushima R. , Fujii T. , Nakamura A. , Okai T. , Juji T. , Taketani Y.
Published: July 2002

Objective: The objective of this study was to gain insight into the pathogenesis of intrauterine growth retardation (IUGR). Method: Immune reactivity against paternal MHC class‐II antigens was determined in 18 women with IUGR pregnancies and 18 normal pregnant women, employing one‐way mixed lymphocyte reaction (MLR). Results: One‐way MLR between the women with IUGR pregnancies and their husbands was significantly suppressed compared with that between the women with IUGR pregnancies and males unrelated to them. However the MLR of normal pregnant women was essentially similar regardless of whether stimulating cells were obtained from their husbands or not. The degree of HLA class‐II antigen shared by IUGR couples was not related to the degree of specific suppression of MLR towards the husbands. Conclusion: IUGR may be associated with specifically suppressed patients' MLR towards their husbands, which may provide insight into the underlying pathophysiology of IUGR.
Transcervical flexible endoscopy for first trimester embryonic/fetal evaluation
Authors: Yin C.‐S. , Liu J.‐Y. , Yu M.‐H.
Published: July 2002

Objective: The aim of the study was to investigate the efficacy of flexible embryofetoscopy for visualization of the first trimester human embryo and fetus. Method: Twelve pregnant women scheduled for legal termination of pregnancy at 6–12 weeks' gestation were included in the study. A flexible fiber optic endoscope with an eyepiece connected to a monitor was used. The sterile endoscope was passed transcervically under ultrasound guidance through the chorion into the chorionic cavity. Embryos/fetuses were observed directly through the intact amniotic membrane. Result: Successful embryofetoscopies with clear visualization of the embryo or fetus were accomplished in 50% of cases. There were no procedure‐related complications. Conclusion: Preliminary experience employing transcervical flexible embryofetoscopy for direct visualization of the first trimester human embryo and fetus suggest that this technique may be used for the early identification of congenital anomalies suspected by ultrasound and is expected to offer opportunities for embryo/fetal tissue sampling as well as for gene and cell therapies.
Binding to 14‐3‐3 proteins is not sufficient to inhibit nitrate reductase in spinach leaves
Authors: Weiner Hendrik , Kaiser Werner M
Published: July 2002

To assess the role of 14‐3‐3 proteins in the magnesium‐dependent inhibition of nitrate reductase (NR) we tested the effect of magnesium on NR binding to 14‐3‐3s by coimmunoprecipitation and gel filtration. The stability of the 14‐3‐3 complex of NR was, unlike its activity, unaffected by magnesium. We therefore conclude that binding to 14‐3‐3s per se does not inhibit NR. Magnesium inhibited 14‐3‐3‐bound NR much more strongly than 14‐3‐3‐free NR. 14‐3‐3s possibly reinforce NR inhibition by magnesium.
MECP2 mutation in male patients with non‐specific X‐linked mental retardation
Authors: Orrico Alfredo , Lam Ching-Wan , Galli Lucia , Dotti Maria Teresa , Hayek Giuseppe , Tong Sui-Fan , Poon Priscilla M.K. , Zappella Michele , Federico Antonio , Sorrentino Vincenzo
Published: July 2002

In contrast to the preponderance of affected males in families with X‐linked mental retardation, Rett syndrome (RTT) is a neurological disorder occurring almost exclusively in females. The near complete absence of affected males in RTT families has been explained by the lethal effect of an X‐linked gene mutation in hemizygous affected males. We report here on a novel mutation (A140V) in the MECP2 gene detected in one female with mild mental retardation. In a family study, the A140V mutation was found to segregate in the affected daughter and in four adult sons with severe mental retardation. These results indicate that MECP2 mutations are not necessarily lethal in males and that they can be causative of non‐specific X‐linked mental retardation.
Regulation of APP cleavage by α‐, β‐ and γ‐secretases
Authors: Nunan Janelle , Small David H
Published: July 2002

Proteolytic cleavage of the amyloid protein from the amyloid protein precursor (APP) by APP secretases is a key event in Alzheimer's disease (AD) pathogenesis. α‐Secretases cleave APP within the amyloid sequences, whereas β‐ and γ‐secretases cleave on the N‐ and C‐terminal ends respectively. The transmembrane aspartyl protease BACE has been identified as β‐secretase and several proteases (ADAM‐10, TACE, PC7) may be α‐secretases. A number of studies have suggested that presenilins could be γ‐secretases, although this remains to be demonstrated conclusively. Inhibition of β‐ and γ‐secretase, or stimulation of α‐secretase, is a rational strategy for therapeutic intervention in AD.
Replacement of Tyr62 by Trp in the designer protein Milk Bundle‐1 results in significant improvement of conformational stability
Authors: Gagnon M.C , Williams M , Doucet A , Beauregard M
Published: July 2002

Protein design is currently used for the creation of new proteins with desirable traits. In our lab, we focus on the synthesis of proteins with high essential amino acid content, having potential application in animal nutrition. One of the limitations we face in this endeavor is the achievement of stable proteins in spite of a highly biased amino acid content. We report here the synthesis and characterization of MB‐1Trp, a protein with a tailored content in selected essential amino acids. The protein is a Tyr62‐Trp mutant of the parent molecule MB‐1 described earlier. The new protein is largely helical as per design, is well folded, and has a melting temperature of 55°C. Its resistance to proteolytic degradation compares to that of cytochrome c, a protein of similar size. Design strategy used for MB‐1Trp is discussed with regards to its applicability toward the creation of efficient nutritional proteins.
Disulfide bridge reorganization induced by proline mutations in maurotoxin
Authors: Carlier E. , Fajloun Z. , Mansuelle P. , Fathallah M. , Mosbah A. , Oughideni R. , Sandoz G. , Di Luccio E. , Geib S. , Regaya I. , Brocard J. , Rochat H. , Darbon H. , Devaux C. , Sabatier J.M. , de Waard M.
Published: July 2002

Maurotoxin (MTX) is a 34‐residue toxin that has been isolated from the venom of the chactidae scorpion Scorpio maurus palmatus, and characterized. Together with Pi1 and HsTx1, MTX belongs to a family of short‐chain four‐disulfide‐bridged scorpion toxins acting on potassium channels. However, contrary to other members of this family, MTX exhibits an uncommon disulfide bridge organization of the type C1–C5, C2–C6, C3–C4 and C7–C8, versus C1–C5, C2–C6, C3–C7 and C4–C8 for both Pi1 and HsTx1. Here, we report that the substitution of MTX proline residues located at positions 12 and/or 20, adjacent to C3 (Cys13) and C4 (Cys19), results in conventional Pi1‐ and HsTx1‐like arrangement of the half‐cystine pairings. In this case, this novel disulfide bridge arrangement is without obvious incidence on the overall three‐dimensional structure of the toxin. Pharmacological assays of this structural analog, [A12,A20]MTX, reveal that the blocking activities on Shaker B and rat Kv1.2 channels remain potent whereas the peptide becomes inactive on rat Kv1.3. These data indicate, for the first time, that discrete point mutations in MTX can result in a marked reorganization of the half‐cystine pairings, accompanied with a novel pharmacological profile for the analog.
Mrp‐dependent Na+/H+ antiporters of Bacillus exhibit characteristics that are unanticipated for completely secondary active transporters
Authors: Ito Masahiro , Guffanti Arthur A. , Krulwich Terry A.
Published: July 2002

The Na+/H+ antiport activity encoded by the seven‐gene mrp operons of Bacillus subtilis and alkaliphilic Bacillus pseudofirmus OF4 were cloned into a low copy plasmid, were expressed in several Escherichia coli mutant strains and compared side‐by‐side with similarly cloned nhaA, a major secondary antiporter from E. coli. All three antiporter systems exhibited electron donor‐dependent antiport in a fluorescence‐based vesicle assay, with NhaA being the most active. In whole cells of the same antiporter‐deficient strain from which the vesicles were made, E. coli KNabc, Mrp‐mediated Na+ exclusion was significantly more protonophore‐resistant than that conferred by NhaA. The Mrp systems were also more efficacious than NhaA: in supporting anaerobic Na+ resistance in wild type and a terminal oxidase mutant strain of E. coli (SBS2115); and in increasing non‐fermentative growth of an NADH dehydrogenase‐minus E. coli mutant (ANN0222). The results suggest the possibility that the Mrp systems may have both secondary and primary energization capacities.
Functional analysis of antibacterial activity of Bacillus amyloliquefaciens phage endolysin against Gram‐negative bacteria
Authors: Morita Masatomo , Tanji Yasunori , Orito Yuji , Mizoguchi Katsunori , Soejima Aya , Unno Hajime
Published: July 2002

To analyze the antibacterial activity of Bacillus amyloliquefaciens phage endolysin, nine deletion derivatives of the endolysin were constructed. Each deletion mutant was overexpressed, purified and characterized. The catalytic domain was located on the N‐terminal region and the C‐terminus had an affinity with the bacterial envelope. The enzymatic activity remained in spite of the deletion of the C‐terminal 116‐amino acid region; however, the antibacterial activity was lost. These results indicate that antibacterial action requires both the C‐terminal cell‐binding and the N‐terminal enzymatic activities.
RNA stimulates aggregation of microtubule‐associated protein tau into Alzheimer‐like paired helical filaments
Authors: Kampers T. , Friedhoff P. , Biernat J. , Mandelkow E.-M. , Mandelkow E.
Published: July 2002

The microtubule‐associated protein tau is the main component of the paired helical filaments (PHFs) of Alzheimer's disease, the most common senile dementia. To understand the origin of tau's abnormal assembly we have studied the influence of other cytosolic components. Here we report that PHF assembly is strongly enhanced by RNA. The RNA‐induced assembly of PHFs is dependent on the formation of intermolecular disulfide bridges involving Cys322 in the third repeat of tau, and it includes the dimerization of tau as an early intermediate. Three‐repeat constructs polymerize most efficiently, two repeat constructs are the minimum number required for assembly, and even all six full‐length isoforms of tau can be induced to form PHFs by RNA.
Association of aminopeptidase N and endopeptidase 24.15 inhibitors potentiate behavioral effects mediated by nociceptin/orphanin FQ in mice
Authors: Noble Florence , Roques Bernard P
Published: July 2002

The behavioral effects induced by central administration in mice of the endogenous ORL1(opioid receptor‐like1) ligand, nociceptin/orphanin FQ, were investigated in the absence or presence of inhibitors of aminopeptidase N (bestatin) and endopeptidase 24.15 (Z‐(l,d)PheΨ(PO2CH2)(l,d)Ala–Arg–Phe) recently shown to be involved in the metabolism of the heptadecapeptide in vitro. A severe reduction in motor activity induced by nociceptin/orphanin FQ was measured in two tests (spontaneous motor activity and open field). This pharmacological effect was shown to be potentiated by the association of bestatin and Z‐(l,d)PheΨ(PO2CH2)(l,d)Ala–Arg–Phe, confirming in vivo the involvement of these peptidases in nociceptin/orphanin FQ inactivation. In our conditions, these inhibitors were devoid of intrinsic effects, suggesting a low tonic regulation by the heptadecapeptide of the measured behaviour.
Anaphylatoxin C5a receptor mRNA is strongly expressed in Kupffer and stellate cells and weakly in sinusoidal endothelial cells but not in hepatocytes of normal rat liver
Authors: Schieferdecker Henrike L , Rothermel Ellen , Timmermann Angela , Götze Otto , Jungermann Kurt
Published: July 2002

Anaphylatoxins (C5a and C3a), which are generated during complement activation, have recently been shown to increase glucose output from hepatocytes (HC) in perfused rat liver. They did not act directly on HC but indirectly by prostanoid release from non‐parenchymal cells (NPC), probably Kupffer cells (KC). In order to corroborate this mechanism, the distribution of anaphylatoxin receptors in the different cell types of rat liver was determined by quantitative RT‐PCR with primers specific for the rat C5a receptor (rC5aR) using RNA isolated from KC, sinusoidal endothelial cells (SEC), hepatic stellate cells (HSC) and HC. In line with functional data, C5aR mRNA was detected in freshly isolated NPC but not in HC of rat liver. Mainly KC but also HSC clearly expressed C5aR mRNA, while SEC did so only weakly. KC expressed up to 10‐fold more C5aR mRNA than HSC and these in turn up to 10‐fold more than SEC. These results support the proposed indirect action of anaphylatoxins on HC.
The modular phosphorylation and activation of p70s6k
Authors: Pullen Nicholas , Thomas George
Published: July 2002

The activation of p70s6k is accompanied by a complex series of phosphorylation events. In this review we propose a model of activation which divides p70s6k into four functional modules that cooperate in leading to full enzyme activity. In the light of the model, we suggest how candidate effectors of p70s6k activation might function by directing the phosphorylation of specific sites.
Periodicity in recA protein‐DNA complexes
Authors: Volodin Alexander A , Smirnova Helen A , Bocharova Tatjana N
Published: July 2002

The reaction of guanine residues with dimethylsulfate was studied for complexes of recA protein with fluorescent dye tagged double stranded oligonucleotides. The patterns of dimethylsulfate modification obtained demonstrate a similarity of DNA states in the complexes with recA protein formed as a result of recA promoted strand exchange and renaturation reactions. The guanine modification efficiency varies periodically as a function of the base position along the oligonucleotide axis, with a period of 3 nucleotides. This effect suggests that the arrangement of recA monomers along the oligonucleotide is strictly ordered, and the dimethylsulfate reactivity of a guanine residue depends on the site of its binding in a recA monomer.
Rolling in the clover: trefoil factor family (TFF)‐domain peptides, cell migration and cancer
Authors: Wright N.A , Hoffmann W , Otto W.R , Rio M.-C , Thim L
Published: July 2002

Trefoil factor family (TFF)‐domain peptides 1–3 are mucin‐associated molecules, largely found in epithelia of gastro‐intestinal tissues. Structurally similar, resistant to enzymatic degradation, they are up‐regulated around areas of epithelial damage such as ulcers. Transgenic expression or exogenous peptide ameliorates or prevents gastric mucosal damage due to indomethacin and some are rapidly up‐regulated after cryogenic burns. A role in promoting cell migration is strongly suggested. Knockout mice lacking TFF1 or TFF3 show significant pathology, with the former developing gastric tumours. A recent Conférence Philippe Laudat agreed upon a new nomenclature for these peptides.
Biochemical evidence that Saccharomyces cerevisiae YGR262c gene, required for normal growth, encodes a novel Ser/Thr‐specific protein kinase
Authors: Stocchetto Silvia , Marin Oriano , Carignani Giovanna , Pinna Lorenzo A
Published: July 2002

Saccharomyces cerevisiae YGR262c gene, whose disruption causes severely defective growth, encodes a putative protein kinase shorter than any other protein kinase biochemically characterized to date and lacking some of the conserved features of these enzymes. Here we show that the product of the YGR262c gene, piD261, expressed in E. coli with a C‐terminal (His)6 tag, is a bona fide Ser/Thr protein kinase as judged from its capability to autophosphorylate and to phosphorylate casein and osteopontin in the presence of [γ‐32P]ATP. In contrast, no phosphorylation of histones, myelin basic protein, phosvitin, bovine serum albumin and poly(Glu/Tyr)4:1 could be detected. Mn2+ or, less effectively, Co2+ are required for piD261 catalytic activity, which is conversely undetectable in the presence of Mg2+, a behaviour unique among Ser/Thr protein kinases.
Molecular cloning and characterization of a human brain ryanodine receptor1
Authors: Nakashima Yasuyo , Nishimura Seiichiro , Maeda Akito , Barsoumian Edward L. , Hakamata Yasuhiro , Nakai Junichi , Allen Paul D. , Imoto Keiji , Kita Toru
Published: July 2002

We have cloned and sequenced the cDNA of the human brain ryanodine receptor (RyR3), which is composed of 4866 amino acids and shares characteristic structural features with the rabbit RyR3. Northern blot analysis shows that the human RyR3 mRNA is abundantly expressed in hippocampus, caudate nucleus and amygdala as well as in skeletal muscle. The human RyR3 mRNA is also detected in several cell lines derived from human brain tumors. Functional expression of RyR3 and a chimeric RyR suggests that RyR3 forms a calcium‐release channel with a very low Ca2+ sensitivity.
Molecular cloning of human homolog of yeast GAA1 which is required for attachment of glycosylphosphatidylinositols to proteins1
Authors: Hiroi Yukio , Komuro Issei , Chen Rui , Hosoda Toru , Mizuno Takehiko , Kudoh Sumiyo , Georgescu Serban P , Medof M.Edward , Yazaki Yoshio
Published: July 2002

Anchoring proteins to cell surface membranes by glycosylphosphatidylinositols (GPIs) is important. We have isolated a component of the putative transamidase machinery, hGaa1p (human GPI anchor attachment protein). hGAA1 cDNA is approximately 2 kb in length and codes 621 amino acids. The amino acid sequence of hGaa1p is 25% identical and 57% homologous to that of yeast Gaa1p. Moreover, Kite‐Dolittle hydrophobicity plots of both proteins show marked similarity. hGAA1 gene is expressed ubiquitously and mRNA levels are higher in the undifferentiated state. Overexpression of antisense hGAA1 in human K562 cells significantly reduced the production of a reporter GPI‐anchored protein.
Pro‐ and anti‐apoptotic effects of K+ in HeLa cells
Authors: Bilney Andrea J , Murray Andrew W
Published: July 2002

The present study determined the effects of osmotic stress induced by sorbitol and KCl on apoptosis. Sorbitol induced apoptosis, activated the mitogen‐activated protein kinase (MAPK) family and stimulated accumulation of cytosolic cytochrome c and procaspase‐3 cleavage. KCl (0.2 M) also activated the MAPKs and induced cytosolic cytochrome c accumulation but did not induce procaspase‐3 cleavage or apoptosis and was protective against sorbitol‐induced apoptosis. However, when cells were exposed to KCl for 1 h, washed and returned to isotonic medium, caspase‐3 was rapidly cleaved and apoptosis induced. We conclude that hyperosmotic KCl initiates early events in apoptosis but blocks caspase‐3 activation by preventing the loss of intracellular K+.
Ancient gene duplication and domain shuffling in the animal cyclic nucleotide phosphodiesterase family1
Authors: Koyanagi Mitsumasa , Suga Hiroshi , Hoshiyama Daisuke , Ono Kanako , Iwabe Naoyuki , Kuma Kei-ichi , Miyata Takashi
Published: July 2002

The animal cyclic nucleotide phosphodiesterases (PDEs) comprise at least seven subtypes, PDE1–7, which differ from each other in domain organization and primary function, and they diverged from an ancestral gene by gene duplication and domain shuffling during animal evolution. To obtain rough estimates for the divergence times of these subtypes, cloning of PDE cDNAs from Ephydatia fluviatilis (freshwater sponge) by RT‐PCR was carried out. We obtained four cDNAs, EFPDE1, EFPDE2, EFPDE3, and EFPDE4, which are possibly homologs of the vertebrate PDE1, PDE2, PDE3, and PDE4, respectively, judging from the sequence similarity, domain organization, and branching pattern in the phylogenetic tree. The phylogenetic tree of the PDE family revealed that most gene duplications and domain shufflings that gave rise to different subtypes had been completed in the early evolution of animals before the separation of sponges and eumetazoans.
Albumin's role in steroid hormone action and the origins of vertebrates: is albumin an essential protein?
Authors: Baker Michael E
Published: July 2002

Albumin, the major serum protein, binds a wide variety of lipophilic compounds including steroids, other lipophilic hormones and phytochemicals that bind to hormone receptors. Albumin has a low affinity for these lipophilic compounds. However, due to albumin's high concentration in serum, albumin is a major carrier of steroids and lipophilic hormones and regulator of their access to their receptors. Moreover, albumin functions as a sink for phytochemicals, which prevents their binding to hormone receptors and other cellular proteins, protecting animals from disruptive phytochemical‐mediated endocrine effects. We propose that these properties of albumin were important in protochordates and vertebrates about 550 to 520 million years ago, just before and during the Cambrian. At that time, animal body sizes and exposure to phytochemicals in food were increasing, and animals in which albumin expression was high had a selective advantage in surviving and reproducing in the presence of toxic phytochemicals. This hypothesis that albumin has essential function(s) in mammalian endocrine physiology can be tested by comparing the effects of phytochemicals in Nagase rats that have 1/1000 the normal albumin concentration or in mice in which the albumin gene is knocked out with those in normal rats and mice.
Uptake by rat liver and intracellular fate of plasmid DNA complexed with poly‐l‐lysine or poly‐d‐lysine
Authors: Laurent Nathanael , Wattiaux-De Coninck Simone , Mihaylova Eugenie , Leontieva Ekaterina , Warnier-Pirotte Marie-Thérèse , Wattiaux Robert , Jadot Michel
Published: July 2002

Efficiency of transfection is probably dependent on the rate of intracellular degradation of plasmid DNA. When a non‐viral vector is used, it is not known to what extent the plasmid DNA catabolism is subordinated to the catabolism of the vector. In the work reported here, the problem was approached by following the intracellular fate in rat liver, of plasmid [35S]DNA complexed with a cationic peptide poly‐l‐lysine that can be hydrolyzed by cellular peptidases or with its stereoisomer, poly‐d‐lysine, that cannot be split by these enzymes. Complexes of DNA with poly‐l‐lysine and poly‐d‐lysine are taken up to the same extent by the liver, mainly by Kupffer cells, but the intracellular degradation of nucleic acid molecules is markedly quicker when poly‐l‐lysine is injected. The association of DNA with the polycations inhibits DNA hydrolysis in vitro by purified lysosomes but similarly for poly‐l‐lysine and poly‐d‐lysine. The intracellular journey followed by [35S]DNA complexed with poly‐l‐ or poly‐d‐lysine was investigated using differential and isopycnic centrifugation. Results indicate that [35S]DNA is transferred more slowly to lysosomes, the main site of intracellular degradation of endocytosed macromolecules, when it is given as a complex with poly‐d‐lysine than with poly‐l‐lysine. They suggest that the digestion of the vector in a prelysosomal compartment is required to allow endocytosed plasmid DNA to rapidly reach lysosomes. Such a phenomenon could explain why injected plasmid DNA is more stable in vivo when it is associated with poly‐d‐lysine.
Somatostatin induces hyperpolarization in pancreatic islet α cells by activating a G protein‐gated K+ channel
Authors: Yoshimoto Yukiko , Fukuyama Yuji , Horio Yoshiyuki , Inanobe Atsushi , Gotoh Mitsukazu , Kurachi Yoshihisa
Published: July 2002

Somatostatin inhibits glucagon‐secretion from pancreatic α cells but its underlying mechanism is unknown. In mouse α cells, we found that somatostatin induced prominent hyperpolarization by activating a K+ channel, which was unaffected by tolbutamide but prevented by pre‐treating the cells with pertussis toxin. The K+ channel was activated by intracellular GTP (with somatostatin), GTPγS or Gβγ subunits. It was thus identified as a G protein‐gated K+ (KG) channel. RT‐PCR and immunohistochemical analyses suggested the KG channel to be composed of Kir3.2c and Kir3.4. This study identified a novel ionic mechanism involved in somatostatin‐inhibition of glucagon‐secretion from pancreatic α cells.
Accelerated filament formation from tau protein with specific FTDP‐17 missense mutations
Authors: Nacharaju Parimala , Lewis Jada , Easson Colin , Yen Samuel , Hackett Jennifer , Hutton Mike , Yen Shu-Hui
Published: July 2002

Tau is the major component of the neurofibrillar tangles that are a pathological hallmark of Alzheimers' disease. The identification of missense and splicing mutations in tau associated with the inherited frontotemporal dementia and Parkinsonism linked to chromosome 17 demonstrated that tau dysfunction can cause neurodegeneration. However, the mechanism by which tau dysfunction leads to neurodegeneration remains uncertain. Here, we present evidence that frontotemporal dementia and Parkinsonism linked to chromosome 17 missense mutations, P301L, V337M and R406W, cause an accelerated aggregation of tau into filaments. These results suggest one mechanism by which these mutations can cause neurodegeneration and frontotemporal dementia and Parkinsonism linked to chromosome 17.
The Qo‐site inhibitor DBMIB favours the proximal position of the chloroplast Rieske protein and induces a pK‐shift of the redox‐linked proton
Authors: Schoepp Barbara , Brugna Myriam , Riedel Astrid , Nitschke Wolfgang , Kramer David M.
Published: July 2002

The interaction of the inhibitor 2,5‐dibromo‐3‐methyl‐6‐isopropylbenzoquinone (DBMIB) with the Rieske protein of the chloroplast b
6
f complex has been studied by EPR. All three redox states of DBMIB were found to interact with the iron‐sulphur cluster. The presence of the oxidised form of DBMIB altered the equilibrium distribution of the Rieske protein's conformational substates, strongly favouring the proximal position close to heme b
L. In addition to this conformational effect, DBMIB shifted the pK‐value of the redox‐linked proton involved in the iron‐sulphur cluster's redox transition by about 1.5 pH units towards more acidic values. The implications of these results with respect to the interaction of the native quinone substrate and the Rieske cluster in cytochrome bc complexes are discussed.
Resveratrol inhibits MAPK activity and nuclear translocation in coronary artery smooth muscle: reversal of endothelin‐1 stimulatory effects
Authors: El-Mowafy Abdalla M. , White Richard E.
Published: July 2002

In porcine coronary arteries, short‐term treatment with resveratrol (RSVL) substantially inhibited MAPK activity (IC50=37 μM); and immunoblot analyses revealed consistent reduction in the phosphorylation of ERK‐1/‐2, JNK‐1 and p38, at active sites. Endothelin‐1 (ET‐1), a primary antecedent in coronary heart diseases, enhanced MAPK activity, phosphorylation and nuclear translocation in a concentration‐responsive but RSVL‐sensitive manner. RSVL had no effect on basal or forskolin‐stimulated cAMP levels, a known downregulator of the MAPK cascade. Likewise, inhibition of MAPK by RSVL was not reversed by the estrogen receptor blockers tamoxifen and ICI‐182,780. Conversely, RSVL remarkably attenuated basal and ET‐1‐evoked protein tyrosine phosphorylation. Because MAPKs promote smooth muscle proliferation and contraction, their current inhibition may contribute to the putative protection by RSVL against coronary heart diseases. These effects apparently do not involve interaction with estrogen receptors.
Enhancement of the UVA induction of haem oxygenase‐1 expression by β‐carotene in human skin fibroblasts
Authors: Obermüller-Jevic Ute C , Francz Pal I , Frank Jürgen , Flaccus Andrea , Biesalski Hans K
Published: July 2002

β‐Carotene has often been discussed as a means to reduce the risk of skin photodamage. We studied the antioxidative potential of β‐carotene in human skin fibroblasts exposed to ultraviolet A light. Surprisingly, we found a pro‐oxidative effect of β‐carotene. Using the induction of haem oxygenase‐1 as a marker for oxidative stress, we found a strong enhancement of gene expression by β‐carotene in ultraviolet A‐irradiated cells. This effect was clearly suppressed by concomitant addition of vitamin E but only moderately by vitamin C. The results show that β‐carotene has pro‐oxidative properties in human skin fibroblasts exposed to ultraviolet‐A light.
Second transmembrane segment of FtsH plays a role in its proteolytic activity and homo‐oligomerization
Authors: Makino Shin-ichi , Makinoa Tomohiro , Abe Kunitake , Hashimoto Junko , Tatsuta Takashi , Kitagawa Masanari , Mori Hirotada , Ogura Teru , Fujii Tomoyuki , Fushinobu Shinya , Wakagi Takayoshi , Matsuzawa Hiroshi
Published: July 2002

The FtsH (HflB) protein of Escherichia coli is a membrane‐bound ATP‐dependent zinc protease. The role(s) of the N‐terminal membrane‐anchoring region of FtsH were studied by fusion with a maltose‐binding protein (MBP) at five different N‐termini of FtsH. The MBP‐FtsH fusions were expressed in the cytoplasm of E. coli, and were purified as soluble proteins. The four longer constructs, which have a second transmembrane segment and the C‐terminal cytoplasmic region in common, retained ATP‐dependent protease activity toward heat‐shock transcription factor σ32, and were found to be homo‐oligomers. In contrast, the shortest construct which has the C‐terminal cytoplasmic region but not the second transmembrane segment showed neither protease activity nor oligomerization. Therefore, the second transmembrane segment, which neighbors the C‐terminal cytoplasmic region of the FtsH, participates in not only its membrane‐anchoring, but also its protease activity and homo‐oligomerization.
The effects of long‐term hormone‐replacement therapy on coagulation, fibrinolysis and inhibitors in postmenopausal women
Authors: Koh S.C.L , Dramusic V , Shan Ratnam S
Published: July 2002

Objective: To study the long‐term effects of hormone‐replacement therapy (HRT). Methods: Twenty‐one menopausal Chinese women who were currently on HRT were studied for the long‐term effects of HRT on hemostasis. Another 21 age‐matched healthy women not on HRT served as controls. Results: Enhanced thrombin generation was seen in women on long‐term HRT. Antithrombin III (ATIII) antigen was significantly reduced but not its activity level and so was FVII. Elevated t‐PA activity and u‐PA antigen were seen with reduced t‐PA antigen. Plasminogen activator inhibitor‐1 (PAI‐1) levels were significantly reduced together with d‐dimer in HRT. Conclusions: Long‐term HRT was associated with enhanced thrombin generation and countered by enhanced systemic t‐PA activity suggesting an equilibrium state. There was no enhanced cardiovascular risk in long‐term HRT as indicated by the observed reduction in t‐PA antigen, PAI‐1 and FVII levels. However, a larger prospective study is required to confirm these findings.
Vacuolar morphology and cell cycle distribution are modified by leucine limitation in auxotrophic Saccharomyces cerevisiae
Authors: Petekçakar Z. , Sauer Uwe , Bailey James E. , Müller Martin , Stolz Martin , Wallimann Theo , Schlattner Uwe
Published: July 2002

Summry— Yeast vacuoles are highly dynamic and flexible organelles. In a previous paper, we have shown that subtle, often unrecognised amino acid limitations lead to much lower final cell densities in cultures of different commonly used auxotrophic Saccharomyces cerevisiae strains (çakar et al., Biotechnol. Lett. 21 (1999) 611). Here, we demonstrate for two of these strains, CEN.PK 113.6B and CBS7752, that such subtle leucine limitations also affect the number and morphology of vacuoles, and that these changes are correlated with the cell cycle in batch cultures in a similar way as is known from synchronized cultures. Morphological aspects were studied by electron microscopy, using advanced high pressure freezing/freeze‐substitution techniques for sample preparation that so far have been barely successful in yeast. Cells of leucine‐limited cultures had single, large vacuoles with a hexagonal tonoplast pattern and were partially arrested in G1 phase. To relieve leucine‐limitation, additional leucine was supplied extracellularly via the medium or intracellularly via enhanced leucine biosynthesis due to plasmid‐based expression of a leucine marker gene. Such cultures reached more than two‐fold higher final optical densities in stationary phase. Cells in later growth phase were characterized by fragmented vacuoles lacking any tonoplast pattern and by a smaller proportion of cells in G1 phase. These drastic effects of subtle leucine limitation on cell physiology, vacuolar morphology and cell cycle distribution present a note of caution for morphological and cell cycle studies in yeast.
In vitro 1H and 31P NMR spectroscopic evidence of multiple aberrant biochemical pathways in murine trisomy 16 brain development
Authors: Yao Faith S. , Caserta Maria T. , Wyrwicz Alice M.
Published: July 2002

Nuclear magnetic resonance (NMR) spectroscopy was used to evaluate cytosolic compounds and membrane phospholipids simultaneously in trisomy 16 (Ts16) and euploid (control) murine brain at fetal day 15 in order to examine the cellular biochemistry that underlies the neurodevelopmental consequences of chromosome triplication in this model of Down syndrome (DS). Proton NMR spectroscopic analysis of brain tissue extracts demonstrated decreased levels of choline and increased levels of myo‐inositol (MI) in Ts16 brains compared with control. These data are consistent with the cholinergic deficits and elevated MI levels previously described in Ts16. Compared with euploid brains, Ts16 brains also possess higher levels of creatine, adenosine, and tyrosine. Increased levels of MI and creatine, compounds that are localized to glia, imply abnormalities in the trophic environment of Ts16 brain. Phosphorus NMR spectroscopic analysis of extracts further revealed elevated levels of anionic phospholipid membrane components, such as phosphatidylinositol (PtdIno) and phosphatidylethanolamine, in Ts16 brains. Since these compounds are confined to the inner leaflet of the membrane, the findings suggest that membrane composition is altered specifically in the cytosolic bilayer at this stage. Together our proton and phosphorus NMR spectroscopic results indicate that multiple biochemical pathways are affected in Ts16 brain development. Understanding the effects of these aberrations may elucidate the processes that lead to neural dysfunction and Alzheimer's disease (AD) neuropathology in DS individuals.
Further evidence for supporting cell conversion in the damaged avian basilar papilla
Authors: Adler Henry J. , Komeda Mototane , Raphael Yehoash
Published: July 2002

Two lines of evidence suggested that a process other than supporting cell divisions may give rise to new hair cells in the bird inner ear injured by either noise or ototoxic drugs. This process, supporting cell conversion, occurs when non‐dividing supporting cells transdifferentiate into hair cells. First, noise‐exposed chicks received zero, one or two daily i.p. injections of cytosine arabinoside (a DNA synthesis blocker), as well as two daily intraperitoneal injections of bromodeoxyuridine, for four days. Following sacrifice, the papillae were processed for bromodeoxyuridine immunocytochemistry. All the ears demonstrated dividing cells, but increasing the number of cytosine arabinoside injections decreased the number of labeled cells. Indeed, two cytosine arabinoside injections per day nearly completely blocked supporting cell divisions in the short hair cell region within the sound‐induced lesion. This suggested that unpaired, immature cells observed in a similar region with scanning electron microscopy, despite the presence of cytosine arabinoside, may have been products of supporting cell conversion. In the second experiment, birds were treated with gentamicin for three days. Upon sacrifice at 6 days post‐treatment, papillae were processed for light and transmission electron microscopy. Several unusual cells were observed with phenotypic features of both hair cells and supporting cells. The peculiar cells may be in a transition from the supporting cell phenotype to that of a hair cell.
Ontogeny of cholinergic amacrine cells in the opossum (Didelphis aurita) retina
Authors: Camargo De Moura Campos Lenira , Hokoç Jan Nora
Published: July 2002

Immunocytochemistry for choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine, was used to determine the onset and to follow the maturation of the cholinergic cells in the retina of a marsupial, the South American opossum (Didelphis aurita). ChAT‐immunoreactivity was first detected in amacrine cells in the ganglion cell layer by postnatal day 15 (P15) and in the inner nuclear layer by P35. Much later, at P50 a second sub‐population of ChAT‐immunoreactive cell bodies was evident in the inner nuclear layer. Processes from ChAT‐immunoreactive amacrine cells were detected in the two bands of the inner plexiform layer before synaptogenesis. In the adult retina, these two bands correspond to sublamina 2 and 4 of the inner plexiform layer. In flat whole‐mounted preparations, cholinergic cell density was 263±13 cells/mm2 in the ganglion cell layer and it was estimated a total of 24,000 cholinergic neurons. ChAT‐immunoreactive somata showed a random pattern of distribution.
Molecular Genetics of Mental Disorders with Particular Reference to Affective Disorders
Authors: Souery D , Lipp O , Mahieu B , Mendlewicz J
Published: July 2002

The present article reviews the recent molecular genetic findings in affective disorders. Results of linkage and association studies are discussed in regard to the main limitations of these approaches in psychiatric disorders. On the whole, linkage and association studies contributed to the localisation of some potential vulnerability genes for Bipolar affective disorder on chromosomes 18, 5, 11, 4, 21 and X. The hypothesis of anticipation in affective disorders is also considered in light of interesting results with trinucleotide repeat mutations.