bluuebunny/crossref_metadata_2025 huggingface dataset:
Salicylic acid‐independent induction of pathogenesis‐related protein transcripts by sugars is dependent on leaf developmental stage
Authors: Herbers Karin , Meuwly Philippe , Métraux Jean-Pierre , Sonnewald Uwe
Published: July 2002

Soluble sugars have been found to regulate a number of genes involved in functions associated with sink metabolism, defense reactions and photosynthesis. As viruses and pathogens induce the expression of pathogenesis‐related (PR) protein genes and have also been reported to lead to localized sugar accumulation in leaves, it was investigated whether a salicylic acid‐independent but sugar‐dependent pathway for PR‐protein gene induction may exist in plant cells. Leaf discs of tobacco plants were floated on different sugar solutions, transcript accumulation and salicylic acid (SA) levels were subsequently determined. PR‐Q and PAR‐1 transcripts were found to be inducible by glucose, fructose and sucrose. No significant change in SA content could be detected, following incubation. On the other hand, SAR8.2 transcripts were repressed by elevated levels of soluble sugars and sorbitol, respectively, suggesting sensitivity to turgor pressure. Since leaves undergo sink to source transition during growth, sugar responsiveness was investigated in leaves of different developmental stages. Interestingly, induction of PR‐Q and PAR‐1 by soluble sugars was essentially restricted to fully expanded leaves and was independent of plant age. Induction by salicylate was not confined to the source capacity of a leaf but was dependent on the age of the respective leaf. Repression of transcripts encoding photosynthetic genes (ribulose‐1,5‐bisphosphate carboxylase/oxygenase(rbcS) and chlorophyll a/b binding protein (cab) by soluble sugars were largely independent from the leaf developmental state. These findings hint to the possibility of salicylic acid‐independent defense reactions of plants against pathogens by induction of a set of PR proteins in source leaves. Furthermore, the data suggest different mechanisms for the induction of PR‐protein genes and the repression of photosynthetic genes by soluble sugars.
The β‐tubulin monomer release factor (p14) has homology with a region of the DnaJ protein
Authors: Llosa Matxalen , Aloria Kerman , Campo Rafael , Padilla Rodolfo , Avila Jésus , Sánchez-Pulido Luis , Zabala Juan Carlos
Published: July 2002

p14 is a molecular chaperone involved in β‐tubulin folding which catalyzes the release of β‐tubulin monomers from intermediate complexes. Here we demostrate that active p14 protein which we have purified from an overproducing Escherichia coli strain can also release ‐tubulin monomers from tubulin dimers in the presence of an additional cofactor (Z). Analysis of p14 secondary structure suggests that this protein may belong to a family of conserved proteins which share structural similarities with the J‐domain of DnaJ. We have constructed deletions and site‐directed mutations in the p14 gene. A single D to E mutation in the region shown in DnaJ to be an essential loop for its function affected the monomer‐release activity of p14. These results support the hypothesis that this p14 loop interacts with β‐tubulin in a similar fashion as DnaJ interacts with DnaK and suggest a possible role of p14 in the folding process.
The α1 and α2 isoforms of the AMP‐activated protein kinase have similar activities in rat liver but exhibit differences in substrate specificity in vitro
Authors: Woods Angela , Salt Ian , Scott James , Hardie D.Grahame , Carling David
Published: July 2002

The AMP‐activated protein kinase (AMPK) is a heterotrimeric complex composed of a catalytic subunit (a) and two regulatory subunits (β and γ). Two isoforms of the catalytic subunit (αl and (α2) have been identified. We show here that the αl‐ and α2‐containing complexes contribute approximately equally to total AMPK activity in rat liver. Furthermore, expression of al or a2 with β and Y in mammalian cells demonstrates that both complexes have equal specific activity measured with the SAMS peptide. Using variant peptides, however, we show that al and a2 exhibit slightly different substrate preferences, which suggest that the two isoforms could play different physiological roles within the cell.
G2 cell cycle arrest and apoptosis are induced in Burkitt's lymphoma cells by the anticancer agent oracin
Authors: Klucar J , Al-Rubeai M
Published: July 2002

The cytotoxic effect of the new potential intercalating anticancer drug oracin was studied on Burkitt's lymphoma cell line that overexpressed bcl‐2 (BL bcl‐2) and a control transfectant without the bcl‐2 gene (BL SV2). Oracin showed a marked cytostatic effect on both BL SV2 and BL bcl‐2 cells. IC50, as measured by the MTT assay, was approx. 5‐times greater for BL bcl‐2 cells (5.0 μmol/l) than for BL SV2 cells (1.0 μmol/l). There was no significant increase in apoptosis after 24 h of treatment with oracin (1.0 μmol/l) in both cell lines. However, after 48 h from the removal of oracin in BL SV2 culture the levels of apoptotic and secondary necrotic cells increased to 20 and 37%, respectively. In contrast, BL bcl‐2 cells treated in a similar manner showed only basal levels of apoptotic and secondary necrotic cells. Analysis of the cell cycle profiles showed a significant increase of S and G2/M phases of the cell cycle in both cell lines after 6 h of drug treatment (1.0 μmol/l). The cells were arrested in G2/M phase of the cell cycle after 24 h, with no significant changes in cell viability. After 72 h, the viable BL SV2 cells were still in G2/M, however, the viability of this culture had fallen to approx. 5%. Flow cytometry analysis of the DNA content revealed the presence of a ‘sub‐G2’ region, which represented the apoptotic cells. The BL SV2 cells died after 72 h while they were in the G2/M phase. Although the treated BL bcl‐2 cells were similarly arrested in the G2/M phase, they nevertheless remained with a relatively high viability (68%).
Interaction of G protein Gβγ dimers with small GTP‐binding proteins of the Rho family
Authors: Harhammer Rainer , Gohla Antje , Schultz Günter
Published: July 2002

Gβγ dimers of heterotrimeric G proteins have been shown to be important for the translocation of cytosolic proteins to membranes. The involvement of Gβγ in those signaling processes mediated by small GTP‐binding proteins of the Rho family was studied using purified proteins. We showed specific binding of bovine brain Gβγ to immobilized GST‐Rho fusion proteins. In addition, brain Gβγ, but not transducin Gβγ, was able to inhibit GTPγS binding to GST‐Rho in a concentration‐dependent manner. GTPγS binding to GST‐Rac was also decreased by brain Gβγ whereas nucleotide binding to GST‐Cdc42 was not changed. We conclude that Gβγ dimers may participate in the process of membrane attachment and/or other regulations of Rho and Rac.
Amino acid sequence of a new type of antifreeze protein, from the longhorn sculpin Myoxocephalus octodecimspinosis
Authors: Deng Gejing , Andrews David W , Laursen Richard A
Published: July 2002

A new type of fish antifreeze protein, designated here type IV, has been isolated from the longhorn sculpin, Myoxocephalus octodecimspinosis. Sequence analysis of the protein (LS‐12) reveals that it contains 108 amino acids, is blocked at the N‐terminus by a pyroglutamyl group and has a high (17%) content of glutamine; it is thus completely unrelated to the earlier described types I, II and III fish antifreeze proteins. Circular dichroism spectra and conformational analysis based on the sequence data indicate that LS‐12 has a high helix content and probably folds as a four‐helix bundle. LS‐12 shows sequence similarity to certain plasma apolipoproteins known to have helix bundle structures, suggesting the possibility that LS‐12 may have arisen by recruitment and mutation of a plasma apolipoprotein.
Direct activation of protein phosphatase‐2A0 by HIV‐1 encoded protein complex NCp7:vpr
Authors: Tung H.Y.Lim , De Rocquigny Hughes , Zhao Ling-Jun , Cayla Xavier , Roques Bernard P , Ozon René
Published: July 2002

The effects of HIV‐1 encoded proteins NCp7, vpr and NCp7:vpr complex on the activity of protein phosphatase‐2A0 have been tested. We report that NCp7 is an activator of protein phosphatase‐2A0 and that vpr activated protein phosphatase‐2A0 only slightly. We also report that NCp7 and vpr form a tight complex which becomes a more potent activator of protein phosphatase‐2A0 than NCp7 alone. The ability of NCp7 to activate protein phosphatase‐2A0 is regulated by vpr. The C‐terminal portion of vpr prevents NCp7 from activating protein phosphatase‐2A0 while the N‐terminal portion of vpr potentiates the effect of NCp7 on the activity of protein phosphatase‐2A0. Our findings indicate that vpr may be acting as a targeting subunit which directs NCp7 to activate protein phosphatase‐2A0. In view of the fact that protein phosphatase‐2A functions as an inhibitor of G2 to M transition of the cell cycle and is involved in other key cellular processes such as the control of RNA transcription, the results presented in this report may explain how HIV‐1 causes cell cycle arrest which may lead to CD4+ T cell depletion and also how it disturbs normal cellular processes of its host cell.
GAK: a cyclin G associated kinase contains a tensin/auxilin‐like domain1
Authors: Kanaoka Yoshihide , Kimura Shinya H , Okazaki Issey , Ikeda Masako , Nojima Hiroshi
Published: July 2002

We have cloned a cDNA encoding a novel association partner of cyclin G by West‐Western blotting. The cDNA encodes a protein that harbors a Ser/Thr protein kinase‐like catalytic domain at the N‐terminal. Hence, we named it GAK (cyclin
‐
ssociated
inase). The long C‐terminal extension shares homology with tensin and auxilin, and contains a leucine zipper region. Co‐immunoprecipitation and Western blotting showed that GAK and cyclin G associate together in vivo. GAK also co‐precipitated with CDK5, and CDK5 was found to be associated with cyclin G. We also showed by BIAcore analysis that the GAK‐cyclin G interaction was direct.
C/EBPα is a major activator for the transcription of rat Cu/Zn superoxide dismutase gene in liver cell
Authors: Kim Young Ho , Yoo Hae Yong , Chang Mun Seog , Jung Guhung , Rho Hyune Mo
Published: July 2002

The rat Cu/Zn superoxide dismutase (SOD1) is expressed in all tissues. Sequence analysis of the SOD1 promoter region showed that none of the cis‐elements of hepatocyte‐specific nuclear factors (HNF) were observed. The cis‐element of C/EBPα in the proximal region of the SOD1 promoter and the high level of C/EBPα in the liver tissue led us to focus on the transcriptional regulation of the SOD1 gene by C/EBPα. Cotransfection assays with the plasmid expressing transcription factor C/EBPα showed that C/EBPα transactivated SOD1 gene by 27 fold. The marked transactivation and direct binding of C/EBPα to the SOD1 promoter were confirmed by deletion analyses and mobility shift assays. These results suggested that C/EBPα plays a major role in the tissue distribution of SOD1.
The protein kinase C inhibitors Ro 318220 and GF 109203X are equally potent inhibitors of MAPKAP kinase‐1β (Rsk‐2) and p70 S6 kinase
Authors: Alessi Dario R
Published: July 2002

The protein kinase C (PKC) inhibitors Ro 318220 and GF 109203X have been used in over 350 published studies to investigate the physiological roles of PKC. Here we demonstrate that these inhibitors are not selective for PKC isoforms as was previously assumed. Ro 318220 inhibited MAPKAP kinase‐1β (also known as Rsk‐2) in vitro (IC50 3 nM) more potently than it inhibited mixed PKC isoforms (IC50 5 nM), and it also inhibited p70 S6 kinase (IC50 15 nM). GF 109203X also potently inhibited MAPKAP kinase‐1β (IC50 50 nM) and p70 S6 kinase (IC50 100 nM) with similar potency to PKC isoforms (IC50 30 nM). The inhibition of MAPKAP kinase‐1β, p70 S6 kinase, and probably other protein kinases, may explain many of the effects previously attributed to PKC.
Proteolytic activation of the precursor of membrane type 1 matrix metalloproteinase by human plasmin
Authors: Okumura Yuushi , Sato Hiroshi , Seiki Motoharu , Kido Hiroshi
Published: July 2002

Membrane type 1 matrix metalloproteinase (MT1‐MMP) was suggested to play a critical role in the regulation of tissue invasion by normal and neoplastic cells by directly mediating the activation of pro‐gelatinase A. Recently, the proteolytic activation of a pro‐MT1‐MMP by an intracellular proprotein convertase, furin, was reported. In this study, we found that plasmin efficiently activates the pro‐MT1‐MMP by cleaving immediately downstream of Arg108 and Arg111 in the multi‐basic motif between its pro‐ and catalytic domains that participates in the activation of pro‐gelatinase A. Our present data suggest that pro‐MT1‐MMP transported to the plasma membrane is activated by plasmin extracellularly and thus it may play an important role in the matrix degradation process.
Depolymerization of malarial hemozoin: a novel reaction initiated by blood schizontocidal antimalarials
Authors: Pandey Amit V , Tekwani Babu L
Published: July 2002

Malaria parasite digests hemoglobin and utilizes the globin part for its nutritional requirements. Heme released as a byproduct of hemoglobin degradation is detoxified by polymerization into a crystalline, insoluble pigment, known as hemozoin. We have identified a novel reaction of depolymerization of hemozoin to heme. This reaction is initiated by the interaction of blood schizonticidal antimalarial drugs with the malarial hemozoin. The reaction has been confirmed, with the purified hemozoin as well as the lysate of the malaria parasite. Pigment breakdown was studied by infrared spectroscopy, thin‐layer chromatography and spectrophotometric analysis. It was complete within 2 h of drug exposure, which explains the selective sensitivity of late stages (trophozoites and schizonts) of malarial parasites loaded with the hemozoin pigment to the toxic action of these drugs. It is suggested that the failure of the parasite heme detoxification system due to this reaction results in the accumulation of toxic heme, which alone, or complexed with the antimalarial leads to the death of malaria parasite. © 1997 Federation of European Biochemical Societies. All rights reserved.
Superoxide anion formation from lucigenin: an electron spin resonance spin‐trapping study
Authors: Vásquez-Vivar Jeannette , Hogg Neil , Pritchard Kirkwood A. , Martasek Pavel , Kalyanaraman B.
Published: July 2002

©1997 Federation of European Biochemical Societies.
At‐GDI1fromArabidopsis thalianaencodes a rab‐specific GDP dissociation inhibitor that complements thesec19mutation ofSaccharomyces cerevisiae
Authors: Žárský Viktor , Cvrčková Fatima , Bischoff Friedrich , Palme Klaus
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Glutamyl endopeptidase of Bacillus intermedius, strain 3‐19
Authors: Leshchinskaya I.B. , Shakirov E.V. , Itskovitch E.L. , Balaban N.P. , Mardanova A.M. , Sharipova M.R. , Viryasov M.B. , Rudenskaya G.N. , Stepanov V.M.
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Primary structure and synthesis of Imperatoxin A (IpTxa), a peptide activator of Ca2+ release channels/ryanodine receptors
Authors: Zamudio Fernando Z. , Gurrola Georgina B. , Arévalo Carolina , Sreekumar Raghava , Walker Jeffery W. , Valdivia Hector H. , Possani Lourival D.
Published: July 2002

We present the complete amino acid sequence of Imperatoxin A (IpTxa), a 33‐amino‐acid peptide from the venom of the scorpion P. imperator which activates Ca2+ release channels/ryanodine receptors (RyR) of sarcoplasmic reticulum (SR). The amino acid sequence of IpTxa shows no homology to any scorpion toxin so far described, but shares some homology to the amino acid sequence of Tx2‐9 and agelenin, two spider toxins that target neuronal P‐type Ca2+ channels. We also describe the total synthesis of IpTxa and demonstrate that it efficiently activates RyRs with potency and affinity identical to those of native IpTxa. The use of synthetic IpTxa should help in the identification of the structural motifs of RyR critical for channel gating.
Direct evidence for the deoxyribonuclease activity of the plant ribosome inactivating protein gelonin
Authors: Nicolas Emmanuelle , Beggs Joseph M. , Haltiwanger Brett M. , Taraschi Theodore F.
Published: July 2002

Several plant ribotoxins, including gelonin, were reported to have additional weak nuclease activities on supercoiled DNA. The potential contribution of this activity to their cytotoxicity has not been given serious consideration due to concerns about contaminating nucleases in the protein preparations. We now report the degradation of single‐stranded DNA by preparations of native plant gelonin and recombinant gelonin produced in E. coli. The DNase activity of both preparations is similarly modulated by zinc. An SDS‐PAGE DNase assay identifies gelonin as the polypeptide responsible for deoxyribonuclease activity.
mRNA distribution and membrane localization of the OAT‐K1 organic anion transporter in rat renal tubules
Authors: Masuda Satohiro , Saito Hideyuki , Nonoguchi Hiroshi , Tomita Kimio , Inui Ken-ichi
Published: July 2002

OAT‐K1, a renal organic anion transporter, which mediates methotrexate uptake, was analyzed for mRNA distribution along microdissected nephron segments and the immunolocalization in isolated plasma membranes from rat kidney. By using a reverse transcription‐coupled PCR, OAT‐K1 mRNA was detected predominantly in the superficial and juxtamedullary proximal straight tubules. Western blotting with antiserum for OAT‐K1 revealed that the transporter protein with the apparent molecular mass of 40 kDa was expressed exclusively in the brush‐border membranes from rat kidney. These findings suggest that the OAT‐K1 is localized in the brush‐border membranes of the renal proximal straight tubules.
A structure–activity study of fatty acid interaction with mitochondrial uncoupling protein
Authors: Ježek Petr , Modrianský Martin , Garlid Keith D
Published: July 2002

Fatty acid (FA) uniport via mitochondrial uncoupling protein (UcP) was detected fluorometrically with PBFI, potassium‐binding benzofuran phthalate and SPQ, 6‐methoxy‐N‐(3‐sulfopropyl)‐quinolinium, indicating K+ and H+, respectively. The FA structural patterns required for FA flip‐flop, UcP‐mediated FA uniport, activation of UcP‐mediated H+ transport in proteoliposomes, and inhibition of UcP‐mediated Cl− uniport by FA, were identical. Positive responses were found exclusively with FA which were able to flip‐flop in a protonated form across the membrane and no responses were found with ‘inactive’ FA lacking the flip‐flop ability. The findings support the existence of FA cycling mechanism.
DNA binding sites recognised in vitro by a knotted class 1 homeodomain protein encoded by the hooded gene, k, in barley (Hordeum vulgare)
Authors: Krusell Lene , Rasmussen Inge , Gausing Kirsten
Published: July 2002

The homeodomain of the knotted classes of transcription factors from plants differs from the well characterized Antp/En type homeodomains from Drosophila at key amino acid residues contributing to the DNA binding. A cDNA, Hvh21, derived from the hooded gene and encoding a full length homolog of knotted1 from maize was isolated from barley seedlings and expressed as a maltose binding protein fusion in E. coli. The purified HvH21‐fusion protein selected DNA fragments with 1–3 copies of the sequence TGAC. Gel shift experiments showed that the TGAC element was required for binding and the results further indicate that the HvH21‐fusion protein binds DNA as a monomer.
Transmembrane topology of the Rieske Fe/S protein of the cytochrome b6/f complex from spinach chloroplasts
Authors: Karnauchov Ivan , Herrmann Reinhold G , Klösgen Ralf Bernd
Published: July 2002

The topology of the Rieske protein of the cytochrome b6/f complex in thylakoids from spinach chloroplasts was examined by protease protection experiments as well as polypeptide extraction assays using solutions of chaotropic salts or alkaline pH. While neither thermolysin nor trypsin cleave any of the Rieske protein when added to the stromal side of the thylakoid membrane, proteinase K is capable of removing approximately four residues from its NH2‐terminus. The protein is resistant to membrane extraction by 0.1 M Na2CO3 or 2 M NaBr but is quantitatively released by 0.1 M NaOH. Treatment of thylakoids with 2 M NaSCN leads to extraction of variable amounts of the protein, depending on the presence or absence of sucrose in the medium which apparently stabilizes the cytochrome complex. From these results we conclude that the Rieske protein is an integral component of the cytochrome complex which spans the thylakoid membrane with a single hydrophobic segment and is anchored predominantly by electrostatic interactions.
A novel gene suppressed in the ventricle of carnitine‐deficient juvenile visceral steatosis mice1
Authors: Masuda Mina , Kobayashi Keiko , Horiuchi Masahisa , Terazono Hiroki , Yoshimura Nozomu , Saheki Takeyori
Published: July 2002

In order to clarify the pathogenesis and pathophysiology of cardiac hypertrophy in carnitine‐deficient juvenile visceral steatosis (JVS) mice, we performed mRNA differential display analysis with total RNA extracted from the ventricles of control and JVS mice at 14 days of age. We identified four up‐regulated genes, two known and two unknown, and a novel down‐regulated gene. Northern blot analysis with a novel cDNA probe derived from the down‐regulated gene fragment 8A2 revealed three mRNA species of 1.1‐, 1.3‐, and 2.6‐kb. The 1.1‐ and 1.3‐kb mRNA species were found only in the heart, and the 2.6‐kb species was found in the heart, kidney and brain, but not in skeletal muscle or liver. The 1.1‐ and 1.3‐kb species were down‐regulated in the ventricles of JVS mice, but not in the auricles, and increased to the control level with carnitine treatment. We isolated cDNA clones from ventricle RNA, termed CDV‐1 (carnitine deficiency‐associated gene expressed in ventricle) and from brain RNA, termed CDV‐1R (CDV‐1‐related gene) by 5′‐ and 3′‐RACE analyses. The entire nucleotide sequence except the 5′‐terminal 64 bp of CDV‐1 cDNA was completely identical to the 992 bp sequence from the 3′‐end of CDV‐1R cDNA. The CDV‐1 cDNA contained an open reading frame predicting a peptide of 107 amino acids, which composed the C‐terminal portion of CDV‐1R peptide consisting of 414 amino acids.
The α‐amylase from the yellow meal worm: complete primary structure, crystallization and preliminary X‐ray analysis
Authors: Strobl Stefan , Gomis-Rüth Franz-Xaver , Maskos Klaus , Frank Gerhard , Huber Robert , Glockshuber Rudi
Published: July 2002

The α‐amylase from Tenebrio molitor larvae (TMA) was purified from a crude larval extract. After removal of the N‐terminal pyroglutamate residue and identification of the following 17 residues by Edman sequencing, the cDNA of mature TMA was cloned from larval mRNA. The encoded enzyme consists of 471 amino acid residues and has 57–79% sequence identity to other insect α‐amylases and also shows high homology to the mammalian enzymes. TMA was crystallized in form of well‐ordered orthorhombic crystals of space group P212121 diffracting beyond 1.6 Å resolution with unit cell dimensions of a=51.24 Å, b=93.46 Å, c=96.95 Å. TMA may serve as model system for the future analysis of interactions between insect α‐amylase and proteinaceous plant inhibitors on the molecular level.
The nodulation gene nolK of Azorhizobium caulinodans is involved in the formation of GDP‐fucose from GDP‐mannose
Authors: Mergaert Peter , Van Montagu Marc , Holsters Marcelle
Published: July 2002

The nolK gene of Azorhizobium caulinodans is essential for the incorporation of a fucosyl group in Nod factors. A NAD(P)‐binding site is present in the NolK amino acid sequence and the gene is homologous to Escherichia coli genes, presumably involved in GDP‐fucose synthesis. Protein extracts of A. caulinodans, overexpressing nolK, have an enzyme activity that synthesizes GDP‐fucose from GDP‐mannose. nolK most probably encodes a 4‐reductase performing the last step in GDP‐fucose synthesis. Wild‐type A. caulinodans produces a population of fucosylated and non‐fucosylated molecules but the nolK‐overexpressing strain produces only fucosylated Nod factors. Thus, the production of activated fucosyl donors is a rate‐limiting step in Nod factor fucosylation.
Human Supt5h protein, a putative modulator of chromatin structure, is reversibly phosphorylated in mitosis
Authors: Stachora Axel A , Enrı́quez Schäfer Ramón , Pohlmeier Matthias , Maier Gernot , Ponstingl Herwig
Published: July 2002

The Saccharomyces cerevisiae proteins Spt4p, Spt5p and Spt6p are involved in transcriptional repression by modulating the structure of chromatin. From HeLa cells we have purified a human homologue of Spt5p, Supt5hp, and show here that the protein is reversibly phosphorylated in mitosis. The cloned cDNA predicts a protein of 1087 residues with 31% identity to yeast Spt5p. It includes an acidic N‐terminus, a putative nuclear localization signal and a C‐terminal region containing two different repeated motifs. One of them, with the consensus sequence P‐T/S‐P‐S‐P‐Q/A‐S/G‐Y, is similar to the C‐terminal domain in the largest subunit of RNA polymerase II.
Non‐iron porphyrins inhibit β‐haematin (malaria pigment) polymerisation
Authors: Basilico Nicoletta , Monti Diego , Olliaro Piero , Taramelli Donatella
Published: July 2002

Infrared spectroscopy was used to evaluate the effect of non‐iron porphyrins (protoporphyrin IX and haematoporphyrin) on haematin polymerisation to β‐haematin at acidic pH. Both molecules effectively inhibited the reaction, with haematoporphyrin 6 times as active as protoporphyrin IX. We postulated that the interaction between the π electron system of porphyrin rings leads to the formation of π–π adducts, which inhibit polymer elongation in the same way as antimalarial drugs (e.g., chloroquine); the presence of hydroxyl groups able to bind haem iron enhances activity.
Structural features of the gene encoding human muscle type carnitine palmitoyltransferase I1
Authors: Yamazaki Naoshi , Yamanaka Yasuhisa , Hashimoto Yoshiko , Shinohara Yasuo , Shima Atsushi , Terada Hiroshi
Published: July 2002

We isolated a human muscle type of carnitine palmitoyltransferase I (CPTI‐M) genomic clone and determined its entire nucleotide sequence. By comparison of the nucleotide sequence of the genomic clone with that of cDNA, we determined the intron/exon junctions. For detection of the exon(s) in the 5′‐region of the CPTI‐M gene, we isolated cDNA clones corresponding to the 5′‐region of its transcript by 5′‐rapid amplification of cDNA ends (5′‐RACE method). Results showed two alternative exons, 1A and 1B, that do not encode amino acids in the 5′‐region of the human CPTI‐M gene. The gene encoding human CPTI‐M was found to consist of two 5′‐non‐coding exons, 18 coding exons and one 3′‐non‐coding exon spanning approximately 10 kbp. Furthermore, on analysis of the 5′‐flanking region, a putative gene encoding a ‘choline kinase homologue’ was found to be located only about 300 bp upstream from exon 1A of the human CPTI‐M gene. Comparison of the gene structure of human CPTI‐M with the reported partial gene structure of human liver type CPTI (CPTI‐L) showed that the intron insertion sites were completely conserved in these two genes.
Expression of trypsin in vascular endothelial cells
Authors: Koshikawa Naohiko , Nagashima Yoji , Miyagi Yohei , Mizushima Hiroto , Yanoma Shunsuke , Yasumitsu Hidetaro , Miyazaki Kaoru
Published: July 2002

Proteinases produced by vascular endothelial cells are expected to play important roles in many biological processes. Here we report that human vascular endothelial cells express trypsinogen‐2 mRNA and its protein product in culture. The trypsinogen production was stimulated by a tumor promoter and associated with cell growth. In situ hybridization analysis showed that the trypsinogen gene was significantly expressed in vascular endothelial cells around gastric tumors and in patients with disseminated intravascular coagulation (DIC). These results suggest the possible roles of endothelial cell‐derived trypsin in tumor angiogenesis and abnormal blood coagulation.
Isolation and characterization of biliprotein aggregates from Acaryochloris marina, a Prochloron‐like prokaryote containing mainly chlorophyll d
Authors: Marquardt Jürgen , Senger Horst , Miyashita Hideaki , Miyachi Shigetoh , Mörschel Erhard
Published: July 2002

Phycobiliprotein aggregates were isolated from the prokaryote Acaryochloris marina, containing chlorophyll d as major pigment. In the electron microscope the biliprotein aggregates appear as rod‐shaped structures of 26.0×11.3 nm, composed of four ring‐shaped subunits 5.8 nm thick and 11.7 nm in diameter. Spectral data indicate that the aggregates contain two types of biliproteins: phycocyanin and an allophycocyanin‐type pigment, with very efficient energy transfer from the phycocyanin‐ to allophycocyanin‐type constituent. The chromophore‐binding polypeptides of the pigments have apparent molecular masses of 16.2 and 17.4 kDa. They crossreact with antibodies against phycocyanin and allophycocyanin from a red alga.
Phage display of Bacillus thuringiensis CryIA(a) insecticidal toxin
Authors: Marzari Roberto , Edomi Paolo , Bhatnagar Raj K , Ahmad Suhail , Selvapandiyan Angamuthu , Bradbury Andrew
Published: July 2002

The display of proteins or peptides on the surface of filamentous phages or phagemids has been shown to be a very powerful technology for the rescue of specific binders from large combinatorial libraries, as well as to select derivatives of known proteins with altered binding properties. The Bacillus thuringiensis (Bt) crystal proteins are a large family of insecticidal toxins which bind to receptors found on the brush border of larval midgut cells, different crystal toxins having different larval specificities. Here we describe the display of different CryIA(a) toxin regions on the surface of phagemids using the display vector pHEN1, the purpose being the identification of toxin sequences suitable for mutagenesis and selection using phage display. We show that CryIA(a) domain II, in which the receptor binding activity is located, is efficiently displayed as well as being secreted as soluble protein into the periplasm of bacterial cell. This forms the basis of a simple means for the modification of toxin specificity and the selection of toxin proteins with novel or expanded host ranges.
Brefeldin A interferes with peroxisomal protein sorting in the yeast Hansenula polymorpha
Authors: Salomons Florian A , van der Klei Ida J , Kram Anita M , Harder Wim , Veenhuis Marten
Published: July 2002

We have studied the effect of brefeldin A (BFA), a fungal toxin that interferes with coated vesicle formation, on the biogenesis of peroxisomes in the yeast Hansenula polymorpha. Addition of BFA (20 μg/ml) to cultures of H. polymorpha partially inhibited the development of peroxisomes and resulted in the reversible accumulation of newly synthesized peroxisomal membrane and matrix proteins at the endoplasmic reticulum. In contrast, BFA did not interfere with the selective degradation of peroxisomes. Taken together, our data suggest that the ER plays a crucial role in peroxisome biogenesis in H. polymorpha, possibly in the biosynthesis of the peroxisomal membrane.
Expression of cartilage oligomeric matrix protein by human synovium
Authors: Di Cesare Paul E , Carlson Cathy S , Stollerman Elliot S , Chen Frank S , Leslie Michael , Perris Roberto
Published: July 2002

Human synovium was analyzed for the possible expression of cartilage oligomeric matrix protein (COMP). Immunostaining with polyclonal antiserum to COMP demonstrated positive staining within the synovial cells and immediately subjacent connective tissue, with less intense staining in the deeper connective tissue. Western blot analysis using either polyclonal or monoclonal antibodies to human COMP confirmed the presence of COMP by immunoreactive bands with the same molecular mass (approximately 110 kDa) as purified articular cartilage COMP. PCR using oligonucleotides that span human COMP exons 7–13 revealed identical amplification products from cDNA prepared from either human chondrocytes or synovium. Northern blot analysis using a biotinylated‐probe to human COMP, spanning exons 12–13, also reveal an identical hybridization product to either human chondrocyte or synovium total RNA. Human synovium should be considered as a potential tissue source of COMP in any investigation of biological markers of cartilage metabolism.
Monoclonal antibodies neutralizing the toxin II from Androctonus australis hector scorpion venom: usefulness of a synthetic, non‐toxic analog
Authors: Devaux Christiane , Clot-Faybesse Olivier , Juin Marianick , Mabrouk Kamel , Sabatier Jean-Marc , Rochat Hervé
Published: July 2002

Scorpion venom contains toxins that act on ion channels. Some are responsible for the noxious effects observed when people are stung by scorpions. The study of the neutralization of these molecules and the production of monoclonal antibodies (mAbs) should prove valuable. Toxin II from Androctonus australis hector scorpion (AahII) is one of the most potent toxins and has been well‐characterized and studied. Producing mAbs against such molecules is often difficult due to their toxicity. We used a synthetic, non‐toxic analog, (Abu)8‐AahII, to obtain mAbs which recognize and neutralize the native toxin AahII. Sets of peptides spanning the entire sequence of AahII were assayed to identify the binding sites of the mAbs. The various mAbs recognized only the largest peptides (12–17 residues). They recognized peptides corresponding to different parts of the AahII sequence, suggesting that several regions of the (Abu)8‐AahII sequence mimic AahII epitopes and then elicit mAbs directed against toxin.
Different distribution of phosphorylated tau protein isoforms in Alzheimer's and Pick's diseases
Authors: Sergeant N , David J.-P , Lefranc D , Vermersch P , Wattez A , Delacourte A
Published: July 2002

Tau proteins aggregate into different neuronal inclusions in several neurodegenerative disorders. In Alzheimer's disease (AD), hyperphosphorylated Tau from paired helical filaments (PHF) of neurofibrillary tangles, named PHF‐Tau, have an electrophoretic profile with four main bands (Tau 55, 64, 69, 74 kDa). In Pick's disease, phosphorylated Tau from Pick bodies are made of two major components (Tau 55, 64 kDa) and a minor 69 kDa. Here we show, using specific antibodies against translated exon 2, 3 or 10 of Tau isoforms, that the set of Tau isoforms engaged in the most insoluble part of PHF in AD is made of Tau isoforms with exon 10 while they are lacking in phosphorylated Tau from Pick's disease. Our results suggest that specific sets of Tau isoforms distinguish between typical neuronal inclusions.
Electrospray ionization mass spectrometry analysis of the apo‐ and metal‐substituted forms of the Fur protein
Authors: Michaud-Soret Isabelle , Adrait Annie , Jaquinod Michel , Forest Eric , Touati Danièle , Latour Jean-Marc
Published: July 2002

Fur has been purified and reconstituted with Co2+ and Mn2+. The ESI‐MS spectra of the apoprotein as well as Mn‐Fur and Co‐Fur under acidic denaturating conditions showed the existence of two species of molecular mass 16 660±3 and 16 792±3 Da, which correspond, respectively, to the N‐terminal methionine `excised' or `non‐excised' forms of the monomer. This result proves the absence of any other post‐translational modification or modification due to metal incorporation. On the other hand, under soft conditions, ESI spectra provided for the first time direct evidence for dimeric metal‐containing forms in solution.
Imaging of cAMP‐dependent protein kinase activity in living neural cells using a novel fluorescent substrate
Authors: Higashi Hideyoshi , Sato Kazuki , Ohtake Atsuko , Omori Akira , Yoshida Sachiyo , Kudo Yoshihisa
Published: July 2002

In order to visualize the activity of the cAMP‐dependent protein kinase (PKA) in living cells, we have constructed a new fluorescence PKA substrate by conjugating a fluorescence probe to a partial amino acid sequence of PKA regulatory domain II which contains a specific autophosphorylation site. The fluorescent peptide was cell‐permeable and became phosphorylated when the intracellular cAMP concentration was increased, resulting in a decrease in its fluorescence intensity. In NG108‐15 cells, PKA activity was localized to the cytosol around the nucleus. In cultured hippocampal neurons, addition of l‐glutamate caused PKA activation associated with increase of the cellular cAMP.
Ceramide‐induced translocation of protein kinase C ζ in primary cultures of astrocytes
Authors: Galve-Roperh Ismael , Haro Amador , Dı́az-Laviada Inés
Published: July 2002

The present research was undertaken to study the possible involvement of the atypical protein kinase C (PKC) ζ in ceramide signal transduction in primary cultures of rat astrocytes. As shown by Western blot analysis, translocation of immunoreactive PKCζ to the particulate fraction occurred upon exposure of astrocytes to cell‐permeable ceramide analogs or to exogenous sphingomyelinase. The particulate fraction may correspond to a perinuclear area, as indicated by immunocytochemical techniques. Furthermore, treatment of cells with N‐octanoylsphingosine led to an increased phosphorylation of PKCζ. Results thus show that stimulation of PKCζ may be one of the intracellular events triggered by activation of the sphingomyelin pathway.
Identification of the human Lewisa carbohydrate motif in a secretory peroxidase from a plant cell suspension culture (Vaccinium myrtillus L.)
Authors: Melo Nuno S , Nimtz Manfred , Conradt Harald S , Fevereiro Pedro S , Costa Júlia
Published: July 2002

This paper reports for the first time the presence of the human Lewisa type determinant in glycoproteins secreted by plant cells. A single glycopeptide was identified in the tryptic hydrolysis of the peroxidase VMPxC1 from Vaccinium myrtillus L. by HPLC/ESI‐MS. The oligosaccharide structures were elucidated by ESI‐MS‐MS and by methylation analysis before and after removal of fucose by mild acid hydrolysis. The major structure determined is of the biantennary plant complex type containing the outer chain motif Lewisa

urn:x-wiley:00145793:media:feb2s0014579397011216:feb2s0014579397011216-fx1

A corresponding fucosyltransferase activity catalyzing the formation of Lewisa type structures in vitro was identified in cellular extracts of the suspension cultures.
Cloning of a functional vesicular GABA and glycine transporter by screening of genome databases
Authors: Sagné Corinne , El Mestikawy Salah , Isambert Marie-Françoise , Hamon Michel , Henry Jean-Pierre , Giros Bruno , Gasnier Bruno
Published: July 2002

The unc‐47 locus of Caenorhabditis elegans has been suggested to encode a synaptic vesicle GABA transporter. Here we used hydropathy plot analysis to identify a candidate vesicular GABA transporter in genomic sequences derived from a region of the physical map comprising unc‐47. A mouse homologue was identified and cloned from EST database information. In situ hybridization in rat brain revealed codistribution with both GABAergic and glycinergic neuronal markers. Moreover, expression in COS‐7 and PC12 cells induced an intracellular, glycine‐sensitive GABA uptake activity. These observations, consistent with previous data on GABA and glycine uptake by synaptic vesicles, demonstrate that the mouse clone encodes a vesicular inhibitory amino acid transporter.
Evidence for a novel function of the CD40 ligand as a signalling molecule in T‐lymphocytes
Authors: Brenner Birgit , Koppenhoefer Ursula , Grassmé Heike , Kun Jutta , Lang Florian , Gulbins Erich
Published: July 2002

The interaction of the CD40 receptor with its ligand has been shown to be crucial for the activation of B‐lymphocytes. Here, we provide evidence that the pg39 molecule/CD40 ligand (gp39/CD40L) also functions as a stimulatory molecule for T‐lymphocytes. Activation of T‐lymphocytes via gp39/CD40L induced a strong activation of Jun‐N‐terminal kinase (JNK) and p38‐K. Activation of these kinases correlates with a stimulation of Rac1 and inhibition of Rac1 prevents gp39/CD40L triggered JNK/p38‐K activation. Further, cellular stimulation via the CD40 ligand results in tyrosine phosphorylation of cellular proteins and the activation of p56lck. Inhibition of src‐like kinases inhibits Rac1 as well as JNK/p38‐K stimulation suggesting a signalling cascade from the gp39/CD40L via p56lck and Rac1 to JNK/p38‐K.
Purification and microsequencing of hyaluronidase isozymes from human urine
Authors: Csóka Tony B , Frost Gregory I , Wong Tim , Stern Robert
Published: July 2002

We recently cloned the major hyaluronidase of human plasma, which we termed HYAL1. All hyaluronidase activity could be purified from human urine on an anti‐HYAL1 monoclonal antibody column. However, urine contains two hyaluronidases of 57 kDa and 45 kDa, whereas plasma only contains the 57 kDa activity. Microsequencing confirmed that both urinary isozymes have N‐termini identical to plasma hyaluronidase, but a second N‐terminus was found in the smaller isozyme, apparently derived from the terminal 25 amino acids of HYAL1, at the C‐terminus. The two polypeptides of the 45 kDa isozyme resulting from endoproteolytic cleavage of the 57 kDa isoform are presumably linked by disulfide bonds. Sperm contains two isozymes of the testicular hyaluronidase, PH‐20, and the lower molecular weight isozyme is believed to be an endoproteolytically processed form of the larger protein. Analogously to PH‐20, the smaller isozyme of HYAL1 is likely to be a proteolytically processed product of the larger isozyme.
Upregulation of MMP‐9 expression in MDA‐MB231 tumor cells by platelet granular membrane
Authors: Lindenmeyer Frédérique , Legrand Yves , Menashi Suzanne
Published: July 2002

The interaction between tumor cells and platelets facilitates the formation of metastasis in a way depending on the platelet aggregating ability of the tumor cell, but the mechanism remains to be elucidated. We have shown, by zymography and Western blot, that platelets greatly increased the secretion to the culture medium of MMP‐9 by human mammary tumor cells MDA‐MB231. This increase, which was dependent on protein synthesis, was caused by the platelet aggregates interacting with the tumor cells and not by the soluble factors released during platelet activation. Platelet subcellular fractionation allowed the localization of the inducing factor to the membrane fraction of the platelet granules, thus requiring platelet aggregation in order to become accessible on the platelet surface.
Cloning of rat uncoupling protein‐3 and uncoupling protein‐2 cDNAs: their gene expression in rats fed high‐fat diet
Authors: Matsuda Junichi , Hosoda Kiminori , Itoh Hiroshi , Son Cheol , Doi Kentaro , Tanaka Tokuji , Fukunaga Yasutomo , Inoue Gen , Nishimura Haruo , Yoshimasa Yasunao , Yamori Yukio , Nakao Kazuwa
Published: July 2002

In order to elucidate energy balance in the skeletal muscle, we cloned cDNA of a homologue of uncoupling protein (UCP) from rat skeletal muscle. We also cloned rat UCP‐2 cDNA from rat brown adipose tissue (BAT). The UCP cloned from rat skeletal muscle showed 57% and 72% identity with rat UCP‐1 and UCP‐2. The mRNA was expressed abundantly in the skeletal muscle, moderately in the BAT, and slightly in the white adipose tissue (WAT) with a major band at 2.5 kb and a minor band at 2.8 kb, while the UCP‐2 gene expression was widely detected in the whole body with substantial levels in the WAT and with slight levels in the skeletal muscle and BAT. The rat UCP cloned in the present study showed 86% identity with the recently cloned human UCP‐3, which was also expressed abundantly in the skeletal muscle with a signal of 2.4 kb. Therefore, the rat UCP was considered to be rat UCP‐3. In rats fed high‐fat diet the UCP‐3 gene expression was augmented 2‐fold in the skeletal muscle while UCP‐2 mRNA levels were increased significantly (1.6‐fold) in the epididymal WAT. Augmented expression of UCPs may provide defense against high‐fat induced obesity and impairment of glucose metabolism.
Regulation of eukaryotic initiation factor eIF2B: glycogen synthase kinase‐3 phosphorylates a conserved serine which undergoes dephosphorylation in response to insulin
Authors: Welsh Gavin I , Miller Christa M , Loughlin A.Jane , Price Nigel T
Published: July 2002

Eukaryotic initiation factor eIF2B catalyses a key regulatory step in mRNA translation. eIF2B and total protein synthesis are acutely activated by insulin, and this requires phosphatidylinositol 3‐kinase (PI 3‐kinase). The ϵ‐subunit of eIF2B is phosphorylated by glycogen synthase kinase‐3 (GSK‐3), which is inactivated by insulin in a PI 3‐kinase‐dependent manner. Here we identify the phosphorylation site in eIF2Bϵ as Ser540 and show that treatment of eIF2B with GSK‐3 inhibits its activity. Ser540 is phosphorylated in intact cells and undergoes dephosphorylation in response to insulin. This is blocked by PI 3‐kinase inhibitors. Insulin‐induced dephosphorylation of this inhibitory site in eIF2B seems likely to be important in the overall activation of translation by this hormone.
Carbamyl‐phosphate synthetase domain of the yeast multifunctional protein Ura2 is necessary for aspartate transcarbamylase inhibition by UTP
Authors: Antonelli Richard , Estevez Laurence , Denis-Duphil Michèle
Published: July 2002

In Saccharomyces cerevisiae, the first two reactions of pyrimidine biosynthesis are catalyzed by the multifunctional protein Ura2 carrying both carbamyl‐phosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase) enzyme activities. In order to study how UTP regulates both of these activities mutant strains were constructed: one strain which expressed the Ura2 protein fused to the green fluorescent protein, and two strains expressed truncated Ura2 proteins. These strains exhibited a phenotype associated with a modified regulation of the pyrimidine pathway. Results presented in this report provide arguments in favor of a single UTP binding site located on the CPSase domain, and support a model in which ATCase activity is inhibited by UTP only when it can interact with the CPSase domain.
Recombinant Plasmodium falciparum glutathione reductase is inhibited by the antimalarial dye methylene blue
Authors: Färber P.M , Arscott L.D , Williams C.H , Becker K , Schirmer R.H
Published: July 2002

Plasmodium falciparum glutathione reductase (PfGR) has emerged as a drug target against tropical malaria. Here we report the expression of PfGR in Escherichia coli SG5(DE3) and isolation procedures for this protein. Recombinant PfGR does not differ from the authentic enzyme in its enzymic properties, the turnover number being 9900 min−1. The dimeric flavoenzyme exhibits redox‐dependent absorption spectra; the single tryptophan residue (per 57.2 kDa subunit) is strongly fluorescent. PfGR can be inhibited by the antimalarial drug methylene blue at therapeutic concentrations; the K
i for non‐competitive inhibition is 6.4 μM. The sensitivity to methylene blue is observed also at high ionic strength so that, by analogy to human GR, analysis of crystalline enzyme‐drug complexes can be envisaged.
Functional dissection of a sugar‐repressed α‐amylase gene (RAmy1A) promoter in rice embryos
Authors: Morita Akiyoshi , Umemura Taka-aki , Kuroyanagi Mitsuyo , Futsuhara Yuzo , Perata Pierdomenico , Yamaguchi Junji
Published: July 2002

The gibberellin‐inducible rice α‐amylase gene, RAmy1A, was demonstrated to be sugar repressed in rice embryos and functional dissection of the promoter of RAmy1A in relation of its sugar‐modulated expression was performed. Gibberellin‐response cis‐elements of GARE (TAACAAA) and pyrimidine box (CCTTTT) were partially involved in the sugar repression.