bluuebunny/crossref_metadata_2025 huggingface dataset:
Binding of uteroglobin to microsomes and plasmatic membranes
Authors: González Keybell Díaz , Nieto Antonio
Published: April 2003

Microsomes and plasmatic membranes from rat liver bind radioactive uteroglobin (UG) in vitro with high affinity (K
d = 1.7 × 10−10 M). The binding is saturable and specific and dependent on previous reduction of UG with dithiothreitol. Microsomes from rat spleen or lung or from rabbit endometrium also possess a similar ability. Binding capacity is not affected by previous treatment of microsomes with phospholipase A2 or peptide‐N‐glycosidase F but is lost after brief treatment with trypsin. The complex formed between UG and the binding component can be solubilized from microsomes with 5 mM CHAPS and it elutes with an apparent M
r of 90,000 in a Sephacryl 200 column. The complex is resistant to 8 M urea but is completely dissociated by Triton X‐100. The UG‐binding protein(s) has been partially purified from solubilized microsomes and membranes by affinity chromatography. The results are discussed in relation to a possible physiological effect of UG on cellular membranes.
Similar substrate recognition motifs for mammalian AMP‐activated protein kinase, higher plant HMG‐CoA reductase kinase‐A, yeast SNF1, and mammalian calmodulin‐dependent protein kinase I
Authors: Dale Susan , Wilson Wayne A. , Edelman Arthur M. , Hardie D.Grahame
Published: April 2003

We have analysed phosphorylation of the synthetic peptide AMARAASAAALARRR, and 23 variants, by mammalian, higher plant and yeast members of the SNF1 protein kinase subfamily (AMP‐activated protein kinase (AMPK), HMG‐CoA reductase kinase (HRK‐A), and SNF1 itself), and by mammalian calmodulin‐dependent protein kinase I (CaMKI). These four kinases recognize motifs which are very similar, although distinguishable. Our studies define the following recognition motifs: AMPK: Φ(X,β)XXS/TXXXΦ; HRK‐A: Φ(X,β)XXSXXXΦ; Snf1: ΦXRXXSXXXΦ; CaMKI: ΦXRXXS/TXXXΦ; where Φ is a hydrophobic residue (M, V, L, I or F) and β is a basic residue (R, K or H).
Purification of an NADPH‐dependent diaphorase from membrane of DMSO‐induced differentiated human promyelocytic leukemia HL‐60 cells
Authors: Otsuka-Murakami Hidetsugu , Nisimoto Yukio
Published: April 2003

NADPH diaphorase activity was found in membrane of DMSO‐induced differentiated human promyelocytic leukemia HL‐60 cells. This membrane‐bound diaphorase activity increased dramatically during differentiation of HL‐60 cells. A dye reductase was extracted from membrane of DMSO‐induced differentiated HL‐60 cells with n‐octyl glucoside and sodium cholate in the presence of several protease inhibitors such as PMSF, DIFP, TLCK, antipain, chymostatin, leupeptin, pepstatin A and trypsin inhibitor. The NADPH diaphorase was highly purified by two‐stage sequential column chromatographies. The purified enzyme, showing both SOD‐insensitive cytochrome c and NBT reductase activities, migrated with an apparent molecular mass of 77 kDa on SDS‐PAGE. When the purification of this diaphorase was carried out in the presence of only three protease inhibitors, PMSF, DIFP and TLCK, a partially proteolyzed form of the diaphorase with a molecular mass of 68 kDa was prepared. The proteolyzed diaphorase exhibited only an NADPH‐dependent cytochrome c reductase. The NADPH diaphorase gave a positive cross‐reaction to polyclonal antibodies raised against microsomal NADPH‐cytochrome P450 reductase from rabbit liver.
Molecular and enzymatic characterization of two stilbene synthases from Eastern white pine (Pinus strobus) A single Arg/His difference determines the activity and the pH dependence of the enzymes
Authors: Raiber Sigrid , Schröder Gudrun , Schröder Joachim
Published: April 2003

Pinus strobus (Eastern white pine) contains stilbenes biosynthetically derived from cinnamoyl‐CoA (pinosylvin) or dihydrocinnamoyl‐CoA (dihydropinosylvin). We screened a P. strobus cDNA library with a stilbene synthase (STS) probe from Pinus sylvestris. The eight isolated cDNAs represented two closely related STS genes with five amino acid differences in the proteins. The enzyme properties were investigated after heterologous expression in Escherichia coli. Both proteins preferred cinnamoyl‐CoA against dihydrocinnamoyl‐CoA and thus represented pinosylvin synthases. Otherwise they revealed large differences. STS1 had only 3–5% of the activity of STS2, its pH optimum was shifted to lower values (pH 6), and it synthesized with cinnamoyl‐CoA a second unknown product. Site‐directed mutagenesis demonstrated that a single Arg‐to‐His exchange in STS1 was responsible for all of the differences. The proton acceptor properties of His are discussed as the reason for the properties of STS1.
The endozepine ODN stimulates polyphosphoinositide metabolism in rat astrocytes
Authors: Patte C. , Vaudry H. , Desrues L. , Gandolfo P. , Strijdveen I. , Lamacz M. , Tonon M.C.
Published: April 2003

Astrocytes synthesize a series of peptides called endozepines which act as endogenous ligands of benzodiazepine receptors. The present study demonstrates that the endozepine ODN causes a dose‐dependent increase in inositol trisphosphate and a parallel decrease in phosphatidylinositol bisphosphate in cultured rat astrocytes. Pre‐incubation of astrocytes with the phospholipase C inhibitor U 73122 or with pertussis toxin totally blocked polyphosphoinositide metabolism. These data show that, in rat astrocytes, ODN stimulates a phospholipase C coupled to a pertussis toxin‐sensitive G protein.
In vitro binding of Bacillus thuringiensis var. israelensis individual toxins to midgut cells of Anopheles gambiae larvae (Diptera: Culicidae)
Authors: Ravoahangimalala Olga , Charles Jean-François
Published: April 2003

Midguts from Anopheles gambiae fourth instars were dissected and processed for immuno‐light microscopy. Cloned insecticidal crystal proteins (ICPs) from Bacillus thuringiensis var. israelensis (Bti) were individually expressed in crystal‐negative strains of Bacillus thuringiensis. Tissue sections of A. gambiae were incubated in vitro with each solubilized and trypsin‐activated ICP. Immunodetection of CryIVA, CryIVB, CryIVD and CytA toxins on sections was performed using purified rabbit IgG directed against BtiICPs, in combination with an anti‐rabbit IgG/peroxidase. CryIVA, CryIVB, CryIVD and CytA toxins were detected on the apical brush border of midgut cells, in the gastric caecae and posterior stomach. CytA was also detected, to a lesser extent, on microvilli of anterior stomach cells.
Differential inhibition of cytosolic and membrane‐derived protein kinase C activity by staurosporine and other kinase inhibitors
Authors: Budworth Joanna , Gescher Andreas
Published: April 2003

The hypothesis was tested that 9 kinase inhibitors with diverse specificities for protein kinase C (PKC), including staurosporine and four of its analogues, interfere differently with PKC derived from either the cytosolic or particulate fractions of MCF‐7 breast carcinoma cells. GF 109203X inhibited the enzyme identically in either preparation. CGP 41251 and calphostin C inhibited cytosolic PKC more effectively than membrane‐derived PKC with ratios of IC50 (cytosolic PKC) over IC50 (membrane‐derived PKC) of 0.07 and 0.04, respectively. The other six agents inhibited membrane‐derived PKC more potently than cytosolic enzyme. Staurosporine and RO 31 8220 exhibited IC50 ratios of 12.3 and 21.6, respectively. The results suggest that there are dramatic differences between kinase inhibitors in their divergent effects on cytosolic and membrane‐derived PKC which should be borne in mind in the interpretation of their pharmacological properties.
Sucrose: a solute that accumulates in the guard‐cell apoplast and guard‐cell symplast of open stomata
Authors: Lu Ping , Zhang Shu Qiu , Outlaw William H. , Riddle Kimberly A.
Published: April 2003

Stomatal conductances of Vicia faba leaves were recorded over a day. Coordinately, (a) guard cells dissected from leaflets were assayed, providing total sucrose content, and (b) guard cells dissected from rinsed epidermes were also assayed, providing symplastic sucrose content. Compared with that of pre‐dawn samples, apoplastic sucrose content increased 4.8 – 5.2 × (2 experiments), reaching 1,130 – 1,300 fmol·guard‐cell‐pair−1 at midday, when conductance was highest (ca. 0.13 mol·m−2·s−1); symplastic sucrose content increased 2.5 – 3.5 ×, reaching 350 – 390 fmol·guard‐cell‐pair−1. Thus, there is a correlation between transpiration and guard‐cell sucrose content, particularly that portion localized to the apoplast. Moreover, apoplastic sucrose is apparently a source of guard‐cell nutrition and, possibly, osmoticum.
Observation of the pre‐steady state process in thermolysin catalysis with a fluorescent displacement probe at low pH
Authors: Kunugi Shigeru , Yokoyama Masahito , Sakamoto Akira
Published: April 2003

The pre‐steady state process in the thermolysin‐catalyzed hydrolysis of Cbz‐Gly‐Phe‐Ala was observed at pH 4.5 by fluorescence stopped‐flow method using Dns‐Phe as a displacement probe. After the confirmation of the pre‐equilibrium hypothesis for the binary interaction, the nonlinear substrate concentration dependence of the apparent kinetic constant for the pre‐steady state process was analyzed and an existence of multi‐intermediates was proposed.
The essential yeast NLT1 gene encodes the 64 kDa glycoprotein subunit of the oligosaccharyl transferase
Authors: Pathak Rahul , Parker Carl S. , Imperiali Barbara
Published: April 2003

The yeast oligosaccharyl transferase catalyzes the glycosylation of asparagine residues in secreted, vesicular, and membrane proteins. A complex of at least four membrane‐bound polypeptides is responsible for oligosaccharyl transferase activity. Amino acid sequences from the 64 kDa glycoprotein subunit of the complex were used to clone the essential NLT1 (N‐linked oligosaccharyl transferase) gene. The Nlt1p gene product is a processed, multiply glycosylated type I membrane protein; it has an extensive amino‐terminal soluble domain, a potential hydrophobic transmembrane domain, and a short carboxy‐terminal soluble domain. The Nlt1p is significantly similar than the mammalian ribophorin I, a component of the mammalian oligosaccharyl transferase complex, and the enzyme is conserved throughout eukaryotic evolution.
Isolation and complete amino acid sequence of the β‐ and α‐polypeptides from the peripheral light‐harvesting pigment‐protein complex II of Rhodobacter sulfidophilus
Authors: Tadros Monier H. , Hagemann Gesine E. , Katsiou Eleni , Dierstein Roland , Schiltz Emile
Published: April 2003

The peripheral light‐harvesting bacteriochlorophyll‐carotenoid‐protein complex B800‐850 (LHII) has been isolated from membranes of semi‐aerobic dark‐grown cells of Rhodobacter sulfidophilus strain W4. A reversed‐phase HPLC system resolved one β‐ and one α‐polypeptide in the ratio 1:1. The material obtained was of high purity and suitable for direct microsequence analysis. The primary structures of the β‐ and α‐polypeptides have been determined. The β‐polypeptide consists of 51 amino acid residues, yielding a molecular mass of 5512 Da and having 64.7% hydrophobicity. The α‐polypeptide consists of 52 amino acid residues, with a calculated molecular mass of 5661 Da and 75% hydrophobicity. The significance of uncommon structure motives with respect to the unusual spectroscopic characteristics of this light‐harvesting complex is discussed.
Direct electrochemistry and EPR spectroscopy of spinach ferredoxin mutants with modified electron transfer properties
Authors: Aliverti Alessandro , Hagen Wilfred R. , Zanetti Giuliana
Published: April 2003

Mutations of the conserved residue Glu‐92 to lysine, glutamine, and alanine have been performed in the recombinant ferredoxin I of spinach leaves. The purified ferredoxin mutants were found twice as active with respect to wild‐type protein in the NADPH‐cytochrome c reductase reaction catalyzed by ferredoxin‐NADP+ reductase in the presence of ferredoxin. Cyclic voltammetry and EPR measurements showed that the mutations cause a change in the [2Fe‐2S] cluster geometry, whose redox potential becomes approximately 80 mV less negative. These data point to a role of the Glu‐92 side‐chain in determining the low redox potential typical of the [2Fe‐2S] cluster of chloroplast and cyanobacterial ferredoxins. Also a ferredoxin/ferredoxin‐NADP+ reductase chimeric protein obtained by gene fusion was overproduced in Escherichia coli and purified. Fusion of the ferredoxin with its reductase causes only minor effects to the iron‐sulfur cluster, as judged by cyclic voltammetry and EPR measurements.
Two‐dimensional gel mapping of the processing of the human amyloid precursor protein in rat hippocampal neurons
Authors: Simons Mikael , Tienari Pentti J. , Dotti Carlos G. , Beyreuther Konrad
Published: April 2003

The proteolytic fragments derived from the amyloid precursor protein (APP) in primary cultures of rat hippocampal neurons were analyzed by two‐dimensional gel electrophoresis. The Semliki Forest Virus expression vector was used to express human APP695 and a mutant form associated with familial Alzheimer's disease (APP‐FAD670/671). Hippocampal neurons expressing wtAPP695 or APP‐FAD670/671 secrete at least six APP fragments of 100–110 kDa with isoelectric focusing points ranging from 4.5 to 4.0. The heterogeneity of the secreted APP forms is shown to be in part due to differences in glycosylation. In contrast to wtAPP695, neurons producing the APP‐FAD670/ 671 variant did not secrete detectable amounts of secretory APP derived from cleavage within the amyloid βA4 domain. This result suggests that there is little α‐secretase cleavage in neurons expressing the APP‐FAD670/671 mutant.
Excitation delocalization over the whole core antenna of photosynthetic purple bacteria evidenced by non‐linear pump‐probe spectroscopy
Authors: Novoderezhkin Vladimir I. , Razjivin Andrei P.
Published: April 2003

Anomalously high values of photoinduced absorption changes were revealed in the antenna of photosynthetic purple bacteria. They were found to be 4–16 times greater at the bleaching peak of the antenna than at the bleaching peak of the BChl dimer of the reaction center. This is direct proof of excitation delocalization over many pigment molecules. Calculations according to the model of exciton delocalization over all core antenna BChls allow one to explain the observed phenomenon.
Extraordinary enhancement of the cleavage activity of a DNA‐armed hammerhead ribozyme at elevated concentrations of Mg2+ ions
Authors: Shimayama Takashi , Nishikawa Satoshi , Taira Kazunari
Published: April 2003

As part of an ongoing effort to characterize structure‐function relationships, activities of all‐RNA and DNA‐armed hammerhead ribozymes were examined. An analysis of the dependence on the concentration of Mg2+ ions of cleavage rates revealed that, whereas the k
cat of the reaction catalyzed by the all‐RNA ribozyme reached a maximum value of about 18 min−1 at a concentration of about 200 mM Mg2+ ions, that of the DNA‐armed ribozyme increased linearly as the concentration of Mg2+ ions was increased above 300 mM, finally reaching a value of more than 100 min−1 at 700 mM Mg2+ ions. These results suggest that the potential activity of a hammerhead ribozyme might be greater than is usually recognized.
Recognition of 2′‐hydroxyl groups by Escherichia coli ribonuclease HI
Authors: Iwai Shigenori , Kataoka Shin , Wakasa Makoto , Ohtsuka Eiko , Nakamura Haruki
Published: April 2003

In order to investigate the hydrogen‐bonding interactions between Escherichia coli ribonuclease HI and the 2′‐hydroxyl functions of the substrate, oligonucleotide duplexes containing 2′‐amino‐2′‐deoxyuridine or 2′‐fluoro‐2′‐deoxyuridine at a specific site were used, and their affinities for the enzyme were determined by kinetic analyses. The results indicate that the hydroxyl groups of the nucleoside 3′‐adjacent to the cleaved phosphodiester linkage and the second nucleoside 5′ to the cleaved phosphodiester act as both a proton donor and an acceptor and as a proton acceptor, respectively, in the enzyme‐substrate complex. A molecular model was constructed using the interactions derived from the results.
Characterization of the interaction of Raf‐1 with ras p21 or 14‐3‐3 protein in intact cells
Authors: Koyama Shinya , Williams Lewis T. , Kikuchi Akira
Published: April 2003

Several deletion mutants of Raf‐1 were expressed with v‐ras p21 or 14‐3‐3 protein in COS‐7 cells and Sf9 cells and the interaction of Raf‐1 with ras p21 or with 14‐3‐3 protein in intact cells was examined. Raf(1–135) (residues 1–135) and Raf(1–322) interacted with v‐ras p21, but other deletion mutants such as Raf(136–322) or Raf(321–648) did not. Raf(1–322) interacted with 14‐3‐3 protein much more efficiently than Raf(321–648) did. While Raf(1–135) did not interact with 14‐3‐3 protein, Raf(136–322) did. These results clearly indicate that Raf‐1 simultaneously interacts with both ras p21 and 14‐3‐3 protein through the distinct binding domains in intact cells.
A role for active oxygen species as second messengers in the induction of alternative oxidase gene expression in Petunia hybrida cells
Authors: Wagner Anneke M.
Published: April 2003

Incubation of Petunia hybrida cells with H2O2 leads to an increase in alternative oxidase activity measured after 24 h. This increased activity is accompanied by an increase in alternative oxidase protein. A model is presented for the regulation of alternative oxidase protein synthesis in which active oxygen species and especially H2O2 play a crucial role as second messengers in the signal transducing pathway from the mitochondria to the nucleus. It is proposed that also the induction of the alternative oxidase by salicylic acid is mediated via H2O2.
Induction of p53 and apoptosis by δ12‐PGJ2 in human hepatocarcinoma SK‐HEP‐1 cells
Authors: Lee Jeong-Hwa , Kim Ho-Shik , Jeong Seong-Yun , Kim In-Kyung
Published: April 2003

Human hepatocarcinoma cells (SK‐HEP‐1) were induced to die through apoptosis by treatment with Δ
12‐prostaglandin (PG)J2, as characterized by the appearance of a typical DNA ladder. The induction of apoptosis by Δ
12‐PGJ2 was specifically blocked by cycloheximide (CHX). Western analysis using anti‐p53 or anti‐WAF1 monoclonal antibodies demonstrated that these two protein levels were increased 3 h after Δ
12‐PGJ2 treatment, and accumulated for up to 12 h. The induction of p53 protein seemed to be dependent on the increase of p53 mRNA level, which was inhibited by CHX treatment. However, delayed addition of CHX after Δ
12‐PGJ2 treatment failed to affect both p53 mRNA levels and DNA fragmentation following Δ
12‐PGJ2 treatment, indicating that the inhibition of p53 synthesis may contribute to the protective effect of CHX against Δ
12‐PGJ2‐mediated cytotoxicity. Therefore, our results suggest that the initial events caused by Δ
12‐PGJ2, leading ultimately to SK‐HEP‐1 cell death, involve a certain process required for p53 induction. However, the finding that Δ
12‐PGJ2 is also active against Hep 3B cells which are devoid of a functional p53 indicates that p53 may not be the critical requirement for inducing apoptosis by Δ
12‐PGJ2.
The Na+‐translocating NADH: ubiquinone oxidoreductase from the marine bacterium Vibrio alginolyticus contains FAD but not FMN
Authors: Pfenninger-Li Xiao Dan , Dimroth Peter
Published: April 2003

The Na+‐translocating NADH: ubiquinone oxidoreductase from Vibrio alginolyticus was extracted from the bacterial membranes and purified by ion exchange chromatographic procedures. The enzyme catalyzed NADH oxidation by suitable electron acceptors, e.g. menadione, and the Na+ and NADH‐dependent reduction of ubiquinone‐1. Four dominant bands and a number of minor bands were visible on SDS‐PAGE that could be part of the enzyme complex. Flavin analyses indicated the presence of FAD but no FMN in the purified enzyme. FAD but no FMN were also present in V. alginolyticus membranes. FAD is therefore a prosthetic group of the Na+‐translocating NADH:ubiquinone oxidoreductase and FMN is not present in the enzyme. The FAD was copurified with the NADH dehydrogenase. The purified enzyme exhibited an absorption spectrum with a maximum at 450 nm that is typical for a flavoprotein. Upon incubation with NADH this absorption disappeared indicating reduction of the enzyme‐bound FAD.
Isolation and characterization of a yeast gene, MPD1, the overexpression of which suppresses inviability caused by protein disulfide isomerase depletion
Authors: Tachikawa Hiroyuki , Takeuchi Yutaka , Funahashi Wataru , Miura Tadashi , Gao Xiao-Dong , Fujimoto Daisaburo , Mizunaga Takemitsu , Onodera Kazukiyo
Published: April 2003

MPD1, a yeast gene the overexpression of which suppresses the inviability caused by the loss of protein disulfide isomerase (PDI) was isolated and characterized. The MPD1 gene product retained a single disulfide isomerase active site sequence (APWCGHCK), an N‐terminal putative signal sequence, and a C‐terminal endoplasmic reticulum (ER) retention signal, and was a novel member of the PDI family. The gene product, identified in yeast extract, contained core size carbohydrates. MPD1 was not essential for growth, but overexpression of the gene suppressed the maturation defect of carboxypeptidase Y caused by PDI1 deletion, indicative of the related function to PDI in the yeast ER.
Selective inhibition of gastrulation in the starfish embryo by albuside B, an inosine analogue
Authors: Shimizu Takahiko , Hamada Keiichi , Isomura Haruhiko , Myotoishi Yuki , Ikegami Susumu , Kaneko Hiroyuki , Dan-Sohkawa Marina
Published: April 2003

External application of 0.2–100 μg/ml albuside B inhibits gastrulation of the starfish (Asterina pectinifera) embryo. Treated embryos retain the late blastula morphology with the vegetal plate. However, the vegetal plate is unreactive to soybean agglutinin, a probe for observing the progenitor cells of the archenteron (mesendoderm) in a normal embryo. The effective period of the treatment is limited from 4 to 6 h after fertilization, a period immediately before the onset of blastulation. RNA synthesis is unaffected during the period of sensitivity. The selectivity of the inhibition shows that albuside B may be a useful tool for studying the mechanisms of mesendoderm differentiation.
Degradation of vitronectin by matrix metalloproteinases‐1, ‐2, ‐3, ‐7 and ‐9
Authors: Imai Kazushi , Shikata Hideo , Okada Yasunori
Published: April 2003

The susceptibility of vitronectin (Vn) purified from human plasma to digestion by matrix metalloproteinases (MMPs) was examined. MMP‐2, ‐3, ‐7 and ‐9 except for MMP‐1 degraded Vn into multiple fragments. MMP‐7 showed the highest activity to the substrate among these MMPs, digesting 8‐, 30‐ and 44‐fold more preferentially than MMP‐2, ‐3 and ‐9, respectively. These data suggest that MMP‐2, ‐3, ‐7 and ‐9 may be responsible for the pathological degradation and/or normal turnover of Vn.
A distal Sp 1‐element is necessary for maximal activity of the human gastrin gene promoter
Authors: R. Bundgaard Jens , Hansen Thomas v.O. , Friis-Hansen Lennart , Rourke Ian J. , van Solinge Wouter W. , Nielsen Finn C. , Rehfeld Jens F.
Published: April 2003

Studies of transgenic mice have shown that transcriptional control of the gastrin gene exhibits significant species differences. Transfection of the human gastrin promoter in murine cells have depicted proximal Sp1, E‐box and CACC elements as the major determinants of transcription. We have examined cis‐regulatory elements of the human promoter in a human gastrin expressing cell line and find that a distal −135 to −142 Sp1 element is necessary for maximal activity. Alignment of the mouse and human promoters shows that the proximal human Sp1 and CACC elements are not conserved, whereas the E‐box element is retained. The distal Spl element is present in mouse but exhibits a C to T transition in the core that is likely to reduce binding affinity of Sp1. We conclude that gastrin gene transcription is regulated by distinct elements in man and rodents.
Selective disulphide linkage of plant thionins with other proteins
Authors: Piñeiro Manuel , Diaz Isabel , Rodriguez-Palenzuela Pablo , Titarenko Elena , Garcia-Olmedo Francisco
Published: April 2003

Thionins are shown to form disulphide linkages with other proteins. The reaction with bacterial enzymes β‐glucuronidase and neomycin phosphotransferase II could be prevented and reversed with dithiothreitol and blocked with N‐ethylmaleimide. Other cysteine‐rich low‐molecular‐weight toxic peptides from plants (LTP‐3 from barley and P19 from potato) did not react as the thionins. Certain systeine‐containing proteins, such bovine serum albumin, ovalbumin and cytochrome c, reacted with thionins, while others, including carbonic anhydrase, soybean trypsin inhibitor, bovine‐lung trypsin inhibitor and phosphorylase B did not. Selectivity of the reaction with a periplasmic component of the phytopathogenic bacterium Pseudomonas solanacearum was also shown.
The control of sialyltransferase activity in tumor‐cell lines derived from different tissues is multifactorial
Authors: Coughlan Christine M. , Breen Kieran C.
Published: April 2003

The activities of the sialyltransferase enzymes and the resulting expression of sialoglycoproteins were examined in tumor cells derived from different tissues in order to gain a greater understanding of the factors controlling the cell glycosylation state. Cell‐cell contact, which is dependent on cell confluency state, was shown to influence glycosylation in the neurally‐derived mouse neuro‐2A neuroblastoma and the C6 glioma cell lines. Both showed a relatively high level of cell sialyltransferase activity under sub‐confluent conditions with activity decreasing upon the formation of cell‐cell contacts associated with confluency. A parallel decrease in the expression of sialoglycoproteins, as determined by lectin blot analysis, was also observed under these conditions. In contrast, the H411e hepatoma cell line showed an increase in enzyme activity with confluency with the susceptibility of the enzyme in this cell line to glucocorticoid induction only being detected in sub‐confluent cell cultures. The number of trypsinisation cycles of the cells was also shown to affect the enzyme activity of the neuro‐2A and C6 cells with an increase in enzyme activity coincident with passage number being observed in the neuro‐2A cells, and a decrease in the C6 glioma cell line. Trypsinisation had no effect on enzyme activity in the H411e cells. These results demonstrate that the control of sialyltransferase activity in tumor cells is multifactorial with the tissue of origin playing a key role.
Nitric oxide regulates mitochondrial respiration and cell functions by inhibiting cytochrome oxidase
Authors: Brown Guy C.
Published: April 2003

Nitric oxide (NO) reversibly inhibits mitochondrial respiration by competing with oxygen at cytochrome oxidase. Concentrations of NO measured in a range of biological systems are similar to those shown to inhibit cytochrome oxidase and mitochondrial respiration. Inhibition of NO synthesis results in a stimulation of respiration in a number of systems. It is proposed that NO exerts some of its main physiological and pathological effects on cell functions by inhibiting cytochrome oxidase. Further NO may be a physiological regulator of the affinity of mitochondrial respiration for oxygen, enabling mitochondria to act as sensors of oxygen over the physiological range.
1H NMR spectroscopy reveals that mouse Hsp25 has a flexible C‐terminal extension of 18 amino acids
Authors: Carver John A. , Esposito Gennaro , Schwedersky Gabriele , Gaestel Matthias
Published: April 2003

The small heat‐shock proteins (Hsps) exist as large aggregates and function by interacting and stabilising non‐native proteins in a chaperone‐like manner. Two‐dimensional 1H NMR spectroscopy of mouse Hsp25 reveals that the last 18 amino acids have great flexibility with motion that is essentially independent of the domain core of the protein. The lens protein, α‐crystallin, is homologous to Hsp25 and its two subunits also have flexible C‐terminal extensions. The flexible region in Hsp25 encompasses exactly that expected from sequence comparison with α‐crystallin implying that both proteins have similar structures and that the C‐terminal extensions could be of functional importance for both proteins.
The features of the spatial structure of the gramicidine A‐cesium complex
Authors: Nekrasov Alexei N. , Stepanov Anatoly V. , Timofeev Vladimir P.
Published: April 2003

Earlier obtained two‐dimensional 1H‐NMR spectroscopy data were used to analyze the spatial structure and conformational mobility of the double right ↑↓ ππ

LD

7.2 helix of the complex formed by gramicidine A and Cs+ ions in an organic solvent (a chloroform‐methanol mixture). Analysis of the experimental data permitted the determination of a set of conformations for each of the high‐mobility residue side chains in the solution. The energy refinement of the most probable conformation of the double right ↑↓ ππ

LD

7.2 helix was made and conformational rearrangements of the tryptophan residue side chain were studied in detail.
A novel β1 integrin isoform produced by alternative splicing: unique expression in cardiac and skeletal muscle
Authors: van der Flier Arjan , Kuikman Ingrid , Baudoin Christian , van der Neut Ronald , Sonnenberg Arnoud
Published: April 2003

The mRNA's of several integrin subunits are alternatively spliced in the region encoding cytoplasmic domains, that may potentially provide alternative integrin‐cytoskeleton interactions and transmembrane signaling pathways. We identified a novel cytoplasmic tail variant of the human β1 subunit by reverse transcriptase polymerase chain reaction. This fourth β1 variant, named β1D, is specific for skeletal and cardica muscle. The determined genomic organization of the 3′‐region of the human β1 gene reveals that β1D is produced by alternative splicing of mRNA. In addition, we show that the expression of β1D is developmentally regulated during murine myoblast differentiation, suggesting a role for β1D in myogenesis.
Poliovirus 2Apro expression inhibits growth of yeast cells
Authors: Barco Angel , Carrasco Luis
Published: April 2003

Poliovirus encodes two proteases, 2Apro and 3Cpro that participate in the processing of the viral polyprotein and cleave a number of host proteins. Both proteases have been cloned and expressed in an inducible manner in Saccharomyces cerevisiae cells. The expression of 2Apro, but not 3Cpro, was highly toxic for yeast cells such that growth was arrested after 5 h of induction and cell survival sharply declined. Cellular morphology was profoundly modified by expression of poliovirus 2Apro, in such a way that electron dense granules and autophagosomic bodies arise in the cytoplasm. Experiments aimed at defining the yeast function affected by 2Apro suggested that translation was not the target of protease toxicity, but showed that RNA synthesis was profoundly blocked.
Microtubule associated protein 1B (MAP1B) promotes efficient tubulin polymerisation in vitro
Authors: Pedrotti Barbara , Islam Khalid
Published: April 2003

The effect of MAP1B on tubulin polymerisation has been examined in reconstitution experiments using purified tubulin and MAP1B. Under the assembly conditions used, tubulin alone was incapable of polymerising, but addition of MAP1B resulted in rapid assembly into microtubules. The kinetics of MAP1B‐promoted microtubule assembly examined using pseudo‐first‐order plots show that assembly is described by a single reaction rate. The calculated association rate constant for MAP1B was about 200 × 106M−1·s−1 and this constant was one order of magnitude higher when compared with that for MAP2‐promoted assembly.
Effect of enzymatic desialylation of human serum amyloid P component on surface exposure of laser photo CIDNP (chemically induced dynamic nuclear polarization) ‐ reactive histidine, tryptophan and tyrosine residues
Authors: Siebert Hans-Christian , André Sabine , Reuter Gerd , Gabius Hans-Joachim , Kaptein Robert , Vliegenthart Johannes F.G.
Published: April 2003

The human pentraxin serum amyloid P component (SAP) exhibits no microheterogeneity in its complex di‐antennary glycan. To elucidate whether the removal of sialic acids from this glycoprotein might affect the accessibility of certain amino acid residues of the protein we employed the laser photo CIDNP approach as a sensitive tool. The CIDNP effect is created by the interaction of a photoexcited dye with reactive amino acids and results in enhanced absorption‐ or emission‐signals which can be observed for the three aromatic amino acids histidine, tryptophan, and tyrosine if they are accessible to the dye. Therefore, this technique can be applied to explore surface exposure of these residues. The respective spectra of SAP and enzymatically desialylated SAP were determined. Six tryptophan/histidine signals and one tyrosine signal are present in the aromatic part of the CIDNP difference spectrum of SAP. The corresponding spectrum of desialylated SAP shows remarkable alterations. The chemical shift of one tryptophan/histidine‐characteristic signal is decreased by 0.1 ppm. One tryptophan/histidine signal disappeared and a new one was formed in the CIDNP difference spectrum of desialylated SAP, while the other signals were unaffected. The tyrosine signal has a clearly enhanced intensity in desialylated SAP. Therefore, the removal of sialic acid moieties from the single N‐glycan of each monomer apparently affects surface presentation of distinct CIDNP‐reactive amino acids of SAP.
Expression of the nir and nor genes for denitrification of Pseudomonas aeruginosa requires a novel CRP/FNR‐related transcriptional regulator, DNR, in addition to ANR
Authors: Arai Hiroyuki , Igarashi Yasuo , Kodama Tohru
Published: April 2003

A gene, designated dnr, was identified in the vicinity of the structural genes for nitrite reductase (nirS) and nitric oxide reductase (norCB), and the gene for activation of the reductases (nirQ) from Pseudomonas aeruginosa. It encodes a protein of 227 amino acids homologous with the CRP/FNR‐family transcriptional regulators. Promoter activities for nirS, nirQ and norCB were considerably reduced in the dnr mutant as well as in the mutant of anr, the other fnr‐like regulatory gene from P. aeruginosa. This is the first finding that two CRP/FNR‐related regulators are involved in denitrification in one strain.
A novel approach to the design of potent bioactive peptides by incorporation of proline brackets: antiplatelet effects of Arg‐Gly‐Asp peptides
Authors: Manjunatha Kini R. , Evans Herbert J.
Published: April 2003

Enhancing the potency of peptides is a critical and important step in the development of peptide drugs. We have proposed that proline residues flanking protein‐protein interaction sites perform a structural role in enhancing their interaction [R.M. Kini and H.J. Evans, Biochem. Biophys. Res. Commun. 212 (1995) 1115–1124]. To test this theory, we incorporated proline residues on either or both sides of the interaction site of an antiplatelet peptide, IARGDMNA and determined the inhibitory potency of the peptides in whole blood aggregation. Inclusion of one proline residue, on either the amino or carboxy terminal side of the interaction site, enhances the antiplatelet activity to approximately the same extent (1.5‐ to 2.5‐fold). Incorporation of proline residues on both sides enhances the activity by 7‐ to 13‐fold. This enhancement of the biological activity of the peptide is probably due to a reduction in the number of possible conformations of the peptide, without introducing the rigidity that would accompany cyclization. Incorporation of proline brackets thus provides a novel approach to the design and development of more potent peptide drugs and ligands.
Resistance of Ehrlich tumor cells to apoptosis can be due to accumulation of heat shock proteins
Authors: Gabai Vladimir L. , Zamulaeva Irina V. , Mosin Alexei F. , Makarova Yulia M. , Mosina Vera A. , Budagova Karina R. , Malutina Yana V. , Kabakov Alexander E.
Published: April 2003

Previously we have found that stationary Ehrlich ascites carcinoma (EAC) cells in vivo accumulated heat shock proteins (HSPs) and became resistant to necrotic heath induced by prolonged energy deprivation of hyperthermia. Here we report that apoptotic death induced by nutrient starvation, transient ATP depletion, heat shock and a microtubule‐disrupting drug, vinblastine, was also suppressed in stationary EAC cells comparing with exponential cells. When exponential (sensitive) cells were subjected to short‐term heating with recovery to accumulate inducible form of HSP70, they also became resistant to all of the employed apoptosis‐inducing exposures, and an inhibitor of cytosolic protein synthesis, cycloheximide, prevented acquisition of the resistance. It is suggested that in vivo accumulation of HSPs in stationary tumor cells can be endogenous protective device against apoptotic death induced by starvation or some anticancer treatments.
A novel chemoenzymatic glycosylation strategy: application to lysozyme modification
Authors: Longo María Asunción , Combes Didier
Published: April 2003

Hen egg lysozyme has been non‐specifically glycosylated using a novel two‐step strategy. First, a number of sucrose molecules have been chemically bound to the protein surface lysines, then the glycosidic chains have been enzymically lengthened, using a glycosyltransferase. For this task, a fructosyltransferase and a levansucrase have been tested, the latter appearing as the most effective one. In all cases, reactions have been optimised and several degrees of modification have been obtained. Finally, the effects of the modifications on lysozyme hydrophobicity, hydrolytic activity, hydrolysis substrate affinity and thermostability have been assessed.
Relationship between muscle myosin isoforms and contractile features in rabbit fast‐twitch denervated muscle
Authors: d'Albis Anne , Couteaux René , Goubel Francis , Janmot Chantal , Mira Jean-Claude
Published: April 2003

The effects of 8‐day‐old rabbit fast‐twitch gastrocnemius denervation on the type of myosin isoforms and on contractile features (maximum velocity V
max and contraction time (CT) of the muscle were followed between 15 and 60 days postnatal. The myosin isoforms and the V
max and CT values of the denervated gastrocnemius displayed large changes during this period. These changes, which led at 2 months postnatal to a muscle displaying the properties of a slow‐twitch muscle did not occur in synchrony: complete conversion to slow‐type myosin isoforms occurred only at 60 days postnatal, whereas complete conversion to slow‐twitch V
max and CT values occurred as soon as 35 days postnatal. The results address a new question concerning the relationship between muscle myosin and contractile features.
Molecular cloning and characterization of a novel truncated form (ClC‐2β) of ClC‐2α (ClC‐2G) in rabbit heart
Authors: Furukawa Tetsushi , Horikawa Saburo , Terai Tomoko , Ogura Takehiko , Katayama Yoshifumi , Hiraoka Masayasu
Published: April 2003

Two cDNAs encoding rabbit heart Cl− channels (rabClC‐2β and rabClC‐2α) were isolated by a PCR cloning strategy. RabClC‐2β is a novel cDNA consisting of 2998 bp and encoding the 822‐amino acid protein, while rabClC‐2α is identical to previously reported ClC‐2G. RabClC‐2β is 68 amino acids truncated from NH2‐terminus of rabClC‐2α, but all 13 putative hydrophobic domains are conserved in rabClC‐2β. Although rabClC‐2α was suggested to be activated by extracellular hypotonicity, expression of rabClC‐2β in Xenopus oocytes induced large Cl− currents even in the absence of extracellular hypotonicity. Induction of external hypotonicity did not further increase the amplitude of membrane currents. On the other hand, as similar to rabClC‐2α, rabClC‐2β current was augmented by PKA activation. Thus, different RNA processing of the same gene appears to provide two highly homologous PKA‐activated Cl− channels with or without responsiveness to cell swelling in rabbit heart.
Cellular distribution of protein kinase C isozymes in CD3‐mediated stimulation of human T lymphocytes with aging
Authors: Fulop Tamàs , Leblanc Chantale , Lacombe Guy , Dupuis Gilles
Published: April 2003

Protein kinase C (PKC) is involved in a variety of cellular responses, such as the expression and secretion of IL‐2, the regulation of cytotoxic killing and cell proliferation. It is known that these immune functions are altered with aging. Here, we show that anti‐CD3‐triggered T cell proliferation is significantly decreased with aging and that H7, an inhibitor of PKC, impairs the anti‐CD3‐induced T cell proliferation in a differential manner, lymphocytes of healthy young subjects being more sensitive to the PKC inhibitor than those of elderly subjects. We examined (Western blot) the presence and the cellular distribution of PKC isozymes in T lymphocytes of healthy young and elderly subjects in the resting state and after anti‐CD3 mAb stimulation using antibodies directed against PKC α, β, δ, ϵ and ζ isoforms in the cytosol and the plasma membrane fractions. These five PKC isotypes were present in human T cells of young and elderly subjects. However, their distribution between the cytosolic and membrane fractions varied according to the isozymes and the age of the subjects. In resting lymphocytes of young subjects, all the PKC isozymes were found in the cytosolic fraction, except PKC‐ζ. In resting lymphocytes of elderly subjects PKC‐δ and ‐ϵ were almost equally distributed between the cytosolic and the membrane fractions, whereas PKC‐α and ‐ζ were mainly found in the membrane fraction and PKC‐β was almost exclusively located in the cytosolic fraction. The translocation of PKC‐α, ‐β, ‐δ and ‐ϵ could be observed under anti‐CD3 mAb stimulation in lymphocytes of young subjects, while in the case of elderly subjects only the PKC β isoform was translocated. Our results suggest that the decreased availability of cytosolic PKC may contribute to the diminished PKC‐dependent responses to CD3‐triggered stimulation of human T lymphocytes with aging.
The recombinant GABA transporter GAT1 is downregulated upon activation of protein kinase C
Authors: Sato Kohji , Betz Heinrich , Schloss Patrick
Published: April 2003

Treatment of human embryonic kidney 293 cells expressing the rat γ‐aminobutyric acid (GABA) transporter 1 (GAT1) with the protein kinase C (PKC) activator phorbol 12‐myristate 13‐acetate (PMA) was found to decrease the velocity of specific [3H]GABA uptake. This downregulation varied with extracellular GABA concentration and was blocked by the PKC inhibitors 1‐(5‐isoquinolinylsulphonyl)‐2‐methylpiperazine (H7) and staurosporine. An about 50% reduction of uptake velocity by PMA treatment was observed at GABA concentrations > 1 μM, whereas only a minor effect was seen at low substrate concentrations. These data indicate that GAT1 activity is downregulated by PKC activation.
NMR identification of calcineurin B residues affected by binding of a calcineurin A peptide
Authors: Anglister Jacob , Ren Hao , Klee Claude B. , Bax Ad
Published: April 2003

Triple resonance 3D NMR methods have been used to study the interaction between calcineurin B and a peptide fragment of calcineurin A for which it has high affinity (K
D ∼4 × 10−7 M). Although calcineurin B aggregates at NMR concentrations of ∼ 1 mM, in the presence of a target peptide fragment of calcineurin A it becomes monomeric and yields NMR spectra that are very similar to those reported previously for calcineurin B solubilized by the zwitterionic detergent CHAPS. Changes in chemical shifts between CHAPS‐ and peptide‐solubilized calcineurin B are small which is indicative of no differences in secondary structure. Residues most affected by binding to target peptide are found primarily on the hydrophobic faces of the four helices, present in each of the two globular domains in calcineurin B, and in the loops connecting helices II and III, IV and V, and possibly in the C‐terminal 12 residues, which also exhibit a change in mobility.
Fast solute release from photosensitive liposomes: an alternative to ‘caged’ reagents for use in biological systems
Authors: Morgan Christopher G. , Bisby Roger H. , Johnson Steven A. , Mitchell Andrew C.
Published: April 2003

The kinetics of release of soluble marker trapped in liposomes of gel phase phospholipid containing a photoisomerisable phospholipid analogue have been investigated. Marker release is triggered by UV laser flash photolysis at 355 nm. A markedly temperature‐dependent release rate is seen, and above 25°C millisecond release kinetics can be achieved. These results suggest that such liposomes might find application as an alternative to conventional ‘caged’ reagents for photo‐triggered reagent release in biological research.
Identification of the pore forming element of Semliki Forest virus spikes
Authors: Spyr Christian A. , Käsermann Fabian , Kempf Christoph
Published: April 2003

Pore formation at mildly acidic pH by SFV spike proteins was investigated using isolated and modified virions. Modification of the virions was performed by limited proteolysis in presence of octylglucoside and resulted in the formation of E1 particles and spikeless particles, respectively. Pore formation was detected by measuring the influx of propidium iodide into the viral particles. The results obtained clearly showed that the presence of E1 alone is sufficient to promote pore formation at mildly acidic pH. Thus E1 represents the pore forming element of the viral spike proteins.
Isolation and characterization of an arylalkylamine N‐acetyltransferase from Drosophila melanogaster
Authors: Hintermann Edith , Jenö Paul , Meyer Urs A.
Published: April 2003

The enzyme arylalkylamine N‐acetyltransferase (aaNAT) catalyzes the rate‐limiting step in melatonin formation in the vertebrate pineal gland. Numerous attempts to purify this highly unstable enzyme from vertebrates have been unsuccessful. Here, we report the purification of an aaNAT enzyme from Drosophila melanogaster, using a radioenzymatic activity assay and column chromatography. The isolated 29.5‐kDa protein acetylates tryptamine, dopamine and serotonin with affinities of 0.89 to 0.97 mM, respectively. This suggests that the identified aaNAT may be involved in melatonin synthesis and sclerotization as well as in neurotransmitter catabolism in insects.
A P2X purinoceptor cDNA conferring a novel pharmacological profile
Authors: Bo Xuenong , Zhang Yi , Nassar Mohammed , Burnstock Geoffrey , Schoepfer Ralf
Published: April 2003

We have cloned P2X4, a member of the P2‐purinoceptor family, which has a new pharmacological profile. Rat P2X4 is distantly related to P2X1, P2X2 and P2X3 and is expressed in brain, spinal cord, lung, thymus, bladder, adrenal, testis and vas deferens. This ligand gated ion channel is activated by ATP and analogs with the potency order of ATP > ATPγS > 2‐methylthio ATP > ADP ≈ αβ‐methylene ATP. However, none of the currently used P2X purinoceptor antagonists suramin, reactive blue 2 and PPADS blocked ATP evoked currents; in contrast their application resulted in potentiation of the agonist response. Due to lack of any known antagonist for P2X4 it is unlikely that native P2X4 has previously been recognized as a P2X purinoceptor.
Characterisation of human cdc2 lysine 33 mutations expressed in the fission yeast Schizosaccharomyces pombe
Authors: Leroy Dorothée , Birck Catherine , Brambilla Paolo , Samama Jean-Pierre , Ducommun Bernard
Published: April 2003

The mammalian p34cdc2 protein kinase, a universal cell cycle regulator, complements cdc2/CDC28 temperature‐sensitive mutations in yeasts. We report the biochemical characterisation of two substitutions of human cdc2 at lysine 33, a residue involved in nucleotide binding, that differently alter the fission yeast cell cycle. K33A‐hscdc2 and K33R‐hscdc2 mutants are both catalytically inactive, but overexpression of K33R‐cdc2 is lethal while K33A‐cdc2 is not. We show that human K33R‐cdc2 acts as a dominant negative allele that associates yeast cdc13/cyclinB and therefore renders endogeneous Schizosaccharomyces pombe cdc2 unactivatable. These results are discussed on the light of the molecular modeling of the mutants in the cdc2 model structure.