bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequence, genomic structure and tissue expression of carp (Cyprinus carpio L.) vertebrate ancient (VA) opsin
Authors: Moutsaki Paraskevi , Bellingham James , Soni Bobby G. , David-Gray Zoë K. , Foster Russell G.
Published: July 2002

We report the isolation and characterisation of a novel opsin cDNA from the retina and pineal of the common carp (Cyprinus carpio L.). When a comparison of the amino acid sequences of salmon vertebrate ancient opsin (sVA) and the novel carp opsin are made, and the carboxyl terminus is omitted, the level of identity between these two opsins is 81% and represents the second example of the VA opsin family. We have therefore termed this C. carpio opsin as carp VA opsin (cVA opsin). We show that members of the VA opsin family may exist in two variants or isoforms based upon the length of the carboxyl terminus and propose that the mechanism of production of the short VA opsin isoform is alternative splicing of intron 4 of the VA opsin gene. The VA opsin gene consists of five exons, with intron 2 significantly shifted in a 3′ direction relative to the corresponding intron in rod and cone opsins. The position (or lack) of intron 2 appears to be a diagnostic feature which separates the image forming rod and cone opsin families from the more recently discovered non‐visual opsin families (pin‐opsins (P), vertebrate ancient (VA), parapinopsin (PP)). Finally, we suggest that lamprey P opsin should be reassigned to the VA opsin family based upon its level of amino acid identity, genomic structure with respect to the position of intron 2 and nucleotide phylogeny.
Regulating the cellular economy of supply and demand
Authors: Hofmeyr Jan-Hendrik S. , Cornish-Bowden Athel
Published: July 2002

Cellular metabolism is a molecular economy that is functionally organised into supply and demand blocks linked by metabolic products and cofactor cycles. Supply–demand analysis allows the behaviour, control and regulation of metabolism as a whole to be understood quantitatively in terms of the elasticities of supply and demand, which are experimentally measurable properties of the individual blocks. The kinetic and thermodynamic aspects of regulation are clearly distinguished. One important result is the demonstration that when flux is controlled by one block, the other block determines to which degree the concentration of the linking metabolite is homeostatically maintained.
Transcriptional regulation of the Saccharomyces cerevisiae DAL5 gene family and identification of the high affinity nicotinic acid permease TNA1 (YGR260w)
Authors: Llorente Bertrand , Dujon Bernard
Published: July 2002

We have studied the transcript levels of YGR260w and YLR004c, two genes encoding members of the yeast Dal5p subfamily of the major facilitator family, and we show that they increase when extracellular nicotinic acid and thiamine, respectively, are absent. The deletion of YGR260w in a bna1 auxotrophic mutant for nicotinic acid prevents growth at low nicotinic acid concentration. This suggests that YGR260w is necessary for nicotinic acid import into the cell. The direct measurement of nicotinic acid uptake on whole cells demonstrates that YGR260w encodes the yeast high affinity nicotinic acid permease. Its apparent K
m of 1.7 μM is low enough to allow the uptake of the low concentrations of nicotinic acid normally secreted by wild type cells.
Functional analysis of proteins involved in Plasmodium falciparum merozoite invasion of red blood cells
Authors: Cowman Alan F. , Baldi Deborah L. , Healer Julie , Mills Kerry E. , O'Donnell Rebecca A. , Reed Michael B. , Triglia Tony , Wickham Mark E. , Crabb Brendan S.
Published: July 2002

Plasmodium falciparum causes the most lethal form of malaria in humans and is responsible for over two million deaths per year. The development of a vaccine against this parasite is an urgent priority and potential protein targets include those on the surface of the asexual merozoite stage, the form that invades the host erythrocyte. The development of methods to transfect P. falciparum has enabled the construction of gain‐of‐function and loss‐of‐function mutants and provided new strategies to analyse the role of parasite proteins. In this review, we describe the use of this technology to examine the role of merozoite antigens in erythrocyte invasion and to address their potential as vaccine candidates.
Plant heterotrimeric G protein β subunit is associated with membranes via protein interactions involving coiled‐coil formation
Authors: Obrdlik Petr , Neuhaus Gunther , Merkle Thomas
Published: July 2002

Gβ subunits from animals are anchored to membranes via Gγ subunits. No Gγ has been identified in plants to date. Using differential centrifugation of Arabidopsis and broccoli extracts, Gβ was highly enriched in the microsomal pellet. Treatment of microsomes with detergents and salts indicates that plant Gβ is located at the membrane surface and attached to membranes by hydrophobic interactions. Analysis of transgenic plants expressing Gβ–GFP fusion proteins showed that mutations in the heptad repeat domain of Gβ severely diminished their membrane association. We propose that plant Gβ is anchored to membranes by an unknown protein similar to animal Gβ by Gγ, via coiled‐coil formation.
Aldehyde dehydrogenase (ALDH) 2 associates with oxidation of methoxyacetaldehyde; in vitro analysis with liver subcellular fraction derived from human and Aldh2 gene targeting mouse
Authors: Kitagawa Kyoko , Kawamoto Toshihiro , Kunugita Naoki , Tsukiyama Tadasuke , Okamoto Kohji , Yoshida Akira , Nakayama Keiko , Nakayama Kei-ichi
Published: July 2002

A principal pathway of 2‐methoxyethanol (ME) metabolism is to the toxic oxidative product, methoxyacetaldehyde (MALD). To assess the role of aldehyde dehydrogenase (ALDH) in MALD metabolism, in vitro MALD oxidation was examined with liver subcellular fractions from Japanese subjects who carried three different ALDH2 genotypes and Aldh2 knockout mice, which were generated in this study. The activity was distributed in mitochondrial fractions of ALDH2*1/*1 and wild type (Aldh2+/+) mice but not ALDH2*1/*2, *2/*2 subjects or Aldh2 homozygous mutant (Aldh2−/−) mice. These data suggest that ALDH2 is a key enzyme for MALD oxidation and ME susceptibility may be influenced by the ALDH2 genotype.
Urokinase‐type plasminogen activator up‐regulates the expression of its cellular receptor
Authors: Montuori Nunzia , Salzano Salvatore , Rossi Guido , Ragno Pia
Published: July 2002

The expression of the receptor for the urokinase‐type plasminogen activator (uPAR) can be regulated by several hormones, cytokines, tumor promoters, etc. Recently, it has been reported that uPAR is capable of transducing signals, even though it is lacking a transmembrane domain and a cytoplasmatic tail. We now report that uPAR cell surface expression can be positively regulated by its ligand, uPA, in thyroid cells. The effect of uPA is independent of its proteolytic activity, since inactivated uPA or its aminoterminal fragment have the same effects of the active enzyme. The increase of uPAR on the cell surface correlates with an increase of specific uPAR mRNA. Finally, uPA up‐regulates uPAR expression also in other cell lines of different type and origin, thus suggesting that the regulatory role of uPA on uPAR expression is not restricted to thyroid cells, but it occurs in different tissues, both normal and tumoral.
Structure of the Bordetella pertussis 1414 endotoxin
Authors: Caroff Martine , Brisson Jean-Robert , Martin Adèle , Karibian Doris
Published: July 2002

The endotoxin (lipopolysaccharide) of Bordetella pertussis, the agent of whooping cough, consists of a lipid A linked to a highly branched dodecasaccharide containing several acid and amino sugars. The elucidation of the polysaccharide structure was accomplished by first analyzing the structures of fragments obtained by hydrolysis and nitrous deamination and then piecing the fragments together. The fine structure of the antigenic distal pentasaccharide, presented here, was determined by chemical analyses as well as by high‐resolution nuclear magnetic resonance and mass spectrometry. The complete structure was reconstituted and confirmed by matrix‐assisted laser desorption/ionization mass spectrometry. The following structure was derived from the combined experimental data:

The detailed structure combined with previously reported serological data now allows the synthesis of its epitopes for potential vaccines.
Influence of the pKa value of the buried, active‐site cysteine on the redox properties of thioredoxin‐like oxidoreductases
Authors: Mössner Ekkehard , Iwai Hideo , Glockshuber Rudi
Published: July 2002

Thioredoxin constitutes the prototype of the thiol‐disulfide oxidoreductase family. These enzymes contain an active‐site disulfide bridge with the consensus sequence Cys‐Xaa‐Xaa‐Cys. The more N‐terminal active‐site cysteine is generally a strong nucleophile with an abnormal low pK
a value. In contrast, the more C‐terminal cysteine is buried and only little is known about its effective pK
a during catalysis of disulfide exchange reactions. Here we have analyzed the pK
a values of the active‐site thiols in wild type thioredoxin and a 400‐fold more oxidizing thioredoxin variant by NMR spectroscopy, using selectively 13Cβ‐Cys‐labeled proteins. We find that the effective pK
a of the buried cysteine (pK
b) of the variant is increased, while the pK
a of the more N‐terminal cysteine (pK
N) is decreased relative to the corresponding pK
a values in the wild type. We propose two empirical models which exclusively require the knowledge of pK
N to predict the redox properties of thiol‐disulfide oxidoreductases with reasonable accuracy.
Activated protein C suppresses tissue factor expression on U937 cells in the endothelial protein C receptor‐dependent manner
Authors: Shu Fang , Kobayashi Hiroshi , Fukudome Kenji , Tsuneyoshi Naoko , Kimoto Masao , Terao Toshihiko
Published: July 2002

The new functional role of activated protein C (APC) in the regulation of tissue factor (TF) expression was investigated using the cultured human monoblastic leukemia U937 cell line. A flow cytofluorometric analysis demonstrated that treatment with APC resulted in time‐ and dose‐dependent decrease in TF expression in unstimulated and phorbol ester‐stimulated cells. The effect was antagonized by the monoclonal antibody (mAb) to endothelial protein C/APC receptor (EPCR), 252, which strongly inhibited the interaction between APC and EPCR. In contrast, mAbs 49 and 379, which bind to EPCR without blocking APC binding, had no or only a modest effect. It is concluded that culturing U937 cells in the presence of APC caused down‐regulation of TF expression through the EPCR‐dependent mechanism, independent of whether induction was triggered by phorbol ester.
Cloning and characterization of rat casein kinase 1ϵ
Authors: Takano Atsuko , Shimizu Kimiko , Kani Shuichi , Buijs Ruud M. , Okada Masato , Nagai Katsuya
Published: July 2002

Genes differentially expressed in the subjective day and night in the rat suprachiasmatic nucleus (SCN) were surveyed by differential display. A gene homologous to human casein kinase 1ϵ (CK1ϵ) was isolated, which initially appeared to be expressed in the suprachiasmatic nucleus (SCN) in a circadian manner. We here describe the cDNA cloning of the rat CK1ϵ and characterization of the protein products. The rCK1ϵ is predominantly expressed in the brain including the SCN, binds and phosphorylates mPer1, mPer2, and mPer3 in vitro, and translocates mPer1 and mPer3, but not mPer2, to the cell nucleus depending on its kinase activity when coexpressed with these Per proteins in COS‐7 cells.
Small proline‐rich protein 1 is the major component of the cell envelope of normal human oral keratinocytes
Authors: Lee Chong Heon , Marekov Lyuben N. , Kim Soo-Youl , Brahim Jaime S. , Park Myung Hee , Steinert Peter M.
Published: July 2002

Oral keratinocytes of buccal and gingival tissues undergo a terminal differentiation program to form a protective epithelial barrier as non‐keratinized or parakeratinized stratified cells. We have examined the protein composition of cell envelopes (CEs) from normal human buccal and gingival tissues as well as keratinocytes from normal human gingival cells grown in culture. Biochemical and sequencing analyses reveal that the CEs contain 60–70% small proline‐rich protein 1a/b (SPR1a/b), together with smaller amounts of involucrin, annexin I and several other known CE proteins. The data imply a specialized role for SPR1 proteins in the unique barrier function requirements of oral epithelia.
Regulation of the activity of caspases by L‐carnitine and palmitoylcarnitine
Authors: Mutomba Martha C. , Yuan Hua , Konyavko Mary , Adachi Souichi , Yokoyama Christopher B. , Esser Victoria , McGarry J.Denis , Babior Bernard M. , Gottlieb Roberta A.
Published: July 2002

L‐Carnitine facilitates the transport of fatty acids into the mitochondrial matrix where they are used for energy production. Recent studies have shown that L‐carnitine is capable of protecting the heart against ischemia/reperfusion injury and has beneficial effects against Alzheimer's disease and AIDS. The mechanism of action, however, is not yet understood. In the present study, we found that in Jurkat cells, L‐carnitine inhibited apoptosis induced by Fas ligation. In addition, 5 mM carnitine potently inhibited the activity of recombinant caspases 3, 7 and 8, whereas its long‐chain fatty acid derivative palmitoylcarnitine stimulated the activity of all the caspases. Palmitoylcarnitine reversed the inhibition mediated by carnitine. Levels of carnitine and palmitoyl‐CoA decreased significantly during Fas‐mediated apoptosis, while palmitoylcarnitine formation increased. These alterations may be due to inactivation of β‐oxidation or to an increase in the activity of the enzyme that converts carnitine to palmitoylcarnitine, carnitine palmitoyltransferase I (CPT I). In support of the latter possibility, fibroblasts deficient in CPT I activity were relatively resistant to staurosporine‐induced apoptosis. These observations suggest that caspase activity may be regulated in part by the balance of carnitine and palmitoylcarnitine.
ζ‐Crystallin catalyzes the reductive activation of 2,4,6‐trinitrotoluene to generate reactive oxygen species: a proposed mechanism for the induction of cataracts
Authors: Kumagai Yoshito , Wakayama Toshihiko , Li Song , Shinohara Azusa , Iwamatsu Akihiro , Sun Guifan , Shimojo Nobuhiro
Published: July 2002

Exposure to 2,4,6‐trinitrotoluene (TNT) has been shown to cause induction of cataract in which oxidative stress plays a critical role. From bovine lens we purified to homogeneity and identified an enzyme that catalyzes the reduction of TNT, resulting in the production of reactive oxygen species. The final preparation of TNT reductase showed a single band with a subunit molecular weight of 38 kDa on SDS–PAGE. Sequence data from peptides obtained by digestion with lysylendopeptidase Achromobacter protease I (API) revealed that TNT reductase is identical to ζ‐crystallin. Superoxide anions were formed during reduction of TNT by ζ‐crystallin, though negligible enzyme activity or protein content for superoxide dismutase, a superoxide scavenging enzyme, was found in the lens. Thus, the present results suggest that the induction of cataracts by TNT may be associated with increased oxidative stress, as a result of reductive activation of TNT generating superoxide anions, there being minimal antioxidant enzyme activity for defense against reactive oxygen species exogenously produced in the lens.
Acid sphingomyelinase is involved in CEACAM receptor‐mediated phagocytosis of Neisseria gonorrhoeae
Authors: Hauck C.R. , Grassmé H. , Bock J. , Jendrossek V. , Ferlinz K. , Meyer T.F. , Gulbins E.
Published: July 2002

The interaction with human phagocytes is a hallmark of symptomatic Neisseria gonorrhoeae infections. Gonococcal outer membrane proteins of the Opa family induce the opsonin‐independent uptake of the bacteria that relies on CEACAM receptors and an active signaling machinery of the phagocyte. Here, we show that CEACAM receptor‐mediated phagocytosis of Opa52‐expressing N. gonorrhoeae into human cells results in a rapid activation of the acid sphingomyelinase. Inhibition of this enzyme by imipramine or SR33557 abolishes opsonin‐independent internalization without affecting bacterial adherence. Reconstitution of ceramide, the product of acid sphingomyelinase activity, in imipramine‐ or SR33557‐treated cells restores internalization of the bacteria. Furthermore, we demonstrate that CEACAM receptor‐initiated stimulation of other signalling molecules, in particular Src‐like tyrosine kinases and Jun N‐terminal kinases, requires acid sphingomyelinase. These studies provide evidence for a crucial role of the acid sphingomyelinase for CEACAM receptor‐initiated signalling events and internalization of Opa52‐expressing N. gonorrhoeae into human neutrophils.
Tissue factor pathway inhibitor‐2 suppresses the production of active matrix metalloproteinase‐2 and is down‐regulated in cells harboring activated ras oncogenes
Authors: Izumi Hideki , Takahashi Chiaki , Oh Junseo , Noda Makoto
Published: July 2002

A human placenta cDNA expression library was screened for genes inducing flat reversion when transfected into a v‐K‐ras‐transformed NIH3T3 cell line, DT. One such gene was found to encode a Kunitz‐type serine protease inhibitor, tissue factor pathway inhibitor‐2 (TFPI‐2). While the TFPI‐2 mRNA can be detected in normal human fibroblasts (MRC‐5), it is down‐regulated in MRC‐5 cells expressing an activated H‐ras oncogene and in the human fibrosarcoma cell line, HT1080. Restored expression of the TFPI‐2 gene in HT1080 cells resulted in the suppression of matrix invasion activity in vitro with concomitant decrease in the relative amount of active matrix metalloproteinase‐2 secreted from the cells. When DT cells were cultured in the presence of conditioned medium and extracellular matrix prepared from TFPI‐2‐transfected HT1080 cells, increased attachment and flat reversion were observed. These results suggest that TFPI‐2 may be required for the maintenance of the integrity of extracellular matrix in normal tissues and its down‐regulation as a result of oncogene activation may contribute to the malignant phenotypes of tumor cells.
M‐type KCNQ2–KCNQ3 potassium channels are modulated by the KCNE2 subunit
Authors: Tinel Norbert , Diochot Sylvie , Lauritzen Inger , Barhanin Jacques , Lazdunski Michel , Borsotto Marc
Published: July 2002

KCNQ2 and KCNQ3 subunits belong to the six transmembrane domain K+ channel family and loss of function mutations are associated with benign familial neonatal convulsions. KCNE2 (MirP1) is a single transmembrane domain subunit first described to be a modulator of the HERG potassium channel in the heart. Here, we show that KCNE2 is present in brain, in areas which also express KCNQ2 and KCNQ3 channels. We demonstrate that KCNE2 associates with KCNQ2 and/or KCNQ3 subunits. In transiently transfected COS cells, KCNE2 expression produces an acceleration of deactivation kinetics of KCNQ2 and of the KCNQ2–KCNQ3 complex. Effects of two previously identified arrhythmogenic mutations of KCNE2 have also been analyzed.
Membrane‐type 6 matrix metalloproteinase (MT6‐MMP, MMP‐25) is the second glycosyl‐phosphatidyl inositol (GPI)‐anchored MMP
Authors: Kojima Shin-ichi , Itoh Yoshifumi , Matsumoto Shun-ichiro , Masuho Yasuhiko , Seiki Motoharu
Published: July 2002

A recently identified membrane‐type 6 matrix metalloproteinase (MT6‐MMP) has a hydrophobic stretch of 24 amino acids at the C‐terminus. This hydrophobicity pattern is similar to glycosyl‐phosphatidyl inositol (GPI)‐anchored MMP, MT4‐MMP, and other GPI‐anchored proteins. Thus, we tested the possibility that MT6‐MMP was also a GPI‐anchored proteinase. Our results showed that MT6‐MMP as well as MT4‐MMP were labeled with [3H]ethanolamine indicating the presence of a GPI unit with incorporated label. In addition, phosphatidyl inositol‐specific phospholipase C treatment released MT6‐MMP from the surface of transfected cells. These results strongly indicate that MT6‐MMP is a GPI‐anchored protein. Since two members of MT‐MMPs are now assigned as GPI‐anchored proteinase, MT‐MMPs can be subgrouped into GPI type and transmembrane type.
Packaging of up to 240 subunits of a 17 kDa nuclease into the interior of recombinant hepatitis B virus capsids
Authors: Beterams Gertrud , Böttcher Bettina , Nassal Michael
Published: July 2002

The icosahedral nucleocapsid of hepatitis B virus (HBV) consists of multiple subunits of a single 183 amino acids (aa) core protein encasing the viral genome. However, recombinant core protein alone also forms capsid‐like particles. We have recently shown that a 238 aa protein centrally inserted into the core protein can be displayed on the particle surface. Here we demonstrate that replacement of the C‐terminal basic domain by the 17 kDaStaphylococcus aureusnuclease also yields particles but that in these the foreign domains are located in the interior. The packaged nuclease is enzymatically active, and the chimeric protein forms mosaic particles with the wild‐type core protein. Hence the HBV capsid is useful as a molecular platform which, dependent on the fusion site, allows foreign protein domains to either be packaged into or be exposed on the exterior of the particle. These results are of relevance for the use of the HBV capsid as a vaccine carrier, and as a target for antiviral therapy.
Structure‐based sequence alignment for the β‐trefoil subdomain of the clostridial neurotoxin family provides residue level information about the putative ganglioside binding site
Authors: Ginalski Krzysztof , Venclovas C̆eslovas , Lesyng Bogdan , Fidelis Krzysztof
Published: July 2002

Clostridial neurotoxins embrace a family of extremely potent toxins comprised of tetanus toxin (TeNT) and seven different serotypes of botulinum toxin (BoNT/A–G). The β‐trefoil subdomain of the C‐terminal part of the heavy chain (HC), responsible for ganglioside binding, is the most divergent region in clostridial neurotoxins with sequence identity as low as 15%. We re‐examined the alignment between family sequences within this subdomain, since in this region all alignments published to date show obvious inconsistencies with the β‐trefoil fold. The final alignment was obtained by considering the general constraints imposed by this fold, and homology modeling studies based on the TeNT structure. Recently solved structures of BoNT/A confirm the validity of this structure‐based approach. Taking into account biochemical data and crystal structures of TeNT and BoNT/A, we also re‐examined the location of the putative ganglioside binding site and, using the new alignment, characterized this site in other BoNT serotypes.
Caspase‐3 and inhibitor of apoptosis protein(s) interactions in Saccharomyces cerevisiae and mammalian cells
Authors: Wright Michael E , Han David K , Hockenbery David M
Published: July 2002

Using a heterologous yeast expression assay, we show that inhibitor of apoptosis proteins (IAPs) suppress caspase‐3‐mediated cytotoxicity in the order of XIAP>c‐IAP2>c‐IAP1>survivin. The same ordering of IAP activities was demonstrated in mammalian cells expressing an auto‐activating caspase‐3. The relative anti‐apoptotic activities of each IAP depended on the particular death stimulus. For IAP‐expressing cells treated with camptothecin, survival correlated with their intrinsic anti‐caspase‐3 activity. However, c‐IAP1‐transfected cells were disproportionately resistant to tumor necrosis factor‐α, suggesting that its anti‐apoptotic activities extend beyond caspase‐3 or ‐7 inhibition. Yeast‐based caspase assays provide rapid, reliable information on specificity and activity of the IAPs and aid in identifying critical targets in mammalian apoptotic pathways.
Geranylgeranylated RhoB is sufficient to mediate tissue‐specific suppression of Akt kinase activity by farnesyltransferase inhibitors
Authors: Liu Ai-xue , Prendergast George C.
Published: July 2002

Farnesyltransferase inhibitors (FTIs) induce apoptosis by elevating the levels of geranylgeranylated RhoB (RhoB‐GG) in cells. However, the mechanism by which RhoB‐GG acts is unclear. Here we report that RhoB‐GG is sufficient to mediate the suppressive effects of FTIs on the activity of the survival kinase Akt‐1 in epithelial cells. This mechanism is tissue‐specific insofar as it does not operate in fibroblasts. We discuss how the cell survival functions of RhoB and Akt may be linked biochemically in certain cell types.
Epoxysuccinyl peptide‐derived affinity labels for cathepsin B
Authors: Schaschke N , Assfalg-Machleidt I , Laßleben Th , Sommerhoff C.P , Moroder L , Machleidt W
Published: July 2002

Extracellular cysteine proteases, in particular cathepsin B, have been implicated in a variety of pathological processes. Selectively targeting labels of this enzyme are important tools to gain more detailed understanding of its specific roles. Starting from our recently developed irreversible epoxysuccinyl‐based inhibitor (R‐Gly‐Gly‐Leu‐(2S,3S)‐tEps‐Leu‐Pro‐OH, R=OMe), we have synthesized two affinity labels, R=NH‐(CH2)6‐NH‐rhodamine B and R=NH‐(CH2)6‐NH‐biotin. Using MCF‐7 cells, the labeled inhibitors were shown to be virtually non‐cell‐permeant. Moreover, affinity blot analysis with the biotinylated inhibitor allowed a highly sensitive and selective non‐radioactive detection of active cathepsin B.
Characterization of Brassica S‐haplotypes lacking S‐locus glycoprotein1
Authors: Suzuki Tohru , Kusaba Makoto , Matsushita Masanori , Okazaki Keiichi , Nishio Takeshi
Published: July 2002

Self‐incompatibility (SI) in Brassica is regulated by a single multi‐allelic locus, S, which contains highly polymorphic stigma‐expressed genes, SLG and SRK. While SRK is shown to be the determinant of female SI specificity, SLG is thought to assist the function of SRK. Here we report that the SLG genes of self‐incompatible S
18 and S
60 homozygotes of Brassica oleracea have an in‐frame stop codon and a 23 bp deletion resulting in a frame‐shift, respectively. The finding that these SLG genes do not encode functional SLG proteins suggests that SLG is not essential for SI. The possible role of SLG in SI was discussed.
Characterisation of new intracellular membranes in Escherichia coli accompanying large scale over‐production of the b subunit of F1Fo ATP synthase
Authors: Arechaga Ignacio , Miroux Bruno , Karrasch Simone , Huijbregts Richard , de Kruijff Ben , Runswick Michael J. , Walker John E.
Published: July 2002

Recombinant membrane proteins in Escherichia coli are either expressed at relatively low level in the cytoplasmic membrane or they accumulate as inclusion bodies. Here, we report that the abundant over‐production of subunit b of E. coli F1Fo ATP synthase in the mutant host strains E. coli C41(DE3) and C43(DE3) is accompanied by the proliferation of intracellular membranes without formation of inclusion bodies. Maximal levels of proliferation of intracellular membranes were observed in C43(DE3) cells over‐producing subunit b. The new proliferated membranes contained all the over‐expressed protein and could be recovered by a single centrifugation step. Recombinant subunit b represented up to 80% of the protein content of the membranes. The lipid:protein ratios and phospholipid compositions of the intracellular membranes differ from those of bacterial cytoplasmic membranes, and they are particularly rich in cardiolipin.
Export of a misprocessed GPI‐anchored protein from the endoplasmic reticulum in vitro in an ATP‐ and cytosol‐dependent manner
Authors: Ali Bassam R.S , Claxton Sheyi , Field Mark C
Published: July 2002

Strict quality control mechanisms within the mammalian endoplasmic reticulum act to prevent misfolded and unprocessed proteins from entering post‐endoplasmic reticulum (ER) compartments. Following translocation into the ER lumen via the Sec61p translocon, nascent polypeptide chains fold and are modified in an environment that contains numerous chaperones and other folding mediators. Recently it has emerged that polypeptides failing to acquire the native state are re‐exported from the ER to the cytosol for ultimate degradation by the proteasome ubiquitin system, apparently mediated again via Sec61p. Substrates for this degradation pathway include proteins destined to become glycosyl phosphatidylinositol (GPI)‐anchored, but which fail to be processed and retain the C‐terminal GPI signal peptide. In order to characterise this process we have used a model GPI‐anchored mutant protein, prepro mini human placental alkaline phosphatase (PLAP) W179, which cannot be processed efficiently on account of being a poor substrate for the transamidase which cleaves the GPI signal peptide and adds the GPI anchor in a coupled reaction. In vitro transcription, translation and translocation into canine pancreatic microsomes resulted in ER‐targeting signal sequence cleavage and formation of prominiPLAP in the ER lumen. We were able to show that prominiPLAPW179 could be exported from the microsomes in a time‐dependent manner and that release requires both ATP and cytosol. Export was not supported by GTP, indicating a biochemical distinction from glycopeptide export which we showed recently requires GTP hydrolysis. The process was not affected by redox, unlike several other GPI‐anchored model proteins. These data demonstrate that misprocessed proteins can be exported in vitro from mammalian microsomes, facilitating identification of factors involved in this process.
The membrane‐bound transferrin homologue melanotransferrin: roles other than iron transport?
Authors: Sekyere Eric , Richardson Des R.
Published: July 2002

Melanotransferrin (MTf) is a membrane‐bound transferrin (Tf) homologue that is found at high levels in melanoma cells. MTf has many characteristics in common with serum Tf and previous studies have shown that it can bind Fe. This has led to speculation that MTf may be involved in Fe transport. Because Fe is required for a variety of metabolic reactions including ATP and DNA synthesis, MTf could play a role in proliferation. However, recently it has been shown that MTf plays very little role in Fe uptake by melanoma cells, and unlike other Fe transport molecules (e.g. the transferrin receptor), its expression is not controlled by Fe. In the present review the function of MTf is discussed in relation to data suggesting other roles apart from Fe uptake.
Abscisic acid plasmalemma perception triggers a calcium influx essential for RAB18 gene expression in Arabidopsis thaliana suspension cells
Authors: Ghelis Thanos , Dellis Olivier , Jeannette Emmanuelle , Bardat Françoise , Miginiac Emile , Sotta Bruno
Published: July 2002

Pretreatment of Arabidopsis thaliana suspension cells with impermeant calcium chelator EGTA inhibited the ABA‐induced RAB18 gene expression. However, extracellular calcium alone, up to 10 mM, did not trigger RAB18 expression. Spectrofluorimetric extracellular Ca2+ measurement with Fluo‐3 showed a fast, within 1 min, Ca2+ influx associated with outer plasmalemma ABA perception. In the presence of the Ca2+ blockers Cd2+ and Ni2+, RAB18 expression was suppressed. Pimozide and fluspirilene inhibited Ca2+ influx and ABA‐induced RAB18 expression. Thus we demonstrated the involvement of specific Ca2+ influx in the ABA signaling sequence leading to RAB18 expression.
Oligomerization of the plasma membrane calcium pump involves two regions with different thermal stability
Authors: Levi Valeria , Rossi Juan P.F.C. , Castello Pablo R. , González Flecha F.Luis
Published: July 2002

Ca2+ pump dimerization was studied by using a combined approach of thermal denaturation and fluorescence resonance energy transfer. The measurement of calcium pump ability to dimerize after the unfolding of individual functional domains of the enzyme demonstrated the existence of two different regions involved in the self‐association process. One of these regions is highly susceptible to thermal unfolding and was identified as the calmodulin (CaM)‐binding domain. The other region whose thermal stability is higher than those of the catalytic and CaM‐binding domains could be related with the previously found C28W‐binding regions.
Induction of synthesis of an antimicrobial peptide in the skin of the freeze‐tolerant frog, Rana sylvatica, in response to environmental stimuli
Authors: Matutte Beverly , Storey Kenneth B. , Knoop Floyd C. , Conlon J.Michael
Published: July 2002

An extract of skin taken from specimens of the freeze‐tolerant wood frog, Rana sylvatica, that were collected from cold (<7°C) ponds and maintained at 5°C lacked detectable antimicrobial activity. In contrast, an extract of skin taken from specimens maintained at 30°C for 3 weeks under laboratory conditions contained a high concentration (approximately 4 nmol/g) of a single antimicrobial peptide of the brevinin‐1 family (FLPVVAGLAAKVLPSIICAVTKKC). The peptide inhibited growth of Escherichia coli (minimum inhibitory concentration 45 μM) and Staphylococcus aureus (minimum inhibitory concentration 7 μM). The data suggest that synthesis of the peptide is induced when the animal is in an environment that promotes the growth of microorganisms consistent with a role in the animal's defense strategy.
The MAT1 cyclin‐dependent kinase‐activating kinase (CAK) assembly/targeting factor interacts physically with the MCM7 DNA licensing factor
Authors: Wang Yan , Xu Fan , Hall Frederick L.
Published: July 2002

MAT1 (ménage à trois1) functions as an assembly/targeting factor of CAK (cyclin‐dependent kinase‐activating kinase). In a search for MAT1‐interacting proteins using yeast two‐hybrid system, MCM7 (minichromosome maintenance 7), a member of a family of DNA licensing factors, was identified. The physical interaction between MAT1 and MCM7 was confirmed in vivo in yeast cells and verified with in vitro protein binding assays. Further studies showed the RING‐finger motif of MAT1 is not required for the interaction with MCM7, while the C‐terminal domain of MAT1 is indispensable. Immunoprecipitation of MCM7 in human osteosarcoma MG63 cells demonstrated that MCM7 associates with the CAK complex in vivo.
Quenching of singlet oxygen by carotenoids produced in Escherichia coli – attenuation of singlet oxygen‐mediated bacterial killing by carotenoids
Authors: Tatsuzawa Hidetaka , Maruyama Tadashi , Misawa Norihiko , Fujimori Ken , Nakano Minoru
Published: July 2002

We examined the viability of Escherichia coli transformants harboring various carotenoids synthesizing genes in a medium containing an enzymatic singlet oxygen generating system, which contained myeloperoxidase, hydrogen peroxide and Br− at pH 4.5. Singlet oxygen quenching activities of various carotenoids in phosphatidyl choline micelles in aqueous media were also studied using the same enzymatic singlet oxygen generating system. Viability of the transformants producing carotenoids was higher than that of the wild type E. coli in the singlet oxygen generation mixture. Of the transformants tested, the viability of zeaxanthin‐diglucoside producing transformant was the highest. Carotenoids in increasing order of k
q values were β‐carotene, a cyclic carotene<zeaxanthin with hydroxy groups≤lycopene, an acyclic carotene<canthaxanthin and astaxanthin with keto groups≪zeaxanthin‐diglucoside. The k
q value of zeaxanthin‐diglucoside was 3.5 times higher than that of β‐carotene. These results suggest that orientation of the carotenoids in lipid layers of micelles and also in phospholipid membrane of bacteria is important for quenching of singlet oxygen. Furthermore, the viability of transformants producing lycopene and phytoene was almost as high as that of the transformant producing zeaxanthin‐glucoside.
Decreased expression of a member of the Rho GTPase family, Cdc42Hs, in cells from Tangier disease – the small G protein may play a role in cholesterol efflux
Authors: Hirano Ken-ichi , Matsuura Fumihiko , Tsukamoto Kosuke , Zhang Zhongyan , Matsuyama Akifumi , Takaishi Kenji , Komuro Ryutaro , Suehiro Tadashi , Yamashita Shizuya , Takai Yoshimi , Matsuzawa Yuji
Published: July 2002

Cholesterol efflux (CE) is the initial and important step of reverse cholesterol transport (RCT), a major protective system against atherosclerosis. However, most of the molecular mechanism for CE still remains to be clarified. In the present study, cDNA subtraction revealed that the expression of a member of the Rho GTPase family, Cdc42Hs, was markedly decreased in both passaged fibroblasts and macrophages (Mφ) from patients with Tangier disease (TD), a rare lipoprotein disorder with reduced CE. This small G protein is known to have many cell biological activities such as rearrangement of actin cytoskeleton and vesicular transport, however the association between this molecule and lipid transport has never been reported. We demonstrate that MDCK cells expressing the dominant negative form of Cdc42Hs had reduced CE, inversely ones expressing the dominant active form had increased CE. From these observations, we would like to raise a novel hypothesis that this type of small G protein may play a role in some steps of CE. To our knowledge, the present study is the first demonstration that the expression of this molecule is altered in cells from human disease.
NMR structure of antibiotics plipastatins A and B from Bacillus subtilis inhibitors of phospholipase A2
Authors: Volpon Laurent , Besson Françoise , Lancelin Jean-Marc
Published: July 2002

Plipastatins A and B are antifungal antibiotics belonging to a family of lipopeptides capable of inhibiting phospholipase A2 (PLA2) and are biosynthesised under certain circumstances by Bacillus subtilis. U‐15N plipastatins A and B were obtained from cultures of the strain NCIB 8872 on a Landy medium modified for stable‐isotope labelling by the substitution of the L‐glutamic acid used as the sole nitrogen source, by 15NH4Cl. These two lipo‐decapeptides, lactonised by esterification of the Ile10 C‐terminus with the phenolic hydroxyl of Tyr3, differ only by a D‐Ala (plipastatin A)/D‐Val (plipastatin B) substitution at the position 6. The 1H‐ and 15N‐nuclear magnetic resonance (NMR) signals of a 4:6 mixture of plipastatins A and B were unambiguously assigned and their structures in dimethylsulfoxide solution were calculated on the basis of a set of NMR‐derived restraints. Plipastatins A and B are well‐defined structures in solution stabilised by a type 1 β‐turn comprising residues 6–9 and several other specific hydrogen bonds. The structures afford a first molecular basis for the future studies of their biological activities both in lipidic layers or on PLA2.
Opioid tolerance/dependence in neuroblastoma×glioma (NG108‐15) hybrid cells is associated with a reduction in spontaneous stimulatory receptor activity
Authors: Ammer Hermann , Schulz Rüdiger
Published: July 2002

Chronic opioid regulation of stimulatory receptor activity was investigated in neuroblastoma×glioma (NG108‐15) hybrid cells stably transfected to express the human β2‐adrenoceptor (β2‐AR). Expressed β2‐ARs are functionally coupled to G proteins and display ligand‐independent signalling activity, as demonstrated by the ability of an inverse agonist to attenuate basal adenylyl cyclase (AC) activity. Despite the relative increase in basal AC activity due to the development of tolerance/dependence, chronic morphine treatment was found to completely abolish spontaneous β2‐AR activity by reducing basal receptor/G protein precoupling. A similar chronic opioid effect was observed in transiently transfected COS‐7 cells. These results indicate that during the state of opioid tolerance/dependence basal levels of AC activity are no longer under the control of spontaneously active stimulatory receptors.
Leukemia‐associated Rho guanine nucleotide exchange factor (LARG) links heterotrimeric G proteins of the G12 family to Rho
Authors: Fukuhara Shigetomo , Chikumi Hiroki , Gutkind J.Silvio
Published: July 2002

A putative guanine nucleotide exchange factor (GEF), termed leukemia‐associated RhoGEF (LARG), was recently identified upon fusion to the coding sequence of the MLL gene in acute myeloid leukemia. Although the function of LARG is still unknown, it exhibits a number of structural domains suggestive of a role in signal transduction, including a PDZ domain, a LH/RGS domain, and a Dbl homology/pleckstrin homology domain. Here, we show that LARG can activate Rho in vivo. Furthermore, we present evidence that LARG is an integral component of a novel biochemical route whereby G protein‐coupled receptors (GPCRs) and heterotrimeric G proteins of the Gα12 family stimulate Rho‐dependent signaling pathways.
The T9176G mutation of human mtDNA gives a fully assembled but inactive ATP synthase when modeled in Escherichia coli
Authors: Carrozzo R. , Murray J. , Santorelli F.M. , Capaldi R.A.
Published: July 2002

A new mutation in human F1F0 ATPase6, T9176G, which changes Leu 217 to an Arg, has been described in two siblings with Leigh syndrome [Carrozzo et al. (2000) Neurology, in press]. This mutation was modeled in Escherichia coli by changing Leu 259 (the equivalent residue) to Arg and the properties of the altered ECF1F0 were compared to those of previously characterized ATPase6 mutants also modeled in the E. coli enzyme. The L259R change produced a fully assembled ECF1F0 which had no significant ATP hydrolysis, ATP synthesis or proton pumping functions. This is very different from previously described human ATPase6 mutations. The presence of Arg at position 259 in subunit a did not make membranes permeable to protons. We conclude that the mutation inhibits functioning by blocking the rotary motor action of the enzyme.
The effects of cysteine to alanine mutations of CD18 on the expression and adhesion of the CD11/CD18 integrins
Authors: Nolan Sheila M. , Mathew Elizabeth C. , Scarth Sarah L. , Al-Shamkhani Aymen , Law S.K.Alex
Published: July 2002

Of the 56 cysteines in the extracellular domain of the CD18 antigen (β2 integrin subunit), corresponding ones are not found in 12 positions in the β4, β7, or β8 integrin subunits. These 12 cysteines were mutated to alanines, either singly or in pairs, in CD18. All these mutants can support the expression of all three CD11/CD18 integrins. Transfectants expressing these variant integrins are generally more adhesive than the wild‐type, suggesting that the cysteine residues, perhaps by engaging in disulphide bonds, may contribute to the maintenance of the CD11/CD18 integrins in a resting state.
Cycloamylose as an efficient artificial chaperone for protein refolding
Authors: Machida Sachiko , Ogawa Setsuko , Xiaohua Shi , Takaha Takeshi , Fujii Kazutoshi , Hayashi Kiyoshi
Published: July 2002

High molecular weight cyclic α‐1,4‐glucan (referred to as cycloamylose) exhibited an artificial chaperone property toward three enzymes in different categories. The inclusion properties of cycloamylose effectively accommodated detergents, which keep the chemically denatured enzymes from aggregation, and promoted proper protein folding. Chemically denatured citrate synthase was refolded and completely recovered it's enzymatic activity after dilution with polyoxyethylenesorbitan buffer followed by cycloamylose treatment. The refolding was completed within 2 h, and the activity of the refolded citrate synthase was quite stable. Cycloamylose also promoted the refolding of denatured carbonic anhydrase B and denatured lysozyme of a reduced form.
Genetics and evolution of ultraviolet vision in vertebrates
Authors: Yokoyama Shozo , Shi Yongsheng
Published: July 2002

Various vertebrates use ultraviolet (UV) vision for such basic behaviors as mating, foraging, and predation. We have successfully interchanged the color‐sensitivities of the mouse UV pigment and the human blue pigment by introducing forward and reverse mutations at five sites. This unveils for the first time the general mechanism of UV vision. Most contemporary UV pigments in vertebrates have maintained their ancestral functions by accumulating no more than one of the five specific amino acid changes. The avian lineage is an exception, where the ancestral pigment lost UV‐sensitivity but some descendants regained it by one amino acid replacement at an entirely different site.
Kidney produces a novel acylated peptide, ghrelin
Authors: Mori Kiyoshi , Yoshimoto Akihiro , Takaya Kazuhiko , Hosoda Kiminori , Ariyasu Hiroyuki , Yahata Kensei , Mukoyama Masashi , Sugawara Akira , Hosoda Hiroshi , Kojima Masayasu , Kangawa Kenji , Nakao Kazuwa
Published: July 2002

Ghrelin is a novel growth hormone‐releasing peptide with a unique acylated structure. Here we reveal that prepro‐ghrelin gene is expressed in the mouse kidney and glomerulus. We also show by reverse‐phase high performance liquid chromatography coupled with radioimmunoassay that the mouse kidney does produce ghrelin. The ghrelin immunoreactivity in the mouse kidney is 6.79±0.48 fmol/mg (n=5), which is much more abundant than that in the mouse plasma of 0.339±0.029 fmol/μl (n=6). Furthermore, prepro‐ghrelin gene is expressed in cultured rat mesangial cells, fibroblast‐like NRK‐49F cells and mouse podocytes, but not in rat epithelial cell‐like NRK‐52E cells. Ghrelin receptor gene is also expressed in the rat kidney. These findings demonstrate that the kidney, glomerulus and renal cells express prepro‐ghrelin gene and ghrelin is produced locally in the kidney, and suggest the endocrine and/or paracrine roles of ghrelin in the kidney.
The formation of chlorophyll from chlorophyllide in leaves containing proplastids is a four‐step process
Authors: Schoefs Benoît , Bertrand Martine
Published: July 2002

The time course of the different esters of chlorophyllide (Chlide) during the formation of chlorophyll a (Chl) in embryonic bean leaves containing proplastids was investigated by HPLC. After the reduction of photoactive Pchlide (Pchlide) to Chlide, three intermediates, i.e. Chlide geranylgeraniol, Chlide dihydrogeranylgeraniol and Chlide tetrahydrogeranylgeraniol were detected before the formation of Chlide phytol, i.e. authentic Chl. The transformation of Chlide to Chl was found to be much faster in leaves containing proplastids than in etiolated leaves with etioplasts.
Insulin selectively stimulates nuclear phosphoinositide‐specific phospholipase C (PI‐PLC) β1 activity through a mitogen‐activated protein (MAP) kinase‐dependent serine phosphorylation
Authors: Martelli Alberto M. , Billi Anna Maria , Manzoli Lucia , Faenza Irene , Aluigi Michela , Falconi Mirella , De Pol Anto , Gilmour R.Stewart , Cocco Lucio
Published: July 2002

Using NIH 3T3 cells, we have investigated nuclear phosphoinositide metabolism in response to insulin, a molecule which acts as a proliferating factor for this cell line and which is known as a powerful activator of the mitogen‐activated protein (MAP) kinase pathway. Insulin stimulated inositol lipid metabolism in the nucleus, as demonstrated by measurement of the diacylglycerol mass produced in vivo and by in vitro nuclear phosphoinositide‐specific phospholipase C (PI‐PLC) activity assay. Despite the fact that nuclei of NIH 3T3 cells contained all of the four isozymes of the β family of PI‐PLC (i.e. β1, β2, β3, and β4), insulin only activated the β1 isoform. Insulin also induced nuclear translocation of MAP kinase, as demonstrated by Western blotting analysis, enzyme activity assays, and immunofluorescence staining, and this translocation was blocked by the specific MAP kinase kinase inhibitor PD98059. By means of both a monoclonal antibody recognizing phosphoserine and in vivo labeling with [32P]orthophosphate, we ascertained that nuclear PI‐PLC‐β1 (and in particular the b subtype) was phosphorylated on serine residues in response to insulin. Both phosphorylation and activation of nuclear PI‐PLC‐β1 were substantially reduced by PD98059. Our results conclusively demonstrate that activation of nuclear PI‐PLC‐β1 strictly depends on its phosphorylation which is mediated through the MAP kinase pathway.