bluuebunny/crossref_metadata_2025 huggingface dataset:
Hyaluronate depolymerization activity induced by progesterone in cultured fibroblasts derived from human uterine cervix
Authors: Tanaka Kanji , Nakamura Toshiya , Ikeya Hitoshi , Higuchi Tsuyoshi , Tanaka Akihiro , Morikawa Akiko , Saito Yoshiharu , Takagaki Keiichi , Endo Masahiko
Published: July 2002

High‐molecular‐weight [14C]hyaluronate was incubated with cultured fibroblasts from human uterine cervix and skin, and then the depolymerization of the hyaluronate was investigated. [14C]Hyaluronate in the medium of skin fibroblasts was depolymerized into a constant molecular weight (M
r about 40,000), whereas that of cervix fibroblasts was not depolymerized, irrespective of incubation period. However, when progesterone was added to the medium of cervix fibroblasts, hyaluronate was depolymerized to the same extent as that in skin fibroblasts. The reducing terminal sugar of the depolymerized hyaluronate was N‐acetylglucosamine. These results suggest that a hyaluronate‐depolymerizing enzyme, endo‐β‐N‐acetylglucosaminidase, was induced by progesterone in cultured fibroblasts derived from human uterine cervix.
Actophorin preferentially binds monomeric ADP‐Actin over ATP‐bound actin: consequences for cell locomotion
Authors: Maciver Sutherland K. , Weeds Alan G.
Published: July 2002

Actophorin from Acanthamoeba castellanii severs actin filaments and sequesters actin monomers. Here we report that actophorin binds ADP‐bound monomers with higher affinity than ATP‐bound monomers. Actophorin is therefore much less efficient at severing actin filaments in the presence of ADP compared to ATP, particularly taking account of the higher critical concentration in ADP. Monomer binding is also reduced in the presence of 25 mM inorganic phosphate (which is assumed to form ADP · Pi‐actin). These findings are discussed in the light of observations on the nucleotide specificity of other monomer binding proteins and related to the role of actin in lamellar protrusion and cell locomotion.
Amyloid precursor protein secretion and βA4 amyloid generation are not mutually exclusive
Authors: Dyrks Thomas , Mönning Ursula , Beyreuther Konrad , Turner Jonathan
Published: July 2002

The cellular factors regulating the generation of βA4 from the amyloid precursor protein (APP) are unknown. Protein phosphorylation by protein kinase C (PKC) has been found to influence the processing and metabolism of APP. In this report, we show that in the human neuroblastoma cell line SY5Y, βA4 generation from full‐length APP is not changed by PKC activation whereas production of the non‐amyloidogenic secretory fragment (APPsec) and of the C‐terminal fragment of βA4 (p3) are stimulated. In addition, βA4 generation from the membrane inserted C‐terminal 100 residues (SPA4CT) of APP is stimulated by PKC activation. Accordingly attempts to divert APP processing from the amyloidogenic, βA4‐generating, to the non‐amyloidogenic, secretory, pathway, have to address the nature and regulation of the two pathways and/or of the process leading to the cleavage of APP at the C‐terminus of the βA4 domain. The data reported here suggest that these mechanisms are cell‐type specific.
Immunochemical identification and translocation of protein kinase C zeta in human neutrophils
Authors: Dang Pham My-Chan , Hakim Jacques , Périanin Axel
Published: July 2002

Western blots of human polymorphonuclear leukocyte (PMN) extracts were immunostained with antibodies specific for various protein kinase C (PKC) isoforms. Two bands corresponding to PKC type ξ with apparent molecular masses of 81 kDa and 76 kDa were identified in the cytosolic fraction of resting cells, in addition to PKC types α and β. PKCξ was apparently abundant, like PKCβ, whereas PKCδ, ‐ε, and ‐γ were not detectable. Following short stimulation (5 min) of PMN with phorbol‐12‐myristate‐13‐acetate (1 μ/ml), physical translocation of PKCξ from the cytosol to the plasma membrane fraction occurred, although this isoform does not bind phorbol esters. These data show that, in addition to the two calcium‐dependent isoenzymes α and β, human PMN express a calcium‐independent isoenzyme ξ which translocates in stimulated cells, suggesting a role in the regulation of antibacterial activities.
Molecular cloning of a novel candidate G protein‐coupled receptor from rat brain
Authors: Song Z.H. , Young W.Scott , Brownstein Michael J. , Bonner Tom I.
Published: July 2002

A PCR cloning strategy using primers designed from sequences selectively conserved among a cannabinoid receptor and two orphan receptors, was used to isolate novel G protein‐coupled receptors. rCNL3, a 1.75 kb cDNA encoding a 363 amino acid protein, was isolated from a rat cerebral cortex library. Sequence analysis showed that rCNL3 possesses a number of structural characteristics of G protein‐coupled receptors and has 61% amino acid identity (from transmembrane region one through the carboxyl‐terminus) with two other candidate G protein‐coupled receptors. Therefore, these three receptors may comprise a receptor subfamily with identical or closely related endogenous ligands. Northern and in situ hybridization experiments demonstrated that rCNL3 mRNA is expressed in the rat brain, with a prominent distribution in striatum.
Calibration and time resolution of lumenal pH‐transients in chromatophores of Rhodobacter capsulatus following a single turnover flash of light: Proton release by the cytochrome bc1‐complex is strongly electrogenic
Authors: Mulkidjanian Armen Y. , Junge Wolfgang
Published: July 2002

The flash‐induced proton release into the lumen of chromatophores from Rhodobacter capsulatus was studied with Neutral red as pH‐indicator. Calibration of the acidification jump after a single flash yielded a much larger figure, at least 0.8 units, than previously thought. A slow kinetic phase of proton release (85–90% of total) was sensitive to inhibitors of the cytochrome bc
1‐complex. Its half‐rise time, about 10 ms, was the same as the rise time of the electrogenic reaction in the cytochrome bc
1‐complex that was recorded by electrochromism of carotenoids. The oxidoreduction of the two b‐hemes was significantly faster (t
½ ≅ 3 ms). Thus the major electrogenic event in the cytochome bc
1‐complex is proton and not electron transfer.
Precursor of cdk5 activator, the 23 kDa subunit of tau protein kinase II: Its sequence and developmental change in brain
Authors: Uchida T. , Ishiguro K. , Ohnuma J. , Takamatsu M. , Yonekura S. , Imahori K.
Published: July 2002

Tau protein kinase II (TPKII) is shown by immunoprecipitation to be a complex composed of two subunits, a catalytic subunit, cdk5, and regulatory subunit, p23. By sequence analysis of p23 CDNA, p23 was found to occupy a region from the 99th amino acid residue to the C‐terminus of a novel protein with a molecular weight of 34,000 Da, suggesting that this 34 kDa protein is a precursor of p23 (pre‐p23). These findings suggest that p23 results from the processing of the precursor protein, pre‐p23. The precursor mRNA was expressed most abundantly in rat brain just before and after birth. Expression of pre‐p23, but not of cdk5, mRNA changed, coinciding with the developmental change of TPKII activity, suggesting that its expression controls the phosphorylation of tau by the TPKII/TPKI system in the neonatal brain. p23 appears to be a cdk5 activator in neuronal cells.
Gallinacins: cysteine‐rich antimicrobial peptides of chicken leukocytes
Authors: Harwig Sylvia S.L. , Swiderek Kristine M. , Kokryakov Vladimir N. , Tan Leonie , Lee Terry D. , Panyutich Elena A. , Aleshina Galina M. , Shamova Olga V. , Lehrer Robert I.
Published: July 2002

We purified three homologous antimicrobial peptides (‘gallinacins’) from chicken leukocytes, examined their antimicrobial activity in vitro, and established their primary sequences by a combination of gas phase microsequencing and on‐line LC‐ESI‐MS analysis of endo‐ and exoprotease peptide digests. The peptides contained 36–39 amino acid residues, were relatively cationic due to their numerous lysine and arginine residues, and each contained 3 intramolecular cystine disulfide bonds. Gallinacins showed primary sequence homology to the recently delineated β‐defensin family, heretofore found only in the respiratory epithelial cells and neutrophils of cattle, suggesting that β‐defensins originated at least 250 million years ago, before avian and mammalian lineages diverged. The 9 invariant residues (6 cysteines, 2 glycines and 1 proline) common to avian gallinacins and bovine β‐defensins are likely to constitute the essential primary structural motif of this ancient family of host‐defense peptides.
Influenza virus M2 protein modifies membrane permeability in E. coli cells
Authors: Guinea Rosario , Carrasco Luis
Published: July 2002

The M2 protein of influenza virus is an integral membrane protein with ion channel activity. This protein has been expressed in E. coli cells in an inducible manner. Expression of the M2 protein causes rapid lysis of BL21(DE3) pLysS E. coli cells upon induction with IPTG. M2 protein increases membrane permeability to a number of hydrophylic molecules, such as ONPG, uridine or impermeant translation inhibitors. The behaviour of M2 in bacteria resembles that of other viral proteins, such as poliovirus 3A and Semliki Forest virus 6K.
Leukotriene A4, and not leukotriene B4, is the main 5‐lipoxygenase metabolite released by bovine leukocytes
Authors: Sala Angelo , Testa Tullio , Folco Giancarlo
Published: July 2002

The production of leukotriene A4 (LTA4)‐derived metabolites, analysed by RP‐HPLC, was studied in purified bovine polymorphonuclear leukocyte (PMNL) preparations and in PMNL‐platelet coincubations after challenge with the calcium ionophore A23187. The results obtained show that in bovine PMNL LTB4 represents the main LTA4 metabolite. When washed platelets were added to PMNL, LTC4 was the main enzymatic metabolite observed, indicating a substantial transfer of PMNL‐derived LTA4 to platelets. The synthesis of LTC4 was accompanied by a significant decrease in LTB4, suggesting that a quota of the LTB4 synthesized in PMNL preparations is the result of transcellular metabolism of released LTA4 by neighbouring PMNL. Reduction of PMNL‐PMNL interactions through dilution of cell incubates allowed us to estimate that most of the leukotriene A4 synthesized by PMNL is indeed released from the cell. LTA4, and not LTB4, represents the main 5‐lipoxygenase metabolite released by bovine PMNL.
Cell‐mediated cytotoxicity can be regulated by p53 tumor suppressor gene activity in vitro
Authors: Mahdi Tarek , Tanzer Joseph , Brizard André , Guilhot François , Babin Philippe , Kitzis Alain
Published: July 2002

Summary— The wild‐type human p53 tumor suppressor gene was tested for its ability to modulate cytotoxic activity of in vitro activated peripheral blood lymphocytes. Peripheral blood mononuclear cells (PBMCs) were stimulated by phytohemagglutinin (PHA), interferon α2b (IFNα2b), interleukin 2 (IL‐2) or their combinations to induce cytotoxicity. This stimulation significantly increased the percentage of cells expressing p53, which was at its maximum when induced by IL‐2 combined with IFNα2b. The role of p53 in the modulation of different aspects of cytotoxic activity of these cells was analyzed by studying the effects of p53 abrogation by antisense oligonucleotide (p53 AS) treatment in comparison with p53 sense or scrambled (missense) oligonucleotide (p53 S or p53 MS) treatment. We show that p53 plays a key role through induction of apoptosis in target cells (tumor necrosis factor pathway) rather than through osmolytic degeneration (perforin pathway) which is only slightly increased by p53 abrogation. Meanwhile, in vitro abrogation of p53 expression in PBL was found to be accompanied by an increase of CD8+ lymphocytes and an important increase of the CD56 ‘bright’ NK cell sub‐population.
DIRECTIONAL NEURITE OUTGROWTH AND AXONAL DIFFERENTIATION OF EMBRYONIC HIPPOCAMPAL NEURONS ARE PROMOTED BY A NEURITE OUTGROWTH DOMAIN OF THE B2‐CHAIN OF LAMININ
Authors: Matsuzawa Mieko , Weight Forrest F. , Potember Richard S. , Liesi Päivi
Published: July 2002

Molecular cues involved in directional neurite outgrowth and axonal differentiation of embryonic hippocampal neurons were studied on substrates coated in a striped 5 μm pattern with synthetic peptides from a neurite outgrowth (RDIAEIIKDI, P1543) and cell attachment (CDPGYIGSR, P364) domain of the B2‐ and B1‐chains of laminin, respectively. Both peptides supported neuronal attachment, but only the B2‐chain‐derived P1543 promoted expression of a mature neuronal phenotype. Directional neurite outgrowth and axonal differentiation of embryonic hippocampal neurons were selectively induced by striped substrates of the B2‐chain‐derived P1543. Axonal differentiation was determined by expression of a phosphorylated epitope of the 200 kDa neurofilament protein in the longer “axonal” neurite of the bipolar embryonic hippocampal neurons. Ethanol (100 mM), a neuroactive compound known to delay neuronal development, impaired both directional neurite outgrowth and expression of a phosphorylated epitope of the 200 kDa neurofilament protein on a patterned P1543 substratum. The present results provide direct evidence that a 10 amino acid peptide (P1543), derived from a neurite outgrowth domain of the B2‐chain of laminin, may be an axonal guidance and differentiation factor for embryonic hippocampal neurons in vitro.
Distinct importin recognition properties of histones and chromatin assembly factors
Authors: Johnson-Saliba Melanie , Siddon Nicole A , Clarkson Michael J , Tremethick David J , Jans David A
Published: July 2002

Synthesis of the protein components of nuclear chromatin occurs in the cytoplasm, necessitating specific import into the nucleus. Here, we report the binding affinities of the nuclear localisation sequence (NLS)‐binding importin subunits for a range of histones and chromatin assembly factors. The results suggest that import of histones to the nucleus may be mediated predominantly by importin β1, whereas the import of the other components probably relies on the conventional α/β1 import pathway. Differences in recognition by importin β1 were observed between histone H2A and the variant H2AZ, as well as between histone H3/4 with or without acetylation. The results imply that different histone variants may possess distinct nuclear import properties, with acetylation possibly playing an inhibitory role through NLS masking.
Purification and characterization of dissimilatory nitrate reductase from a denitrifying halophilic archaeon, Haloarcula marismortui
Authors: Yoshimatsu Katsuhiko , Sakurai Takeshi , Fujiwara Taketomo
Published: July 2002

Dissimilatory nitrate reductase was purified from a denitrifying halophilic archaeon, Haloarcula marismortui, to an electrophoretically homogeneous state. The purified enzyme was inferred to be a homotetramer composed of a 63 kDa polypeptide. The electron paramagnetic resonance spectrum of the purified enzyme revealed typical rhombic signals which were ascribed to Mo(V) in the Mo–molybdopterin complex. Like the bacterial membrane‐bound (Nar‐) enzyme, the purified enzyme supported the catalysis of chlorate. The enzyme was activated in extreme saline conditions and the values of k
cat and K
m toward nitrate were 145 s−1 and 79 μM, respectively, in the presence of 2.0 M NaCl.
Activation of a capacitative Ca2+ entry pathway by store depletion in cultured hippocampal neurones
Authors: Bouron Alexandre
Published: July 2002

Intracellular Ca2+ ([Ca2+]i) changes were measured in cell bodies of cultured rat hippocampal neurones with the fluorescent indicator Fluo‐3. In the absence of external Ca2+, the cholinergic agonist carbachol (200 μM) and the sarco‐endoplasmic reticulum Ca2+ pump inhibitor thapsigargin (0.4 μM) both transiently elevated [Ca2+]i. A subsequent addition of Ca2+ into the bathing medium caused a second [Ca2+]i change which was blocked by lanthanum (50 μM). Taken together, these experiments indicate that stores depletion can activate a capacitative Ca2+ entry pathway in cultured hippocampal neurones and further demonstrate the existence of such a Ca2+ entry in excitable cells.
Direct association of LIS1, the lissencephaly gene product, with a mammalian homologue of a fungal nuclear distribution protein, rNUDE
Authors: Kitagawa Mayumi , Umezu Makiko , Aoki Junken , Koizumi Hiroyuki , Arai Hiroyuki , Inoue Keizo
Published: July 2002

LIS1 is a product of the causative gene for type I lissencephaly characterized by a smooth brain surface due to a defect in neuronal migration during brain development and a regulatory subunit of platelet‐activating factor acetylhydrolase (PAF‐AH). It is also a mammalian homologue of the fungal nuclear distribution (nud) gene, nudF, which controls the migration of fungal nuclei. Using the two‐hybrid system, we identified a novel LIS1‐interacting protein, rat NUDE (rNUDE), and found that it is a mammalian homologue of another fungal nud gene product, NUDE, and Xenopus mitotic phosphoprotein 43 which is phosphorylated in a cell cycle‐dependent manner. rNUDE and the catalytic subunits of PAF‐AH interact with the N‐ and C‐termini of LIS1, respectively. However, these proteins, instead of simultaneously binding to LIS1, appeared to bind to LIS1 in a competitive manner. These results suggest that LIS1 functions in nuclear migration by interacting with multiple intracellular proteins in mammals.
The human homologue of the yeast mitochondrial AAA metalloprotease Yme1p complements a yeast yme1 disruptant
Authors: Shah Zahid H. , Hakkaart Gerrit A.J. , Arku Benedict , de Jong Lisbeth , van der Spek Hans , Grivell Leslie A. , Jacobs Howard T.
Published: July 2002

In yeast, three AAA superfamily metalloproteases (Yme1p, Afg3p and Rca1p) are localized to the mitochondrial inner membrane where they perform roles in the assembly and turnover of the respiratory chain complexes. We have investigated the function of the proposed human orthologue of yeast Yme1p, encoded by the YME1L gene on chromosome 10p. Transfection of both HEK‐293EBNA and yeast cells with a green fluorescent protein‐tagged YME1L cDNA confirmed mitochondrial targeting. When expressed in a yme1 disruptant yeast strain, YME1L restored growth on glycerol at 37°C. We propose that YME1L plays a phylogenetically conserved role in mitochondrial protein metabolism and could be involved in mitochondrial pathologies.
Protection against oxidant‐mediated lysosomal rupture: a new anti‐apoptotic activity of Bcl‐2?
Authors: Zhao Ming , Eaton John W. , Brunk Ulf T.
Published: July 2002

Bcl‐2 antagonizes apoptosis through mechanisms which are not completely understood. We have proposed that apoptosis is initiated by minor lysosomal destabilization followed some time later by secondary massive lysosomal rupture. In J774 cells over‐expressing Bcl‐2, early oxidant‐induced lysosomal destabilization is unaffected but secondary lysosomal rupture and apoptosis are suppressed, despite the fact that wild‐type and Bcl‐2 over‐expressing cells degrade hydrogen peroxide at similar rates. It may be that Bcl‐2 directly blocks the effects of released lysosomal enzymes and/or prevents downstream activation of unknown cytosolic pro‐enzymes by released lysosomal hydrolases, suggesting a new and heretofore unknown activity of Bcl‐2.
The asp‐rich region at the carboxyl‐terminus of calsequestrin binds to Ca2+ and interacts with triadin
Authors: Shin Dong Wook , Ma Jianjie , Kim Do Han
Published: July 2002

Calsequestrin (CSQ) is a high capacity Ca2+ binding protein in the junctional sarcoplasmic reticulum of striated muscles, and has been shown to regulate the ryanodine receptor (RyR) through triadin and junctin. In order to identify the functional roles of specific regions on CSQ, several CSQ deletion mutants were prepared by molecular cloning and Escherichia coli expression. 45Ca2+ overlay assay using a native gel system revealed that the major Ca2+ binding motif of CSQ resides in the asp‐rich region (amino acids 354–367). In an in vitro binding assay using a glutathione‐S‐transferase affinity column, the interaction between CSQ and triadin was found to be Ca2+‐dependent, and the site of interaction was confined to the asp‐rich region of CSQ. Our results suggest that the asp‐rich region of CSQ could participate in the RyR‐mediated Ca2+ release process by offering a direct binding site to luminal Ca2+ as well as triadin.
Potential role of group X secretory phospholipase A2 in cyclooxygenase‐2‐dependent PGE2 formation during colon tumorigenesis
Authors: Morioka Yasuhide , Ikeda Minoru , Saiga Akihiko , Fujii Noriko , Ishimoto Yoshikazu , Arita Hitoshi , Hanasaki Kohji
Published: July 2002

Although the cyclooxygenase‐2 (COX‐2) pathway of the arachidonic acid cascade has been suggested to play an important role in colon carcinogenesis, there is little information concerning the identity of phospholipase A2 (PLA2) involved in the arachidonic acid release in colon tumors. Here, we compared the potencies of three types of secretory PLA2s (group IB, IIA and X sPLA2s) for the arachidonic acid release from cultured human colon adenocarcinoma cells, and found that group X sPLA2 has the most powerful potency in the release of arachidonic acid leading to COX‐2‐dependent prostaglandin E2 (PGE2) formation. Furthermore, immunohistological analysis revealed the elevated expression of group X sPLA2 in human colon adenocarcinoma neoplastic cells in concert with augmented expression of COX‐2. These findings suggest a critical role of group X sPLA2 in the PGE2 biosynthesis during colon tumorigenesis.
Alternative splice variants of hTrp4 differentially interact with the C‐terminal portion of the inositol 1,4,5‐trisphosphate receptors
Authors: Mery Laurence , Magnino Fabrice , Schmidt Karin , Krause Karl-Heinz , Dufour Jean-François
Published: July 2002

The molecular basis of capacitative (or store‐operated) Ca2+ entry is still subject to debate. The transient receptor potential proteins have been hypothesized to be structural components of store‐operated Ca2+ channels and recent evidence suggests that Trp3 and its closely related homolog Trp6 are gated by the N‐terminal region of the inositol 1,4,5‐triphosphate receptors (InsP3R). In this study, we report the existence of two isoforms of the human Trp4 protein, referred to as α‐hTrp4 and β‐hTrp4. The shorter variant β‐hTrp4 is generated through alternative splicing and lacks the C‐terminal amino acids G785–S868. Using a yeast two‐hybrid assay and glutathione‐S‐transferase‐pulldown experiments, we found that the C‐terminus of α‐hTrp4, but not of β‐hTrp4, associates in vitro with the C‐terminal domain of the InsP3 receptors type 1, 2 and 3. Thus, we describe a novel interaction between Trp proteins and InsP3R and we provide evidence suggesting that the formation of hTrp4–InsP3R complexes may be regulated by alternative splicing.
ADP‐insensitive phosphoenzyme intermediate of sarcoplasmic reticulum Ca2+‐ATPase has a compact conformation resistant to proteinase K, V8 protease and trypsin
Authors: Danko Stefania , Daiho Takashi , Yamasaki Kazuo , Kamidochi Mika , Suzuki Hiroshi , Toyoshima Chikashi
Published: July 2002

Sarcoplasmic reticulum Ca2+‐ATPase was digested with proteinase K, V8 protease and trypsin in the absence of Ca2+. Unphosphorylated enzyme was rapidly degraded. In contrast, ADP‐insensitive phosphoenzyme formed with Pi and phosphorylated state analogues produced by the binding of F− or orthovanadate, were almost completely resistant to the proteolysis except for tryptic cleavage at the T1 site (Arg505). The results indicate that the phosphoenzyme and its analogues have a very compact form in the cytoplasmic region, being consistent with large domain motions (gathering of three cytoplasmic domains). Results further show that the structure of the enzyme with bound decavanadate is very similar to ADP‐insensitive phosphoenzyme. Thapsigargin did not affect the changes in digestion time course induced by the formation of the phosphorylated state analogues.
Extracellular Ba2+ and voltage interact to gate Ca2+ channels at the plasma membrane of stomatal guard cells
Authors: Hamilton David W.A. , Hills Adrian , Blatt Michael R.
Published: July 2002

Ca2+ channels at the plasma membrane of stomatal guard cells contribute to increases in cytosolic free [Ca2+] ([Ca2+]i) that regulate K+ and Cl− channels for stomatal closure in higher‐plant leaves. Under voltage clamp, the initial rate of increase in [Ca2+]i in guard cells is sensitive to the extracellular divalent concentration, suggesting a close interaction between the permeant ion and channel gating. To test this idea, we recorded single‐channel currents across the Vicia guard cell plasma membrane using Ba2+ as a charge carrying ion. Unlike other Ca2+ channels characterised to date, these channels activate at hyperpolarising voltages. We found that the open probability (P
o) increased strongly with external Ba2+ concentration, consistent with a 4‐fold cooperative action of Ba2+ in which its binding promoted channel opening in the steady state. Dwell time analyses indicated the presence of a single open state and at least three closed states of the channel, and showed that both hyperpolarising voltage and external Ba2+ concentration prolonged channel residence in the open state. Remarkably, increasing Ba2+ concentration also enhanced the sensitivity of the open channel to membrane voltage. We propose that Ba2+ binds at external sites distinct from the permeation pathway and that divalent binding directly influences the voltage gate.
Complex formation and submembranous localization of annexin 2 and S100A10 in live HepG2 cells
Authors: Zobiack Nicole , Gerke Volker , Rescher Ursula
Published: July 2002

The Ca2+ and membrane binding protein annexin 2 can form a heterotetrameric complex with the S100A10 protein and this complex is thought to serve a bridging or scaffolding function in the membrane underlying cytoskeleton. To elucidate which of the subunits targets the complex to the subplasmalemmal region in live cells we employed YFP/CFP fusion proteins and live cell imaging in HepG2 cells. We show that monomeric annexin 2 is targeted to the plasma membrane whereas non‐complexed S100A10 acquires a general cytosolic distribution. Co‐expression of S100A10 together with annexin 2 and the resulting complex formation, however, lead to a recruitment of S100A10 to the plasma membrane thus identifying annexin 2 as the membrane targeting subunit.
CYP98A6 from Lithospermum erythrorhizon encodes 4‐coumaroyl‐4′‐hydroxyphenyllactic acid 3‐hydroxylase involved in rosmarinic acid biosynthesis1
Authors: Matsuno Michiyo , Nagatsu Akito , Ogihara Yukio , Ellis Brian E , Mizukami Hajime
Published: July 2002

Rosmarinic acid is the dominant hydroxycinnamic acid ester accumulated in Boraginaceae and Lamiaceae plants. A cytochrome P450 cDNA was isolated by differential display from cultured cells of Lithospermum erythrorhizon, and the gene product was designated CYP98A6 based on the deduced amino acid sequence. After expression in yeast, the P450 was shown to catalyze the 3‐hydroxylation of 4‐coumaroyl‐4′‐hydroxyphenyllactic acid, one of the final two steps leading to rosmarinic acid. The expression level of CYP98A6 is dramatically increased by addition of yeast extract or methyl jasmonate to L. erythrorhizon cells, and its expression pattern reflected the elicitor‐induced change in rosmarinic acid production, indicating that CYP98A6 plays an important role in regulation of rosmarinic acid biosynthesis.
Cloning, expression and CNS distribution of Kv4.3, an A‐type K+ channel α subunit
Authors: Tsaur Meei-Ling , Chou Chaio-Chaio , Shih Yang-Hsin , Wang Hung-Li
Published: July 2002

A full‐length K+ channel cDNA of Kv4.3, with an open reading frame of 611 amino acids, was isolated from rat hippocampus. Functional expression of Kv4.3 cDNA in Xenopus oocytes revealed an A‐type K+ channel. In the central nervous system, Kv4.3 is most prominently expressed in the retrosplenial cortex, medial habenula, anterior thalamus, hippocampus, cerebellum, as well as lateral geniculate and superior colliculus, which are important for vision. The abundant expression of Kv4.3 in many CNS neurons supports its important role as a major component of subthreshold A currents in the control of action potentials and thus neuronal excitability.
Mapping of a potent transcriptional repression region of the human homeodomain protein EVX1
Authors: Briata Paola , Ilengo Cristina , Van DeWerken Rinke , Corte Giorgio
Published: July 2002

The human homeodomain protein EVX1 is a transcriptional repressor in transfected mammalian cells and this function depends on a region carboxyl‐terminal to the homeodomain. In this study, we transiently expressed several deletions of the EVX1 C‐terminal region in mammalian cells and investigated their effect on the transcription of a reporter gene directed by different promoters. We show that the repressor activity maps to a region of 51 amino acids with a high abundance of alanine and proline residues. This region is able to transfer the repressor function to either the entire HOXC6 or CREB transcription factors, or to the GAL4 DNA binding domain.
Newly delivered transferrin iron and oxidative cell injury
Authors: Breuer William , Greenberg Eran , Cabantchik Z.Ioav
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Bcl‐2 prevents nitric oxide‐mediated apoptosis and poly(ADP‐ribose) polymerase cleavage
Authors: Mělková Zora , Lee Sean Bong , Rodriguez Dolores , Esteban Mariano
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Single molecular assay of individual ATP turnover by a myosin‐GFP fusion protein expressed in vitro
Authors: Iwane Atsuko Hikikoshi , Funatsu Takashi , Harada Yoshie , Tokunaga Makio , Ohara Osamu , Morimoto Sayuri , Yanagida Toshio
Published: July 2002

Fusion proteins of a truncated mutant of myosin subfragment‐1 (S1dC) and green fluorescent protein (GFP) were expressed in vitro by T7 RNA polymerase and rabbit reticulocyte lysate. Single S1dC‐GFP fusion proteins were clearly seen and their individual ATP turnovers were directly monitored using low background total internal reflection fluorescence microscopy (LBTIRFM), recently developed by our laboratory. LBTIRFM using GFP as a fluorescent tag allowed us to assay functions of single protein molecules expressed in vitro. Thus, the results suggested that this method may be particularly useful to analyze functions of proteins that cannot be produced in an active form and/or in large quantities in conventional heterologous expression systems.
Activation of Stat1 and subsequent transcription of inducible nitric oxide synthase gene in C6 glioma cells is independent of interferon‐γ‐induced MAPK activation that is mediated by p21ras
Authors: Nishiya Tadashi , Uehara Takashi , Edamatsu Hiroki , Kaziro Yoshito , Itoh Hiroshi , Nomura Yasuyuki
Published: July 2002

Rat C6 glioma cells have been used to characterize molecular events involved in the regulation of inducible nitric oxide synthase (iNOS) gene expression stimulated by interferon‐γ (IFN‐γ) plus lipopolysaccharide (LPS). IFNs induce a signaling event which involves activation of Stat1 transcription factor. Previous studies have shown that IFNs also induce extracellular signal‐regulated kinase/mitogen‐activated protein kinase (ERK/MAPK) activation. However, the mechanisms by which IFNs stimulate MAPK activation remain elusive. Here we show that in C6 glioma cells, transiently expressing the dominant‐negative form of c‐Ha‐Ras (Asn‐17) abrogated IFN‐γ‐induced ERK1 and ERK2 activation. Furthermore, PD98059, a specific MEK1 inhibitor, also blocked this activation. These results indicate that p21
ras
and MEK1 are required for IFN‐γ‐induced ERK1 and ERK2 activation. Recent studies have reported that MAPK is responsible for serine phosphorylation of Stat1 which is required for Stat1's DNA binding and maximal transcriptional activity. Thus, we examined the role of the Ras‐MAPK pathway in Stat1 activation and subsequent iNOS induction in C6 glioma cells. Further experiments showed that neither Asn‐17 Ras expression nor concentrations of PD98059, which completely abrogated IFN‐γ‐induced ERK1 and ERK2 activation, affected Stat1 DNA binding activity or iNOS induction, indicating that the Ras‐MAPK pathway does not appear to be involved in the activation of Stat1 and subsequent iNOS induction in C6 glioma cells.
Regulation of the DNA binding of p53 by its interaction with protein kinase CK2
Authors: Prowald Alexandra , Schuster Norbert , Montenarh Mathias
Published: July 2002

Some of the numerous functions of the growth suppressor protein p53 are regulated by its interaction with viral and cellular proteins. C‐terminal sequences of p53 are implicated in binding to the regulatory β‐subunit of protein kinase CK2. Using a p53‐specific DNA binding element we found that the β‐subunit of CK2 inhibited the DNA binding of p53 whereas the α‐subunit had no influence. The CK2 holoenzyme consisting of two α‐ and two β‐subunits led to a supershift in DNA binding of p53 similar to the p53‐specific monoclonal antibody PAb421 as well as the C‐terminus of p53. Thus, our results showed an individual role of the free β‐subunit of CK2 on the DNA binding activity of p53.
Regulation of proliferation and apoptosis by Ras and Rho GTPases through specific phospholipid‐dependent signaling
Authors: Lacal Juan Carlos
Published: July 2002

Small GTPases are molecular switches that control signaling pathways critical for diverse cellular functions. Recent evidence indicates that multiple effector molecules can be activated by small GTPases. As a result, complex biological processes such as cell proliferation and apoptosis are turned on. Thus, rather than a single linear pathway from the membrane to the nucleus, the integration of complementary signals is required for these events to occur. In fact, the coordinated activation of small GTPases may constitute some of the critical modulators of those signals triggering either proliferation or cell death. In addition to the activation of specific kinases cascades, phospholipid‐derived messengers are candidates to compose some of the most critical elements associated to regulation of signaling cascades capable of discerning among life and death. Both proliferation and apoptosis needs competence and progression signals. Phospholipase D and sphingomyelinase may be important players in this decision‐maker step.
Mutagenesis of nitrite reductase from Pseudomonas aeruginosa: tyrosine‐10 in the c heme domain is not involved in catalysis1
Authors: Cutruzzolà Francesca , Arese Marzia , Grasso Sabrina , Bellelli Andrea , Brunori Maurizio
Published: July 2002

In Pseudomonas aeruginosa, conversion of nitrite to NO in dissimilatory denitrification is catalyzed by the enzyme nitrite reductase (NiR), a homodimer containing a covalently bound c heme and a d1 heme per subunit. We report the purification and characterization of the first single mutant of P. aeruginosa cd1 NiR in which Tyr10 has been replaced by Phe; this amino acid was chosen as a possibly important residue in the catalytic mechanism of this enzyme based on the proposal (Fülöp, V., Moir, J.W.B., Ferguson, S.J. and Hajdu, J. (1995) Cell 81, 369–377) that the topologically homologous Tyr25 plays a crucial role in controlling the activity of the cd1 NiR from Thiosphaera pantotropha. Our results show that in P. aeruginosa NiR substitution of Tyr10 with Phe has no effect on the activity, optical spectroscopy and electron transfer kinetics of the enzyme, indicating that distal coordination of the Fe3+ of the d1 heme is provided by different side‐chains in different species.
Chloroplastic prenylated proteins
Authors: Parmryd Ingela , Shipton Catherine Ann , Swiezewska Ewa , Dallner Gustav , Andersson Bertil
Published: July 2002

By in vivo [3H]mevalonate labelling of spinach combined with biochemical analysis, evidence is provided for the existence of protein prenylation in chloroplasts. Approximately 20 prenylated polypeptides were resolved by SDS‐PAGE followed by autoradiography. Thermolysin treatment of intact chloroplasts revealed that about 40% of the prenylated polypeptides were associated with the cytoplasmic surface of the outer envelope membrane. The remaining portion was present in thylakoids and/or the inner envelope membrane. The majority of the prenylated polypeptides were associated with larger membrane protein complexes. A farnesyl protein transferase activity was found to be associated with the thylakoid membrane.
Sequential assignments and secondary structure of the RNA‐binding transcriptional regulator NusB1
Authors: Altieri Amanda S. , Mazzulla Marie J. , Zhou Hongjun , Costantino Nina , Court Donald L. , Byrd R.Andrew
Published: July 2002

The NusB protein is involved in transcriptional regulation in bacteriophage λ. NusB binds to the RNA form of thenutsite and along with N, NusA, NusE and NusG, stabilizes the RNA polymerase transcription complex and allows stable, persistent antitermination. NusB contains a 10 residue Arg‐rich RNA‐binding motif (ARM) at the N‐terminus but is not sequentially homologous to any other proteins. In contrast to other known ARM‐containing proteins, NusB forms a stable structure in solution in the absence of RNA. NMR spectroscopy was used to determine that NusB contains six α‐helices: R10–Q21, I27–F34, V45–L65, Q79–S93, Y100–F114 and D118–L127. The structure of NusB makes it a member of a newly emerging class of α‐helical RNA‐binding proteins.
Small heat shock proteins inhibit in vitro Aβ1–42 amyloidogenesis
Authors: Kudva Yogish C , Hiddinga Henry J , Butler Peter C , Mueske Cheryl S , Eberhardt Norman L
Published: July 2002

We demonstrate that small heat shock proteins (sHsp) inhibit in vitro amyloid formation by the Alzheimer's Aβ1–42 polypeptide as detected by a thioflavine T fluorescence assay and electron microscopy. Human sHsp27 (0.50–3.0 μM) inhibited amyloid formation from 20 μM Aβ1–42 by 23–75% in 24 h. In contrast, treatment of pre‐formed amyloid with 0.5–3.0 μM sHsp27 only reduced the fluorescence signal by 6–36%. The data suggest that ordered fibril formation may represent a form of off‐pathway aggregation that can be prevented by chaperone action. The data raise the possibility that age‐related changes in chaperone function could contribute toward the pathogenesis of Alzheimer's and other amyloid‐associated diseases.
Glucose regulates the promoter activity of aldolase A and pyruvate kinase M2 via dephosphorylation of Sp1
Authors: Schäfer Doris , Hamm-Künzelmann Brigitte , Brand Karl
Published: July 2002

Proliferating cells and tumour cells maintain a high glycolytic rate even under aerobic conditions. FTO2B cells, a rat hepatoma cell line, show high activities of glycolytic enzymes. Within a culture period of 48 h the cell number increases 5‐fold. Replacement of glucose by pyruvate in the culture medium lowers glycolytic enzyme activity and prevents proliferation. Transfection assays revealed that glucose deprivation dramatically decreases the transcriptional activities of the Sp1‐dependent aldolase and pyruvate kinase promoters leading to reduced reporter gene expression. Sp1 binding activity is also inhibited by ocadaic acid, an inhibitor of protein phosphatase 1. Western blot analyses with nuclear extracts from FTO2B cells cultured in the presence or absence of glucose revealed differences in the phosphorylation state of Sp1. From these results we conclude that glucose increases the amount of the dephosphorylated form of Sp1 which has a higher DNA binding activity. As a consequence gene expression of the glycolytic enzymes is increased which is a prerequisite for cell proliferation.
Conversion of cysteine to formylglycine in eukaryotic sulfatases occurs by a common mechanism in the endoplasmic reticulum
Authors: Dierks Thomas , Lecca Maria Rita , Schmidt Bernhard , von Figura Kurt
Published: July 2002

Sulfatases undergo an unusual protein modification leading to conversion of a specific cysteine residue into α‐formylglycine. This conversion is essential for catalytic activity. In arylsulfatase A the α‐formylglycine is generated inside the endoplasmic reticulum at a late stage of protein translocation. Using in vitro translation in the presence of transport‐competent microsomes we found that arylsulfatase B is also modified in a similar way by the formylglycine‐generating machinery. Modification depended on protein transport and on the correct position of the relevant cysteine. Arylsulfatase A and B did not compete for modification, as became apparent in co‐expression experiments. This could argue for an association of the modification machinery with the protein translocation apparatus.
Elderberry (Sambucus nigra) contains truncated Neu5Ac(α‐2,6)Gal/GalNAc‐binding type 2 ribosome‐inactivating proteins
Authors: Peumans Willy J , Roy Soma , Barre Annick , Rouge Pierre , van Leuven Fred , van Damme Els J.M
Published: July 2002

Analysis of affinity‐purified preparations of the fetuin‐binding proteins from elderberry bark and fruits revealed besides the previously reported Neu5Ac(α‐2,6)Gal/GalNAc‐specific type 2 ribosome‐inactivating proteins (RIP) the occurrence of single chain proteins of 22 kDa, which according to their N‐terminal amino acid sequence correspond to the second part of the B chain of the respective type 2 RIP. Both proteins are very similar except that the polypeptides of the fruit lectin are 10 amino acid residues longer than these from the bark lectin. Our findings not only demonstrate the occurrence of carbohydrate‐binding fragments of type 2 RIP but also provide further evidence that type 2 RIP genes give rise to complex mixtures of type 2 RIP/lectins in elderberry.
Flash‐induced voltage changes in halorhodopsin from Natronobacterium pharaonis
Authors: Kalaidzidis Inna V. , Kalaidzidis Yannis L. , Kaulen Andrey D.
Published: July 2002

The flash‐induced voltage response of halorhodopsin at high NaCl concentration comprises two main kinetic components. The first component with τ≈1 μs does not exceed 4% of the overall response amplitude and is probably associated with the formation of the L (hR520) intermediate. The second main component with τ≈1–2.5 ms which is independent of Cl− concentration can be ascribed to the transmembrane Cl− translocation during the L intermediate decay. The photoelectric response in the absence of Cl− has the opposite polarity and does not exceed 6% of the overall response amplitude at high NaCl concentration. A pH decrease results in substitution of the Cl−‐dependent components by the photoresponse which is similar to that in the absence of Cl−. Thus, the difference between photoresponses of chloride‐binding and chloride‐free halorhodopsin forms resembles that of bacteriorhodopsin purple neutral and blue acid forms, respectively. The photovoltage data obtained can hardly be explained within the framework of the photocycle scheme suggested by Varo et al. [Biochemistry 34 (1995), 14490–14499]. We suppose that the O‐type intermediate belongs to some form of halorhodopsin incapable of Cl− transport.
Simulation of the HIV‐1 Vpu transmembrane domain as a pentameric bundle
Authors: Moore Preston B , Zhong Qingfeng , Husslein Thomas , Klein Michael L
Published: July 2002

The transmembrane domain of oligomeric protein Vpu encoded by HIV‐1 has been studied by means of a molecular dynamics simulation. A pentameric bundle of unconstrained helices (residues 6–28 of Vpu) with a water filled pore was initially assembled in a membrane mimetic octane/water system. This system was simulated, using the CHARMm19 and OPLS united atom force fields with no constraints at a temperature of 300 K and a pressure of 1 atm. For these forcefields and the initial conditions tested, the oligomeric bundle expelled most of the pore water molecules. The resulting bundle and residual waters adopt a conical structural motif with some resemblance to a potassium channel.
A residue in the middle of the M2‐M3 loop of the β4 subunit specifically affects gating of neuronal nicotinic receptors
Authors: Rovira José Carlos , Ballesta Juan José , Vicente-Agulló Francisco , Campos-Caro Antonio , Criado Manuel , Sala Francisco , Sala Salvador
Published: July 2002

An aspartate residue in the M2‐M3 loop of neuronal nicotinic receptor α7 subunits is a major determinant of the channel functional response. This residue is conserved in most β4 subunits, e.g. human and rat, but not in others, e.g. bovine. We have used these differences to examine the mechanism by which this residue alters the functional properties of α3β4 receptors. Having ruled out an effect on the macroscopic binding ability of the agonist, the level of receptor expression, or the single channel conductance, the results suggest that receptors lacking that residue have a deficient coupling between binding and gating.
Ca2+‐independent interaction of annexin I with phospholipid monolayers
Authors: Rosengarth Anja , Wintergalen Andreas , Galla Hans-Joachim , Hinz Hans-Jürgen , Gerke Volker
Published: July 2002

At pH 6.0, the interaction of annexin I, a proteolytic fragment of annexin I and annexin V, was studied with monolayers composed of dipalmitoylphosphatidylserine (DPPS), dipalmitoylphosphatidylcholine (DPPC) or DPPS/DPPC mixtures (molar ratio 1:4). The measurements reveal that only annexin I shows a significant increase in the surface pressure at constant surface area in the absence of Ca2+ ions. We interpret these pressure changes as reflecting penetration of the protein. Kinetic analyses of the annexin I/monolayer interaction at pH 6.0 in the presence and absence of Ca2+ ions show differences between the interaction mechanisms that support the occurrence of a pH‐regulated process. At pH 7.4, Ca2+ ions are required for the interaction.
Gibberellic acid stabilises microtubules in maize suspension cells to cold and stimulates acetylation of α‐tubulin1
Authors: Huang Rong Feng , Lloyd Clive W
Published: July 2002

Gibberellic acid is known to stabilise microtubules in plant organs against depolymerisation. We have now devised a simplified cell system for studying this. Pretreatment of a maize cell suspension with gibberellic acid for just 3 h stabilised protoplast microtubules against depolymerisation on ice. In other eukaryotes, acetylation of α‐tubulin is known to correlate with microtubule stabilisation but this is not established in plants. By isolating the polymeric tubulin fraction from maize cytoskeletons and immunoblotting with the antibody 6‐11B‐1, we have demonstrated that gibberellic acid stimulates the acetylation of α‐tubulin. This is the first demonstrated link between microtubule stabilisation and tubulin acetylation in higher plants.
A tentative mechanism of the ternary complex formation between phosphorylase kinase, glycogen phosphorylase b and glycogen
Authors: Andreeva Iraida E. , Makeeva Valentina F. , Kurganov Boris I. , Chebotareva Nataliya A. , Livanova Nataliya B.
Published: July 2002

The kinetics of rabbit skeletal muscle phosphorylase kinase interaction with glycogen has been studied. At pH 6.8 the binding of phosphorylase kinase to glycogen proceeds only in the presence of Mg2+, whereas at pH 8.2 formation of the complex occurs even in the absence of Mg2+. On the other hand, the interaction of phosphorylase kinase with glycogen requires Ca2+ at both pH values. The initial rate of the complex formation is proportional to the enzyme and glycogen concentrations, suggesting the formation of the complex with stoichiometry 1:1 at the initial step of phosphorylase kinase binding by glycogen. According to the kinetic and sedimentation data, the substrate of the phosphorylase kinase reaction, glycogen phosphorylase b, favors the binding of phosphorylase kinase with glycogen. We suggest a model for the ordered binding of phosphorylase b and phosphorylase kinase to the glycogen particle that explains the increase in the tightness of phosphorylase kinase binding with glycogen in the presence of phosphorylase b.