bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of protein kinase C in the induction of VCAM‐1 expression on human umbilical vein endothelial cells
Authors: Deisher Theresa A. , Haddix Terri L. , Montgomery Kevin F. , Pohlman Timothy H. , Kaushansky Kenneth , Harlan John M.
Published: July 2002

The role of protein kinase C (PKC) in interleukin‐1β‐ (II‐ 1β)‐, tumor necrosis factor‐α‐ (TNF‐α)‐, and lipopolysaccharide‐(LPS)‐induced vascular cell adhesion molecule‐1 (VCAM‐1) expression on human umbilical vein endothelial cells (HUVEC) was studied. PKC inhibition or downregulation diminished VCAM‐1 mRNA accumulation and protein expression. Interleukin‐1β, TNF‐α, and LPS induce nuclear factor (NF)‐kB‐like binding activity, which precedes VCAM‐1 transcription. PKC inhibition did not prevent NF‐xB‐like binding activity, indicating that this is PKC‐independent, and NF‐kB‐like binding activity is insufficient for transcription of VCAM‐1.
Cytokine‐induced expression of an inducible type of nitric oxide synthase gene in cultured vascular smooth muscle cells
Authors: Koide Masanobu , Kawahara Yasuhiro , Tsuda Terutaka , Yokoyama Mitsuhiro
Published: July 2002

In unstimulated cultured vascular smooth muscle cells (VSMC), mRNA of an inducible macrophage‐type of nitric oxide synthase (iNOS) was barely detectable. Interferon γ (IFNγ) and tumor necrosis factor α (TNFα) markedly increased iNOS mRNA levels in time‐ and dose‐dependent manners. The induction of iNOS mRNA paralleled the cytokine‐induced nitrite production. Actinomycin D abolished the IFNγ‐ and TNFγ‐induced increases in iNOS mRNA and nitrite production. Cycloheximide, which abolished both the IFNγ‐ and TNFα‐induced increases in nitrite production, had no effect on the IFNγ‐induced increase in iNOS mRNA but markedly inhibited the TNFα‐induced one. These results suggest that IFNγ directly induces the expression of the iNOS gene whereas TNFα mainly induces it via the induction of an intermediary protein in cultured VSMC.
Termination of right handed helices in proteins by residues in left handed helical conformations
Authors: Nagarajaram H.A. , Sowdhamini R. , Ramakrishnan C. , Balaram P.
Published: July 2002

An analysis of 636 helical segments, ranging in length from 4 to 32 residues, from 123 independent protein crystal structures reveals that helix termination by residues in left handed (αL) helical conformations is a common occurrence. Gly and Asn residues are the most frequent αL helix terminators, with the former having a very high propensity to adopt such conformations. The αR‐αR‐αR‐αL segment at the C termini of protein helices often possesses a 6 → 1 (π‐type) hydrogen bond between the CO of residue i and the NH of residue i + 5 with residue i + 4 occurring in the αL conformation. A stereochemical analysis of 216 examples shows that in 62 cases the 6 → 1 hydrogen bond is absent. The present analysis provides a quantitative measure of the propensity of the 20 amino acids to adopt αL helix terminating conformations.
Cloning, expression and sequence analysis of cDNA for the Ca2+‐binding photoprotein, mitrocomin
Authors: Fagan Thomas F. , Ohmiya Yoshihiro , Blinks John R. , Inouye Satoshi , Tsuji Frederick I.
Published: July 2002

The primary structure of mitrocomin consists of 190 amino acid residues, with three Ca2+‐binding sites and a tyrosine residue at the C‐terminus. Mitrocomin shows an amino acid sequence homology of 67.9% and 60.7% when compared with aequorin and clytin, respectively. The amino acid residues Cys152, His58, His169, Trp12, Trp86, Trp108, Trp129 and Trp173 are conserved in all three photoproteins, suggesting that they play a role in light emission.
Human islet amyloid polypeptide transgenic mice as a model of non‐insulin‐dependent diabetes mellitus (NIDDM)
Authors: Fox Niles , Schrementi James , Nishi Masahiro , Ohagi Shinyo , Chan Shu Jin , Heisserman Judith A , Westermark Gunilla T. , Leckström Arnold , Westermark Per , Steiner Donald F.
Published: July 2002

To model islet amyloidogenesis in NIDDM and explore the glucoregulatory role of islet amyloid polypeptide (IAPP), we have created transgenic micye containing a rat insulin‐I promoter‐human IAPP fusion gene. Expression of human IAPP was localized to the islets of Langerhans, anterior pituitary and brain in transgenic animals; blood IAPP levels were elevated 5‐fold while fasting glucose levels remained normal. Amyloid deposits have not been detected in transgenic islets suggesting that other co‐existing abnormalitites in NIDDM may be required for the formation of islet amyloid. These animals provide a unique model for exploring this hypothesis and other proposed functions of IAPP.
Evidence for an [Fe]‐type hydrogenase in the parasitic protozoan Trichomonas vaginalis
Authors: Payne Martin J. , Chapman Alan , Cammack Richard
Published: July 2002

The hydrogenase of the pathogenic protozoan Trichomonas vaginalis was extracted and partially purified. The catalytic and spectroscopic properties of the enzyme indicate that it belongs to the class of [Fe]‐hydrogenases, rather than the [NiFe]‐hydrogenases. The hydrogenase activity was highly sensitive to carbon monoxide, 50% inhibition being attained by 1 μM CO. The EPR spectrum of the most active fractions from chromatography, after reduction by hydrogen and partial reoxidation under argon, showed an EPR spectrum at g = 2.10, 2.04, 2.00. This unusual spectrum is characteristic of the ‘H‐cluster’, as seen in [Fe]‐hydrogenases of anaerobic bacteria such as Clostridium spp.
Interleukin‐10 inhibits endotoxin‐induced tissue factor mRNA production by human monocytes
Authors: Ramani M. , Ollivier V. , Khechai F. , Vu T. , Ternisien C. , Bridey F. , de Prost D.
Published: July 2002

In Gram‐negative septic shock, human monocytes synthesize and express on their cytoplasmic membrane tissue factor (TF), a potent activator of the coagulation cascades. The role of TF in triggering disseminated intravascular coagulation (DIC) in these patients appears to be clear. We report the suppressive effect of interleukin‐10 (IL‐10) on endotoxin‐induced TF activity and antigen levels, and on the expression of TF mRNA levels in human monocytes. These results emphasize the potential therapeutic value of this cytokine in septic shock, a condition still associated with a high mortality rate.
Localization of rap1 and rap2 proteins in the gelatinase‐containing granules of human neutrophils
Authors: Mollinedo Faustino , Perez-Sala Dolores , Gajate Consuelo , Jimenez Benilde , Rodriguez Pablo , Lacal Juan Carlos
Published: July 2002

The subcellular localization of rap proteins in resting human neutrophils was investigated by immunoblot analysis with specific anti‐rap2 and anti‐rap1 antibodies of the membrane proteins obtained from distinct subcellutar fractions. Rap2 protein was mainly located in gelatinase‐containing granules, whereas rap1 protein was detected both in gelatinase‐containing granules and in fractions enriched in plasma membrane. Neither rap1 nor rap2 proteins were found in the cytosol or in azurophilic granules. Rap2B, not rap2A, appeared to be the major rap2 protein in human neutrophils. The identification and subcellular localization of rap1 and rap2 proteins at the membranes of gelatinase‐rich granules suggest that these proteins could play a role in the regulation of the rapid and selective mobilization of gelatinase‐containing granules in human neutrophils.
Alzheimer's β‐amyloid peptide specifically interacts with and is degraded by insulin degrading enzyme
Authors: Kurochkin Igor V. , Goto Sataro
Published: July 2002

Cerebral deposition of β‐amyloid peptide (βA) is a hallmark of Alzheimer's disease. Concentration of βA could play a critical role in the rate of amyloid deposition. It is therefore of considerable importance to identify proteases involved in processing of βA. 125I‐labeled synthetic βA specifically cross‐linked to a single protein with M
r = 110,000 in cytosol fractions from rat brain and liver. This protein was identified as insulin degrading enzyme (IDE) since the labeling of the 110 kDa protein was completely blocked by an excess of insulin, and anti‐IDE monoclonal antibodies precipitated the labeled protein. Purified rat IDE effectively degraded βA.
Functional role of a consensus peptide which is common to α‐, β‐, and γ‐tubulin, to actin and centractin, to phytochrome A, and to the TCP1α chaperonin protein
Authors: Burns Roy G. , Surridge Christopher D.
Published: July 2002

The TRiC (TCP1 Ring Complex) chaperonin complex participates in the functional folding of actin, centractin, α‐,β‐,γ‐tubulin, and phytochrome. Each of the cytoskeletal proteins contain a peptide, RK(A,C,T)F/KRAF, located towards the C‐terminus, which is homologous to a TCP1α peptide, while the equivalent phytochrome peptide (RLKAF in certain isoforms) is very similar to the KLRAF peptide of TCP1α. We propose that this TCP1α peptide binds to the nascent polypeptides as they emerge from the ribosome, that this binding restricts the folding pathway, and that the TCP1α peptide is subsequently displaced by the synthesis of the consensus peptide. This hypothesis is strongly supported by the crystallographic structure of actin.
Retinoic acid up‐regulates erythropoietin production in hepatoma cells and in vitamin A‐depleted rats
Authors: Okano Masaki , Masuda Seiji , Narita Hiroshi , Masushige Shoichi , Kato Shigeaki , Imagawa Shigehiko , Sasaki Ryuzo
Published: July 2002

Retinoic acid (RA) stimulated the production of erythropoietin (Epo) in a human hepatoma cell line, HepG2 cells. The stimulation was due to the accumulation of Epo mRNA. The Epo production in HepG2 cells was also dependent on O2 tension for cell culture but the enhancement of Epo production by RA was independent of O2 tension, indicating that RA exerts its effect through a pathway different from O2. Oral administration of RA to the vitamin A‐depleted rats elevated the concentration of Epo in serum. These results suggest that RA up‐regulates EPO production in vivo as well as in vitro.
The lipocalin protein family: A role in cell regulation
Authors: Flower Darren R.
Published: July 2002

The lipocalins, a large, diverse, but relatively poorly understood family of small extracellular proteins, are characterized by the ability to bind small hydrophobic molecules, such as retinol, and by their binding to specific cell surface receptors. These general properties suggest such proteins as appropriate transporters transferring biologically hazardous molecules in a safe and controlled manner between cells. Moreover, many lipocalins have been implicated in the regulation of cell homeostasis: apolipoprotein D, quiescience specific protein, purpurin, α‐1‐microglobulin, and NGAL. This combination of direct and indirect evidence suggests that the lipocalin protein family may be involved, in a quite general way, in the mediation of cell regulation and that many presently functionless family members might act in this way.
Palmitoylation but not the extreme amino‐terminus of Gqα is required for coupling to the NK2 receptor
Authors: Edgerton Michael D. , Chabert Christian , Chollet Andre , Arkinstall Steve
Published: July 2002

Gqα and G11α differ from other G protein α subunits in that they have unique, conserved 6 residue amino‐terminal extensions. Wild‐type and amino‐terminal mutants of Gqα expressed in COS cells were analyzed for their ability to functionally couple with co‐expressed neurokinin NK2 receptor. Wild‐type, T2A and Δ2–7 Gqα were able to stimulate agonist driven phospholipase C (PLC) activity in identical manners. Other activities of these two amino‐terminal mutants including aluminum fluoride stimulated PLC activity, palmitoylation, interaction with Gβγ subunits and GTPγS‐induced trypsin resistance are also similar to the wild‐type α subunit. This demonstrates that the NK2 receptor is able to functionally interact with the α subunit of Gq and that the first seven amino‐acids of Gqα are not required for any of the α subunit functions tested. In contrast to the T2A and Δ2–7 mutants, a C9,10A Gqα mutant was not able to couple to either the NK2 receptor or PLC, as assessed by high‐affinity agonist binding and activation of PLC either in intact cells or in vitro. The C9,10A protein was able to assume a GTPγS‐induced trypsin‐resistant conformation and partitioned primarily to the pelletable fraction in a manner similar to the wild‐type protein. However, it was not labeled with [3H]palmitic acid. This suggests that blocking palmitoylation at the amino‐terminus of Gqα results in a loss of functional activity which reflects an inability to interact with both the receptor and downstream signaling targets.
Very low density lipoprotein receptor from mammary gland and mammary epithelial cell lines binds and mediates endocytosis of Mr, 40,000 receptor associated protein
Authors: Wiborg Simonsen Anna Carina , Heegaard Christian W. , Rasmussen Lone K. , Ellgaard Lars , Kjøller Lars , Christensen Anni , Etzerodt Michael , Andreasen Peter A.
Published: July 2002

We here report that the M
r 40,000 receptor associated protein (RAP), previously found to bind to α2‐macroglobulin receptor/low density lipoprotein receptor related protein (α2MR/LRP) and glycoprotein 330 (gp330), binds to an M
r, 105,000 membrane protein from bovine mammary gland, human mamma tumors and mammary epithelial cell lines. We have purified this protein from bovine and human sources. N‐terminal amino acid sequencing and immunoblotting analyses showed that the protein was identical or closely related to very low density lipoprotein receptor (VLDL‐R). Experiments with the human mamma carcinoma cell line MCF‐7 showed that this receptor was able to mediate an efficient endocytosis of RAP. These novel findings strongly suggest that RAP functions as a modulator of ligand binding to VLDL‐R, similarly to α2MR/LRP and gp330.
A new human leukemia cell 8.2 kDa differentiation factor: Isolation and primary structure
Authors: Kostanyan Irina A. , Astapova Mariya V. , Starovoytova Elena V. , Dranitsina Svetlana M. , Lipkin Valery M.
Published: July 2002

A new 8.2 kDa differentiation factor has been purified to homogeneity from the cultural media of human myelogenous HL‐60 leukemia cells induced by retinoic acid. cDNA clones encoding this factor were isolated from a cDNA library prepared from HL‐60 differentiated cells and their nucleotide sequence has been determined. The deduced amino acid sequence of the differentiation factor molecule consists of 54 amino acid residues. The protein is shown to be glycosylated. It was shown by Northern blot experiments that the level of poly(A)+ RNA with a length of 450 nucleotides was higher in differentiated cells than in non‐differentiated cells.
RT‐PCR cloning of Rab3 isoforms expressed in peritoneal mast cells
Authors: Oberhauser Andreas F. , Balan Vijayan , Fernandez-Badilla Carmen L. , Fernandez Julio M.
Published: July 2002

Rab proteins are ras‐like low molecular mass GTP‐binding proteins, which are postulated to act as specific regulators of membrane trafficking in exocytosis and endocytosis. We have previously shown that synthetic peptides, corresponding to the effector domain of Rab3 proteins, stimulate a complete exocytotic response in mast cells. We have used a PCR‐eloning strategy to investigate the presence of mRNA encoding Rab3 in mast cells. RNA based PCR was then performed on mast cell RNA using degenerate oligonucleotide primers based on two conserved sequences among Rab3 proteins. However, no PCR products were obtained, even for proteins known to be expressed in high copy numbers in mast cells (β‐actin and Fc receptor). We have found that the problem resides in the presence of mast cell secretory granule derived heparin, that copurifies with the RNA; heparin has been shown to inhibit the activity of reverse transcriptase and Taq polymerase in PCR. After treating the RNA (obtained from about 500 mast cells) with heparinase, several PCR products of varying size were obtained using primers specific for Rab3 proteins. These products were cloned and sequenced. We have found clones containing sequences that had a 100% homology at the deduced amino acid level to a portion of Rab3B and Rab3D (amino acids 16 to 83).
Characterization of a novel protein‐binding module — the WW domain
Authors: Sudol Marius , Chen Henry I. , Bougeret Cecile , Einbond Aaron , Bork Peer
Published: July 2002

We have identified, characterized and cloned human, mouse and chicken cDNA of a novel protein that binds to the Src homology domain 3 (SH3) of the Yes proto‐oncogene product. We subsequently named it YAP for Yes‐associated protein. Analysis of the YAP sequence revealed a protein module that was found in various structural, regulatory and signaling molecules. Because one of the prominent features of this sequence motif is the presence of two conserved tryptophans (W), we named it the WW domain. Using a functional screen of a cDNA expression library, we have identified two putative ligands of the WW domain of YAP which we named WBP‐1 and WBP‐2. Peptide sequence comparison between the two partial clones revealed a homologous proline‐rich region. Binding assays and site‐specific mutagenesis have shown that the proline‐rich motif binds with relatively high affinity and specificity to the WW domain of YAP, with a preliminary consensus that is different from the SH3‐binding PXXP motif. This suggests that the WW domain has a role in mediating protein‐protein interactions via proline‐rich regions, similar but distinct from Src homology 3 (SH3) domains. Based on this finding, we hypothesize that additional protein modules exist and that they could be isolated using proline‐rich peptides as functional probes.
Furin/PACE/SPC1: A convertase involved in exocytic and endocytic processing of precursor proteins
Authors: Denault Jean-Bernard , Leduc Richard
Published: July 2002

One of the most exciting breakthroughs of the 90's in the fields of biochemistry, cell biology and neuroendocrinology is the identification of a novel family of proteolytic enzymes called mammalian subtilisin‐like convertases. This family is comprised so far of seven distinct endoproteases responsible for the proteolytic excision of biologically active polypeptides from inactive precursor proteins. Six years after the initial observation of a structural conservation between a characterized yeast enzyme (kexin) and a human gene product (furin), it is now well accepted that one of these convertases, furin, has the enzymatic capabilities to efficiently and correctly process a great variety of precursors. Furin's ability to cleave precursors within both the exocytic and endocytic pathways will require sustained efforts in order to delineate all of its physiological roles.
A mechanism for β‐amyloid overproduction in Alzheimer's disease: precursor‐independent generation of β‐amyloid via antisense RNA‐primed mRNA synthesis
Authors: Volloch Vladimir
Published: July 2002

The overproduction of β‐amyloid (Aβ) appears to be a primary cause of Alzheimer's disease (AD). Aβ can be generated by proteolytic cleavage of precursor protein (βAPP) at β‐ and γ‐secretase sites in both disease and normal cells. There is, however, no evidence that proteolytic processing of βAPP in sporadic AD‐affected tissues differs qualitatively or quantitatively from that occurring in normal cells, and additional pathways for the enhanced production of Aβ in sporadic AD which constitutes the majority of all AD cases should be considered. The major factor limiting the production of Aβ in normal cells is cleavage at the α‐secretase site within the Aβ sequence. But, whereas the intact βAPP is a substrate for cleavage at the α‐secretase site, the immediate precursor of Aβ, 12‐kDa C‐terminal βAPP fragment, is not susceptible to the α‐secretase cleavage but it can be cleaved by γ‐secretase thus generating Aβ. Moreover, the γ‐secretase cleavage is not the rate‐limiting step in the production of Aβ. Therefore, the increase in production of the 12‐kDa C‐terminal βAPP fragment may be an efficient way to overproduce Aβ. A mechanism for the generation of the 12‐kDa fragment independently of βAPP is proposed. It postulates an additional step of amplification of mRNA, namely the antisense RNA‐mediated generation of a truncated mRNA encoding 12‐kDA C‐terminal fragment. Initiation of translation at the first AUG in the truncated mRNA results in a polypeptide that is cleaved by γ‐secretase generating Aβ. The proposed model makes several verifiable predictions and suggests new directions of experimentation that may lead to a better understanding of the mechanisms involved in AD.
Identification of N‐acetylglucosamine binding residues in Griffonia simplicifolia lectin II
Authors: Zhu Keyan , Bressan Ray A. , Hasegawa Paul M. , Murdock Larry L.
Published: July 2002

Primary structure and crystallographic data of several legume lectins were used to predict the involvement in carbohydrate binding of six amino acid residues (Asp88, Glu108, Tyr134, Asn136, Leu226 and Gln227) in Griffonia simplicifolia lectin II (GS‐II). The functional involvement of these residues was evaluated by assessing GlcNAc binding of modified forms of GS‐II in which these residues were eliminated in truncated peptides or systematically substituted with other amino acids by site‐specific mutations. Mutations at (Asp88, Tyr134 or Asn136 eliminated GlcNAc binding activity by GS‐II, while those at Glut108, Leu226 or Gln227 did not alter the activity. The former three amino acids were functionally essential for carbohydrate binding by GS‐II presumably through hydrogen bonding to and hydrophobic interactions with GlcNAc. Although an Asp or Gly substitution for Tyr134 eliminated GlcNAc affinity, substitution with Phe did not appreciably affect binding. Despite the fact that mutations to Leu226 and Gln227 did not alter carbohydrate binding, a truncated form of GS‐II lacking these residues no longer exhibited carbohydrate binding affinity.
Both action potentials and variation potentials induce proteinase inhibitor gene expression in tomato
Authors: Stanković Bratislav , Davies Eric
Published: July 2002

Tomato plants (Lycopersicon esculentum) accumulate proteinase inhibitor 2 (pin2) mRNA in response to insect attack, crushing and flaming in leaves distant from those treated. Most earlier work suggests that the systemic wound signals are chemical; here we try to determine whether electrical or physical (hydraulic) signals can also evoke pin expression. We used a mild flame to evoke a systemic hydraulic signal and its local electrical aftermath, the variation potential (VP), and we used an electric stimulus to evoke a systemic electrical signal, the action potential (AP). We determined the kinetic parameters of both the VP and AP. Flame‐wounded plants essentially always exhibited major electrical responses throughout the plant and a several‐fold increase in pin2 mRNA within 1 h. Electrically stimulated plants that generated and transmitted a signal (AP) into the analyzed leaf exhibited similarly large, rapid increases in pin2 mRNA levels. Plants which generated no signal, or signals of just a few microvolts, had unchanged levels of pin2 mRNA. Since the AP and VP both arrived in the receiving leaf before accumulation of pin2 mRNA began, we conclude that, in addition to the previously shown chemical signals, both hydraulically induced VPs and electrically induced APs are capable of evoking pin2 gene expression.
Cloning and characterization of a bifunctional metabotropic receptor activated by both extracellular calcium and glutamate
Authors: Kubokawa Kaoru , Miyashita Tomoyuki , Nagasawa Hiromichi , Kubo Yoshihiro
Published: July 2002

A cDNA from salmon brain which induces a consistent activation of Ca2+ dependent Cl− (Ca2+−Cl−) current in Xenopus oocytes in the saline solution was isolated by an expression cloning method. The primary structure showed a high homology with the metabotropic glutamate receptors (mGluR), and a limited homology with the Ca2+/polyvalent cation receptors (CaR). The encoded protein was functionally characterized for the sensitivity to extracellular Ca2+ (Cao
2+), and the consistent activation of Ca2+−Cl− current was determined to be due to Ca2+ contained in the saline solution. It was also confirmed that the encoded protein has a function to sense glutamate. An increase in inositol 1,4,5‐triphosphate turnover was observed when either Ca2+ or glutamate was applied. Thus, the encoded protein was identified as a bifunctional metabotropic receptor for both Ca2+ and glutamate. In addition, rat mGluR1α was also shown to be activated by Cao
2+. This is the first report of a G‐protein coupled receptor which is activated by two totally different physiological agonists.
Transient expression of S‐100 by melanotropes of the rat pituitary intermediate lobe during development
Authors: Sands Scott A. , Gary Keith A. , Chronwall Bibie M.
Published: July 2002

S‐100, an acidic calcium‐binding protein, is co‐localized with vimentin in glial‐like cells in the adult rat pituitary intermediate lobe. S‐100 and melanotrope markers were not co‐localized in the adult. During development, S‐100 and vimentin were not co‐localized but appeared in cells with different morphological characteristics. S‐100 was co‐localized with POMC mRNA and β‐endorphin during prenatal time and the first three postnatal weeks. This was demonstrated by double‐label immunohistochemistry, using combinations of antisera against S‐100, vimentin and β‐endorphin, and in situ hybridization histochemistry for POMC mRNA combined with immunohistochemistry for S‐100. In the second and third weeks of postnatal development, S‐100 was observed in fewer melanotropes and more frequently in stellate cells, which also expressed vimentin. Thus, S‐100 appeared to be transiently expressed in melanotropes during prenatal and early postnatal development. S‐100 serves as a neurotrophic and glial maturation factor in the CNS. Since S‐100 expression in melanotropes coincides with the onset of dopaminergic innervation and morphological changes in glial‐like cells of the lobe, it could have similar functions in the rat pituitary intermediate lobe.
The platelet cytoskeleton regulates the aggregation‐dependent synthesis of phosphatidylinositol 3,4‐bisphosphate induced by thrombin
Authors: Torti Mauro , Bertoni Alessandra , Sinigaglia Fabiola , Balduini Cesare , Payrastre Bernard , Plantavid Monique , Chap Hugues , Mauco Gerard
Published: July 2002

Pretreatment of intact platelets with cytochalasin D prevented actin polymerization and cytoskeleton reorganization induced by thrombin, but did not affect platelet aggregation. Under these conditions, synthesis of phosphatidylinositol 3,4‐bisphosphate (PtdIns(3,4)P2) stimulated by thrombin was strongly inhibited, while production of phosphatidic acid was unaffected. The inhibitory effect of cytochalasin D was not observed when platelet aggregation was prevented by the RGDS peptide. We also found that cytochalasin D did not affect PtdIns(3,4)P2 synthesis induced by concanavalin A (ConA), which is known to occur through an aggregation‐independent mechanism. Moreover, thrombin, but not ConA, induced the translocation of phosphatidylinositol 3‐kinase to the cytoskeleton. This process was equally inhibited by both the RGDS peptide and cytochalasin D. These results demonstrate that the cytoskeleton represents a functional link between thrombin‐induced aggregation and synthesis of PtdIns(3,4)P2.
Mitochondrial Isa2p plays a crucial role in the maturation of cellular iron–sulfur proteins
Authors: Pelzer Winfried , Mühlenhoff Ulrich , Diekert Kerstin , Siegmund Kerstin , Kispal Gyula , Lill Roland
Published: July 2002

The assembly of iron–sulfur (Fe/S) clusters in a living cell is mediated by a complex machinery which, in eukaryotes, is localised within mitochondria. Here, we report on a new component of this machinery, the protein Isa2p of the yeast Saccharomyces cerevisiae. The protein shares sequence similarity with yeast Isa1p and the bacterial IscA proteins which recently have been shown to perform a function in Fe/S cluster biosynthesis. Like the Isa1p homologue, Isa2p is localised in the mitochondrial matrix as a soluble protein. Deletion of the ISA2 gene results in the loss of mitochondrial DNA and a strong growth defect. Simultaneous deletion of the ISA1 gene does not further exacerbate this growth phenotype suggesting that the Isa proteins perform a non‐essential function. When Isa2p was depleted by regulated gene expression, mtDNA was maintained, but cells grew slowly on non‐fermentable carbon sources. The maturation of both mitochondrial and cytosolic Fe/S proteins was strongly impaired in the absence of Isa2p. Thus, Isa2p is a new member of the Fe/S cluster biosynthesis machinery of the mitochondrial matrix and may be involved in the binding of an intermediate of Fe/S cluster assembly.
Intersectin 2, a new multimodular protein involved in clathrin‐mediated endocytosis
Authors: Pucharcos Carles , Estivill Xavier , de la Luna Susana
Published: July 2002

Intersectin 1 (ITSN1) is a binding partner of dynamin that has been shown to participate in clathrin‐mediated endocytosis. Here we report the characterization of a new human gene, ITSN2, highly similar to ITSN1. Alternative splicing of ITSN2 generates a short isoform with two EH domains, a coiled‐coil region and five SH3 domains, and a longer isoform containing extra carboxy domains (DH, PH and C2 domains), suggesting that it could act as a guanine nucleotide exchange factor for Rho‐like GTPases. ITSN2 expression analysis indicates that it is widely expressed in human tissues. Intersectin 2 isoforms show a subcellular distribution similar to other components of the endocytic machinery and co‐localize with Eps15. Moreover, their overexpression, as well as the corresponding ITSN1 protein forms, inhibits transferrin internalization.
Differential modulation of nitric oxide production by curcumin in host macrophages and NK cells
Authors: Bhaumik Sraboni , Jyothi M.Divya , Khar Ashok
Published: July 2002

Curcumin, the yellow pigment from Curcuma longa, has been shown to possess tumoricidal activity. We have earlier reported the induction of apoptosis in AK‐5, rat histiocytic cells by curcumin leading to the inhibition of tumor growth in vivo. In this study we have observed differential activation status in host macrophages and NK cells induced by curcumin during the spontaneous regression of subcutaneously transplanted AK‐5 tumors. Closer scrutiny of the cytokine profile and nitric oxide (NO) production by immune cells showed an initial downregulation of Th1 cytokine response and NO production by macrophages, and their upregulation in NK cells, which picked‐up upon prolonged treatment with curcumin, culminating in a stronger tumoricidal effect. These studies suggest that the host macrophages and NK cells play an important modulatory role in the remission of AK‐5 tumor.
Identification of mouse Jun dimerization protein 2 as a novel repressor of ATF‐2
Authors: Jin Chunyuan , Ugai Hideyo , Song Jun , Murata Takehide , Nili Feuerstein , Sun Kailai , Horikoshi Masami , Yokoyama Kazunari K.
Published: July 2002

A mouse cDNA that encodes a DNA‐binding protein was identified by yeast two‐hybrid screening, using activating transcription factor‐2 (ATF‐2) as the bait. The protein contained a bZIP (basic amino acid‐leucine zipper region) domain and its amino acid sequence was almost identical to that of rat Jun dimerization protein 2 (JDP2). Mouse JDP2 interacted with ATF‐2 both in vitro and in vivo via its bZIP domain. It was encoded by a single gene and various transcripts were expressed in all tested tissues of adult mice, as well as in embryos, albeit at different levels in various tissues. Furthermore, mouse JDP2 bound to the cAMP‐response element (CRE) as a homodimer or as a heterodimer with ATF‐2, and repressed CRE‐dependent transcription that was mediated by ATF‐2. JDP2 was identified as a novel repressor protein that affects ATF‐2‐mediated transcription.
Unbalanced expression of VEGF and PEDF in ischemia‐induced retinal neovascularization
Authors: Gao Guoquan , Li Yan , Zhang Dongchang , Gee Stephen , Crosson Craig , Ma Jian-xing
Published: July 2002

Retinal levels of vascular endothelial growth factor (VEGF) and pigment epithelium‐derived factor (PEDF), an angiogenic inhibitor, were measured and correlated with the ischemia‐induced retinal neovascularization in rats. The retinas with neovascularization showed a 5‐fold increase in VEGF while 2‐fold decrease in PEDF, compared to the age‐matched controls, resulting in an increased VEGF/PEDF ratio. The time course of the VEGF/PEDF ratio change correlated with the progression of retinal neovascularization. Changes in the VEGF and PEDF mRNAs preceded their protein level changes. These results suggest that an unbalance between angiogenic stimulators and inhibitors may contribute to retinal neovascularization.
Local DNA demethylation in vertebrates: how could it be performed and targeted?
Authors: Kress Clémence , Thomassin Hélène , Grange Thierry
Published: July 2002

In vertebrates, cytosine methylation is an epigenetic DNA modification that participates in genome stability and gene repression. Methylation patterns are either maintained throughout cell division, or modified by global or local de novo methylation and demethylation. Site‐specific demethylation is a rather elusive process that occurs mainly in parallel to gene activation during development. In light of our studies of the glucocorticoid‐dependent DNA demethylation of the tyrosine aminotransferase gene, we discuss the potential biochemical mechanisms allowing DNA demethylation and its targeting to specific sequences by transcription factors as well as possible links to DNA replication and chromatin remodelling.
Topographical structure of membrane‐bound Escherichia coli F1F0 ATP synthase in aqueous buffer
Authors: Singh Seema , Turina Paola , Bustamante Carlos J. , Keller David J. , Capaldi Roderick
Published: July 2002

Scanning force microscope images of membrane‐bound Escherichia coli ATP synthase F0 complexes have been obtained in aqueous solution. The images show a consistent set of internal features: a ring structure which surrounds a central dimple and contains an asymmetric lateral mass. Images of trypsin‐treated F0 complexes, which have lost part of their b subunits, show a reduced asymmetric mass, while images of c‐subunit oligomers, which lack both the a and b subunits, show a ring and dimple but do not have an asymmetric mass. These results support models in which the F0 complex contains a ring of 9–12 c subunits with the b subunits located outside this ring, and show that scanning force microscopy is able to provide structural information on membrane proteins of molecular mass less than 200 000 Da.
Effect of zeta potential of cationic liposomes containing cationic cholesterol derivatives on gene transfection
Authors: Takeuchi Ken-ichiro , Ishihara Minoru , Kawaura Chiyo , Noji Masahide , Furuno Tadahide , Nakanishi Mamoru
Published: July 2002

Cationic liposomes are known to be useful tools for gene transfection. However, the relation between transfection efficiency and physicochemical properties of liposomes has not been well understood. Here, we synthesized eight cationic derivatives of cholesterol which contain a tertiary amino head group with a different spacer arm. Transfection of plasmid pSV2CAT DNA into cells was done by cationic liposomes made of a mixture of dioleoylphosphatidylethanolamine (DOPE) and each cationic cholesterol derivative. At the same time we measured zeta potential of cationic liposomes by laser Doppler spectroscopy. The present results indicated that zeta potentials of cationic liposomes were well related to transfection activity of pSV2CAT DNA. This suggested that zeta potential of cationic liposomes is one of important factors which control gene transfection.
The pH‐dependent reciprocal changes in contributions of ADP/ATP antiporter and aspartate/glutamate antiporter to the fatty acid‐induced uncoupling
Authors: Samartsev Victor N , Mokhova Elena N , Skulachev Vladimir P
Published: July 2002

The pH effect on carboxyatractylate‐, glutamate‐ and aspartate‐induced recoupling of palmitate‐uncoupled rat liver mitochondria has been studied. Stimulation of respiration by low palmitate concentrations (5–20 μM) in the presence of 3 mM MgCl2 is shown to be pH‐independent within the 7.0–7.8 range. The recoupling effect of glutamate (or aspartate) decreases and that of carboxyatractylate increases with increase in pH. The recoupling effect of a combination of carboxyatractylate and glutamate (aspartate) appears to be constant at these pH values, being as high as about 80%. It is concluded that uncoupling by low palmitate in liver mitochondria is mediated mainly by ATP/ADP and aspartate/glutamate antiporter.
Proadrenomedullin N‐terminal 20 peptide (PAMP) inhibits proliferation of human neuroblastoma TGW cells
Authors: Ando Katsuyuki , Omi Naomi , Shimosawa Tatsuo , Fujita Toshiro
Published: July 2002

We investigated the effects of proadrenomedullin N‐terminal 20 peptide (PAMP) and adrenomedullin (AM) on the growth of human neuroblastoma TGW cells. Both PAMP and AM inhibited growth and DNA synthesis in neuroblastoma cells. Calcitonin gene‐related peptide (CGRP)8–37, an antagonist to CGRP, abolished the inhibitory effect of AM on growth and DNA synthesis of neuroblastoma cells but did not affect that of PAMP. AM22–52, an antagonist to AM, also reversed the effect of AM. On the other hand, pertussis toxin (PTX) and ω‐conotoxin GIVA blocked the effect of PAMP alone. Thus, PAMP inhibits the growth of neuroblastoma cells by inhibiting N‐type Ca2+ channels through PTX‐sensitive G protein‐coupled receptors, which is different mechanism of AM‐induced inhibition of the cell growth.
Sequence analysis and bacterial production of the anti‐c‐myc antibody 9E10: the VH domain has an extended CDR‐H3 and exhibits unusual solubility
Authors: Schiweck Wolfram , Buxbaum Britta , Schätzlein Christian , Neiss Hans Günther , Skerra Arne
Published: July 2002

The cDNAs for the two variable domains of the antibody 9E10 were cloned from the hybridoma cell line. A chimeric 9E10 Fab fragment was produced in E. coli under control of the tightly controlled tetracycline promoter. The functional Fab fragment was isolated in a single step via a His6‐tag, which also served for its recognition by a nickel chelate‐alkaline phosphatase conjugate. Thus, the recombinant Fab fragment permitted the immunochemical detection of the myc tag in a sandwich ELISA. The dissociation constant for the interaction with the myc tag peptide was determined as 80±5 nM by fluorescence titration. In an attempt to produce the smaller 9E10 Fv fragment it was found that its VH domain alone can be readily isolated from E. coli as a soluble protein. This unusual behaviour may be explained by the 18 amino acid‐long CDR‐H3 and could be of value in the design of `single domain' antibodies.
Insulin dependent tyrosine phosphorylation of the tyrosine internalisation motif of TGN38 creates a specific SH2 domain binding site
Authors: Stephens D.J , Banting G
Published: July 2002

Tyrosine‐based motifs are involved in both protein targeting and, via SH2 domain binding, intracellular signalling. To date there has only been one example of such a motif acting as both an intracellular sorting signal and SH2 binding determinant, namely that of the T cell costimulation receptor, CTLA‐4. We show that insulin stimulation of cultured rat hepatoma cells results in increased cell surface expression of TGN38. Furthermore, the cytosolic domain of TGN38 can be phosphorylated by the insulin receptor in vitro and tyrosine phosphorylated TGN38 can specifically bind to the SH2 domains of the spleen tyrosine kinase Syk. These data imply that tyrosine‐based motifs may play a broader role than has previously been accepted and could help to integrate trafficking and signalling events.
Apolipoprotein E, transthyretin and actin in the CSF of Alzheimer's patients: relation with the senile plaques and cytoskeleton biochemistry
Authors: Merched Aksam , Serot Jean-Marie , Visvikis Sophie , Aguillon Dominique , Faure Gilbert , Siest Gérard
Published: July 2002

We measured the levels of two β‐amyloid (Aβ)‐sequestering proteins, apolipoprotein (Apo) E and transthyretin (TTR), in ventricular human cerebrospinal fluid (CSF) of Alzheimer's disease (AD) patients and controls in relation to brain histological findings. We also studied actin levels in CSF as a marker of the biochemical role of these two proteins in the cytoskeleton. We show that TTR levels in CSF were significantly decreased in AD patients compared to controls and negatively correlated with the senile plaque (SP) abundance. Moreover, actin levels were positively linked to TTR levels and increased in CSF samples of patients homozygous for the ApoE ϵ4‐allele. We propose that TTR and ApoE4 may have competition in the aggregation of Aβ and its deposition in the SP of AD brain. The relationships between ApoE, TTR and actin could suggest a metabolic implication of ApoE genetics and TTR levels in cytoskeletal biochemistry which may be relevant to the pathogenesis of AD.
DNAs with unusual properties revealed by field inversion gel electrophoresis of agarose‐encapsulated DNA from mammalian cells
Authors: Sukhova Tatjana , Popenko Vladimir , Serdjuk Ol'ga , Alekhina Raisa , Botezatu Irina , Shelepov Valery , Lichtenstein Anatoly
Published: July 2002

Distinct DNA fractions (fr‐DNAs), moving separately from bulk DNA, were revealed by field inversion gel electrophoresis of DNA from intact cells lysed and deproteinized in agarose plugs. These fr‐DNAs (∼2% of the total DNA) were ubiquitously present in nuclei of all mammalian cells studied, including human normal and tumor tissues, and showed a typical electrophoretic pattern (three bands with constant mobilities termed a‐, b‐, and c‐DNA). Characteristic mobility shifts induced by gamma irradiation of a‐ and b‐DNAs suggest their non‐linear conformation. In fact, electron microscopy of a‐ and b‐DNAs from Namalwa cells revealed rosette‐like structures stabilized by a central protease‐resistant knob. Comparative PCR analysis revealed qualitative differences between genomic fingerprints of a‐ and b‐DNAs on the one hand and chromosomal DNA on the other. The results obtained suggest that fr‐DNAs originate either from some specific chromatin regions due to non‐random cleavages or from an autonomous intranuclear structure, not identified as yet.
Overexpression of hsp70i facilitates reactivation of intracellular proteins in neurones and protects them from denaturing stress
Authors: Beaucamp N. , Harding T.C. , Geddes B.J. , Williams J. , Uney J.B.
Published: July 2002

Transfection of neurones with an adenoviral vector (Ad) expressing high levels of hsp70i was shown to protect primary hippocampal cultures from heat stress. To investigate one of the molecular mechanisms which may underlie hsp70i's neuroprotective effects we measured luciferase activity in the presence and absence of hsp70i following heat or chemical inactivation. Luciferase activity was recovered to 80% of control levels in the presence of recombinant hsp70i in vitro. Luciferase activity was also maintained in primary hippocampal neurones exposed to a denaturing stress if transfected with Ad‐hsp70i. These results support the hypothesis that hsp70i protects neurones from stress by interacting with cytosolic proteins and thereby protecting them from inactivation.
Identification of a long variant of mRNA encoding the NR3 subunit of the NMDA receptor: its regional distribution and developmental expression in the rat brain
Authors: Sun Lixin , Margolis Frank L. , Shipley Michael T. , Lidow Michael S.
Published: July 2002

A longer variant of rat mRNA encoding the NR3 subunit of the NMDA receptor has been identified. It contains a 60‐bp insertion at the nucleotide position 3007 in the intracellular domain of the C‐terminal of the previously cloned variant. Therefore, the NR3 mRNA exists in at least two variants – with the insert (NR3‐long; NR3‐l) and without the insert (NR3‐short; NR3‐s). The NR3‐l variant is expressed throughout the adult rat brain. Moreover, this variant predominates in the occipital and entorhinal cortices, thalamus and cerebellum. Analysis of NR3‐l development indicates that it is regulated in a region‐specific manner.
The extracellular processing of HIV‐1 envelope glycoprotein gp160 by human plasmin
Authors: Okumura Yuushi , Yano Mihiro , Murakami Meiko , Mori Sachie , Towatari Takae , Kido Hiroshi
Published: July 2002

Cleavage of the envelope glycoprotein precursor gp160 of HIV‐1 is a prerequisite for the infectivity of HIV‐1, and occurs at least in part before gp160 reaches the cell surface. Kexin/subtilisin‐related endopeptidases are proposed enzyme candidates for this intracellular processing. In this study, we reveal the possibility that plasminogen binds to the cell surface and part of gp160 escaping intracellular processing is cleaved by plasmin extracellularly. Plasmin cleaves gp160 precisely at the C‐terminal arginine residue of gp120, and the processing is effectively inhibited by an analogue peptide of the cleavage motif (RXK/RR) and by plasmin inhibitors.
Distortion of the L→M transition in the photocycle of the bacteriorhodopsin mutant D96N: a time‐resolved step‐scan FTIR investigation
Authors: Rödig C , Siebert F
Published: July 2002

The D96N mutant of bacteriorhodopsin has often been taken as a model system to study the M intermediate of the wild type photocycle due to the long life time of the corresponding intermediate of the mutant. Using time‐resolved step‐scan FTIR spectroscopy in combination with a sample changing wheel we investigated the photocycle of the mutant with microsecond time resolution. Already after several microseconds an intermediate similar to the MN state is observed, which contrasts with the M state of the wild type protein. At reduced hydration M and N intermediates similar to those of wild type BR can be detected. These results have a bearing on the interpretation of the photocycle of this mutant. A mechanism is suggested for the fast rise of MN which provides some insight into the molecular events involved in triggering the opening of the cytosolic channel also of the wild type protein.
Antitumor activity of curcumin is mediated through the induction of apoptosis in AK‐5 tumor cells
Authors: Khar Ashok , Ali A.Mubarak , Pardhasaradhi B.V.V , Begum Zareena , Anjum Rana
Published: July 2002

Curcumin, the yellow pigment of turmeric (Curcuma longa), used commonly as a spice, has been shown to possess anticarcinogenic activity. Curcumin inhibited AK‐5 tumor growth and induced apoptosis in AK‐5 cells. Curcumin induced apoptosis is mediated through the activation of caspase‐3, which is specifically inhibited by the tetrapeptide Ac‐DEVD‐CHO. In addition, curcumin induced tumor cell death is caused through the generation of reactive oxygen intermediates which is inhibited by N‐acetyl‐l‐cysteine. Our studies suggest that the apoptotic process induced by curcumin is the mechanism mediating AK‐5 tumor cell death.
β‐Carotene enhances hydrogen peroxide‐induced DNA damage in human hepatocellular HepG2 Cells
Authors: Woods Julie A. , Bilton Rodney F. , Young Andrew J.
Published: July 2002

In this study, the alkaline version of the comet assay has been used to determine the effect of β‐carotene supplementation (10 μM) on peroxide‐initiated free radical‐mediated DNA damage in human HepG2 hepatoma cells. In supplemented cells, β‐carotene failed to afford any protection against hydrogen peroxide‐induced DNA strand breaks. Indeed, levels of strand breaks in supplemented cells were significantly higher than in cells exposed to hydrogen peroxide alone, especially after a long incubation period. In contrast, β‐carotene afforded significant levels of protection against DNA strand breaks when cells were treated with tert‐butyl hydroperoxide. In this case, the level of protection increased as supplementation continued.