bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of rat α‐fetoprotein and albumin with polyunsaturated and other fatty acids: Determination of apparent association constants
Authors: Anel Alberto , Calvo Miguel , Naval Javier , Iturralde María , Alava María A. , Piñeiro Andrés
Published: July 2002

The interaction of fatty acids with rat α‐fetoprotein and albumin was measured using a partition equilibrium method. α‐Fetoprotein (AFP) displays one high‐affinity binding site for fatty acids and albumin near two binding sites. The AFP association constants for most fatty acids were similar to those of albumin (in the 107 M−1 range) whereas for docosa‐hexaenoic acid it was 9.7 × 108 M−1, about 50‐fold higher than that corresponding to albumin. This difference justifies docosahexaenoic acid in fetal or neonatal serum being mainly bound to AFP and can indicate a highly specific role of AFP in the transport of this fatty acid.
New herbicide‐binding site in the photosynthetic electron‐transport chain
Authors: Itoh Shigeru , Iwaki Masayo
Published: July 2002

The binding of herbicides to the phylloquinone‐(primary electron acceptor A1)‐binding site in green plant photosystem (PS) I reaction centers is shown. Dissociation constants (Kd) of various herbicides to the phylloquinone‐binding site were estimated by analyzing their competitive inhibition of the reconstitution of the phylloquinone analogue, menadione (vitamin K3), to the phylloquinone‐extracted spinach PS I particles. The phylloquinone‐binding site was found to bindo‐phenanthroline (Kd= 1.2 × 10−4M), but only weak binding was observed with atrazine (Kd> 10−2M), although both are known to bind specifically to the quinone‐(QB)‐binding site in reaction centers of purple photosynthetic bacteria or PS II. The inhibitors of the cytochromeb/c1(ƒ) complex, myxothiazol (Kd=9.5 × 10−6M) or antimycin A (Kd= 2.8 × 10−6M), also strongly bound to the phylloquinone site. This is the first report showing that the PS I reaction center complex also has a herbicide‐binding site, although the site is probably not sensitive in vivo to these herbicides due to its higher affinity for phylloquinone than herbicides. The inhibitor specificity of the PS I phylloquinone site is different from that of the other quinone‐functioning sites in the photosynthetic or respiratory electron‐transfer chain, suggesting it to have a unique structure.
Inhibition of protein kinase C induces differentiation of neuroblastoma cells
Authors: Miñana Maria-Dolores , Felipo Vicente , Grisolia Santiago
Published: July 2002

It is shown that 1‐(5‐isoquinolinylsulfonyl)‐2‐methylpiperazine (H7), a specific inhibitor of protein kinase C, induces neuritogenesis in neuro 2a cells. The percentage of differentiated cells was 9%, 20%, 59% and 85% at 0, 17, 85 and 500 μM H7, respectively. The number of neurites/cell increased 2‐, 8‐ and 14‐fold over the controls for 17, 85 and 500 μM H7, respectively. These results indicate that protein kinase C plays a key role in the control of differentiation of neural cells and that its specific inhibition may be of basic as well as of practical importance.
Chicken spermatogenesis is accompanied by a genomic‐wide loss of DNA methylation
Authors: Rocamora Nativitat , Mezquita Cristóbal
Published: July 2002

Renaturation kinetics of DNA obtained from chicken testis cell nuclei separated by sedimentation at unit gravity showed that the undermethylation, previously observed in meiotic and postmeiotic cells, is not a peculiarity of repetitive sequences, but is also a feature of unique sequences. The large proportion of slowly renaturing, intermediately renaturing and rapidly renaturing DNAs contain 27, 32 and 31% less methylcytosines in meiotic and postmeiotic cells than the corresponding fractions of premeiotic cells. DNA methyltransferase activity is lower in meiotic and postmeiotic cells containing undermethylated DNA than in immature testis, enriched in spermatogonia, with higher levels of DNA methylation.
Regulation of cGMP‐dependent conductance in cytoplasmic membrane of rod outer segments by transducin
Authors: Krapivinsky G.B. , Filatov G.N. , Filatova E.A. , Lyubarsky A.L. , Fesenko E.E.
Published: July 2002

A preparation of the photoreceptor G‐protein, transducin, containing mainly the Tα‐subunit in a GTP‐γ‐S‐bound form, has been used for perfusion of the intracellular surface of excised patches of rod outer segment cytoplasmic membrane from frog retina. The preparation has been shown to result in the complete suppression of the cGMP‐activated ionic conductance of the cytoplasmic membrane patch. The effect is entirely reversible after the protein has been washed out and is not observed in the absence of CGMP. The degree of conductance inhibition depends on the protein concentration, half‐maximal inhibition occurring at 1 αM Tα—GTP‐γ‐S.
Amino acid sequences of cytotoxin‐like basic proteins derived from cobra venoms
Authors: Inoue Seiji , Okumura Kohji , Tsujino Michiyo , Ohkura Kazuto , Ikeda Kiyoshi , Takechi Masayuki , Tanaka Yasuo , Hayashi Kyozo
Published: July 2002

Amino acid sequences of cytotoxin‐like basic proteins (CLBPs), purified from the venoms of Formosan cobra (Naja naja atra) and Indian cobra (Naja naja), were reinvestigated. The determined sequences differed from those reported previously by Takechi et al. [(1985) Biochem. Int. 11, 795‐802; (1987) Biochem. Int. 14, 145‐152]. The sequence of CLBPs at residues 25‐30 was found to be hydrophilic as compared with those of cytotoxins. The difference in the hydrophobicity of this region might be responsible for the difference in their cytotoxic activities.
Irradiation of the template with high‐intensity (pulse‐laser) ultraviolet light results in DNA‐polymerase termination events at deoxyguanosine residues
Authors: Panyutin I.G. , Kovalsky O.I. , Budowsky E.I.
Published: July 2002

During primer elongation byEscherichia coliDNA‐polymerase I large fragments on the template were irradiated with UV laser pulses at an intensity ⩾ 1010W/m2. In addition to the termination events at photoproducts typical of low‐intensity UV irradiation, termination is observed before deoxyguanosine residues. The effect of the UV light intensity on the ratio of termination efficiencies before dPy and dG suggests that the termination of polymerization before deoxyguanosine residues results from the formation of photoproducts yielded by two‐quantum reactions. The results obtained herein, together with data published previously, imply that photomodification of dG residues is the major two‐quantum reaction under the action of high‐intensity UV radiation on DNA.
Isolation and characterization of the gene encoding phosphoenolpyruvate carboxykinase from Saccharomyces cerevisiae
Authors: Valdés-Hevia Marta D. , de la Guerra Raquel , Gancedo Carlos
Published: July 2002

The yeast PCK1 gene coding for phosphoenolpyruvate carboxykinase (PEPCK) was isolated by functional complementation of pck1 strains from S. cerevisiae. Only one copy of the gene was found per haploid yeast genome. An RNA of about 2 kb which hybridized with a DNA probe internal to the PCK1 gene was found only in cells growing in non‐fermentable carbon sources. Yeast strains carrying multiple copies of the PCK1 gene showed normal catabolite repression of PEPCK except those carrying the shortest insertion complementing the mutation (2.2 kb) that presented an altered kinetics of derepression. Catabolite inactivation was decreased in strains transformed with multicopy plasmids carrying the PCK1 gene.
Electron transfer in spinach photosystem I reaction center containing benzo‐, naphtho‐ and anthraquinones in place of phylloquinone
Authors: Iwaki Masayo , Itoh Shigeru
Published: July 2002

Quinone; Phylloquinone; Vitamin K1; Photosystem I; Reaction center; Electron transfer; Photosynthesis
Bacterial expression of human cysteine proteinase inhibitor stefin A
Authors: Fong Dunne , Kartasova Tonja , Sloane Bonnie F. , Chan Marion Man-ying
Published: July 2002

Gene expression system; DNA, recombinant; Stefin A; Cystatin A
Fluorescence properties of the Ca2+Mg2+‐ATPase protein of sarcoplasmic reticulum labeled with 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐ diazole
Authors: Bailin Gary , Huang John R.
Published: July 2002

The fluorescence intensity of the Ca2+,Mg2+‐ATPase protein of rabbit skeletal sarcoplasmic reticulum that incorporated about 2 mol of 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1, 3‐diazole (NBD‐Cl) was enhanced at high MgATP concentrations with or without 50 μM calcium. The observed enhancement indicates that the fluorophore, NBD‐Cl, can detect conformational changes in the ATPase protein.
Sequence analysis of the promoter region of the rat vasopressin gene
Authors: Mohr Evita , Richter Dietmar
Published: July 2002

The vasopressin gene is highly transcribed in magnocellular neurons of the supraoptic (SON) and paraventricular nucleus (PVN) in the rat hypothalamus. In order to identify cis‐acting elements involved in the expression of the vasopressin gene, approximately 1 kb upstream of the transcription start site has been sequenced. Several putative regulatory elements have been detected, including a glucocorticoid response element (GRE), a cAMP response element (CRE), and four AP2 binding sites. In gel shift assays performed with a labelled DNA fragment corresponding to nucleotide residues −214 to −36 and nuclear proteins extracted from SON‐derived tissue enriched in magnocellular neurons, three specific protein‐DNA complexes have been detected. Complex formation is effectively competed by addition of an excess of unlabelled fragment.
Photoreactions of retinochrome at very low temperatures
Authors: Tokunaga Fumio , Watanabe Takahiro , Uematsu Jun , Hara Reiko , Hara Tomiyuki
Published: July 2002

Retinochrome is one of the retinal proteins found in the retina of cephalopods. It catalyses the light isomerization of retinal from the all‐transto the 11‐cisform. On cooling to 25 K, the absorption peak of retinochrome (λmax490 nm) was broadened with a shoulder, showing the spectrum steepened on the long wavelength side. On irradiation with yellow‐green light (550 nm), retinochrome produced an intermediate with λmaxat a shorter wavelength, around 465 nm, and a lower extinction coefficient than lumiretinochrome. It changed to lunuretinochrome (λmax475 nm) in the dark on warming to liquid nitrogen temperature. We shall call this new intermediate prelunuretinochrome.
DNA binding and intercalation by novel porphyrins: role of charge and substituents probed by DNase I footprinting and topoisomerase I unwinding
Authors: Kuroda Reiko , Takahashi Eitaro , Austin Caroline A. , Fisher L.Mark
Published: July 2002

Porphyrins carrying four charged sidechains, e.g., meso‐tetrakis[4‐N‐methylpyridiniumyl]‐ and meso‐tetrakis[4‐N‐(2‐hydroxyethyl)pyridiniumyl]‐porphyrin, bound and intercalated similarly into DNA as measured by helix stabilization and DNA unwinding studies in the presence of DNA topoisomerase I. Despite their different bulky sidechains, these complexes gave essentially identical DNase I footprinting patterns. In contrast, tetrasubstituted porphyrins carrying three phenyl rings and a single positively charged pyridiniumyl sidechain did not intercalate and exhibited little affinity for DNA. Thus, the presence of charged sidechains on the porphyrin rather than their identity appears to be critical for efficient DNA intercalation. The results are discussed in regard to current models for the porphyrin‐DNA intercalation complex.
Evidence for aggregation of endothelin 1 in water
Authors: Bennes René , Calas Bernard , Chabrier Pierre-Etienne , Demaille Jacques , Heitz Frédéric
Published: July 2002

In this report it is shown by CD spectroscopy that endothelin 1, when dissolved in water, is able to present intermolecular interactions leading to formation of aggregates. Surface tension and conductivity measurements suggest that the aggregation occurs through formation of micelles with a CMC of about 2.2 × 10−5 M.
Characterization of a cytotoxin‐like basic protein from the cobra (Naja naja naja) venom
Authors: Shafqat Jawed , Zaidi Zafar H. , Jörnvall Hans
Published: July 2002

A cytotoxin‐like basic protein has been isolated from the venom of the nominate race of cobra (Naja naja naja from Pakistan) by a single step of high‐performance liquid chromatography. The primary structure was determined and consists of 62 amino acid residues in a single polypeptide chain. It is highly similar to that of the cytotoxin‐like basic proteins isolated from other Naja species, but differs in two of the SS‐loop structures from that of cytotoxins.
Parathyroid hormone increases thiol proteinase activity by activation of protein kinase C in cultured kidney tubule cells (OK)
Authors: Denek Liliana , Schwegler Johann S. , Schaefer Roland M. , Heidland August
Published: July 2002

Chronic exposure (24 h) to parathyroid hormone (PTH) increases the intracellular proteolytic activity in cultured opossum kidney cells 2‐fold at physiological PTH concentrations (10−12
. This increase can be blocked by E‐64, an inhibitor of thiol proteinases. The phorbol ester TPA mimicks the effect of PTH, whereas the calcium ionophore A23187 reduces the intracellular proteinase activity. Forskolin and dibutyrylic cAMP do not elevate proteinase activity. The protein kinase C inhibitor staurosporine is equally effective in blocking the TPA‐ and PTH‐induced proteinase activity increase. These data indicate that PTH increases the intracellular thiol proteinase activity by an activation of protein kinase C and not by the cAMP dependent way.
Effect of 7β‐hydroxycholesterol on astrocyte primary cultures and derived spontaneously transformed cell lines Cytotoxicity and cholesterogenesis
Authors: Behr P. , Kupferberg A. , Leray C. , Urban P.F. , Mersel M.
Published: July 2002

The correlation between the lethal effect of 7β‐hydroxycholesterol (7β‐OH‐CH) on spontaneously transformed cell lines derived from rat astrocyte primary cultures (normal cells) and de novo cholesterogenesis was investigated. Both 7β‐OH‐CH and 7‐keto‐CH were not cytotoxic on normal cells but 7β‐OH‐CH affected markedly the viability of the transformed cells. The use of [14C]acetate or [14C] mevalonate indicated that 7‐keto‐CH inhibits de novo cholesterogenesis upstream of 3‐hydroxy‐3‐methylglutaryl CoA reductase (HMGR) in both cell types whereas 7β‐OH‐CH also inhibits downstream of HMGR. The accumulation of two radiolabelled products X1 and X2 between mevalonate and CH was found in unsaponifiable neutral lipids extracted from 7β‐OH‐CH treated transformed cells. HPLC and GC‐MS revealed that X1 and X2 are not lanosterol anti 24.25‐epoxylanosterol, respectively. Incubation of the transformed cells with X1 and X2 did not affect their viability. Our data demonstrate that, under our experimental conditions, 7β‐OH‐CH cytotoxicity is not linked to the inhibition of de novo cholesterogenesis in cultured glial transformed cells.
Nucleotide sequence and chromosomal location of Cab11 and Cab12, the genes for the fourth polypeptide of the photosystem I light‐harvesting antenna (LHCI)
Authors: Schwartz Egbert , Shen Dingren , Aebersold Ruedi , McGrath J.Mitchell , Pichersky Eran , Green Beverley R.
Published: July 2002

Tryptic peptide sequences from the 22 kDa polypeptide of tomato LHCI were used to construct a probe for gene cloning. The two genes cloned,cab11 andcab12, encode proteins of 251 and 250 residues that are 88% identical in overall amino acid sequence and 93% identical in the deduced mature protein. Each gene is present in a single copy per haploid genome;cab11 on chromosome 3 andcab12 on chromosome 6, and each has 2 introns located in similar positions to introns in other members of the Chla/b‐binding (CAB) protein gene family. Comparison of the amino acid sequences of LHCI, LHCII, CP29 and CP24 polypeptides confirms that all CABs share two regions of very high similarity which include the first and third transmembrane helices and the stroma‐exposed sequences preceding them. However, near the N‐terminus and between the conserved regions, the LHCI polypeptides have sequence motifs which appear to be PSI‐specific.
Characterization of glucagon‐like peptide‐I(7–36)amide receptors of rat lung membranes by covalent cross‐linking
Authors: Richter Gerd , Göke Rüdiger , Göke Burkhard , Schmidt Harald , Arnold Rudolf
Published: July 2002

125I‐labelled GLP‐I(7–36)amide was cross‐linked to a specific binding protein in rat lung membranes using disuccinimidyl suberate. A single radio‐labelled band at M
r 66000 was identified by SDS‐PAGE after solubilization of the ligand‐binding protein complex which is consistent with the presence of a single class of binding sites on rat lung membranes. The band was undetectable when 1 μmol/1 GLP‐I(7–36)amide was included in the binding assay. No change in the mobility of the band was observed under reducing conditions suggesting that the binding protein in the receptor is not part of a larger disulphide‐liked protein. The intensity of the radiolabelled protein band was reduced when the incubation with 125I‐labelled GLP‐I(7–36)amide was carried out in the presence of guanine nucleotides suggesting that the GLP‐I(7–36)amide receptor is coupled to the adenylate cyclase system.
Site‐directed mutagenesis of alanine‐382 of human antithrombin III
Authors: Austin Richard C. , Rachubinski Richard A. , Blajchman Morris A.
Published: July 2002

Antithrombin III Hamilton is a structural variant of antithrombin III (AT‐III) with normal heparin affinity but impaired scrine protcase inhibitory activity. The molecular defect of AT‐III‐Hamilton is a substitution of threonine for alanine at amino acid residue 382. Recently it has been shown that both plasma‐derived and cell‐free‐derived AT‐III‐Hamilton polypeptides act as substrates rather than inhibitors of thrombin and factor Xa. In the present study, the cell‐free expression phagemid vector pGEM‐32f(+)‐AT‐III1–433 was mutated at amino acid residue 382 of AT‐III to generate 7 cell‐free‐derived variants. All these cell‐free‐derived AT‐III variants were able to bind heparin as effectively as cell‐free‐derived normal AT‐III. In terms of α‐thrombin inhibitory activity each variant reacted differently. Variants could be grouped into 3 categories with respect to thrombin‐AT‐III complex formation: (1) near normal activity (glycine, isolcucine, leucine, valine): (2) low activity (threonine, glutamine): (3) no detectable activity (lysine). These data suggest that mutations at position 382 of AT‐III may have a variable effect on protease inhibitory activity, depending on either the stability of the P12‐P9 region of the exposed loop of AT‐III, or the inability of the amino acid residue at position 382 to interact with a conserved hydrophobic pocket consisting of phenylalanine (at positions 77,221 and 422) and isolcucine (position 412) residues.
Generation of interleukin‐8 by plasmin from AVLPR‐interleukin‐8, the human fibroblast‐derived neutrophil chemotactic factor
Authors: Nakagawa Hideo , Hatakeyama Shinji , Ikesue Atsutoshi , Miyai Hisato
Published: July 2002

Plasmin mainly cleaved the Arg5‐Ser6 bond of Arg‐Val‐Leu‐Pro‐Arg‐interleukin‐8 (AVLPR‐IL‐8) produced by human dermal fibroblasts, which resulted in the conversion of AVLPR‐IL‐8 to IL‐8 and the inactive pentapeptide, though a minor cleavage of AVLPR‐IL‐8 by plasmin at Lys8‐Glu9 bond occurred.
Amphibian myocardial angiotensin II receptors are distinct from mammalian AT1 and AT2 receptor subtypes
Authors: Sandberg Kathryn , Ji Hong , Millan Monica A. , Catt Kevin J.
Published: July 2002

High‐affinity receptors for angiotensin II were identified on Xenopus laevis cardiac membranes and characterized by binding‐inhibition studies with peptide and non‐peptide AII antagonists. Scatchard analysis of the binding data identified a high‐affinity site with K
d1
=1.6 nM and B
max1=3.7 pmol/mg protein and a low‐affinity site with K
d2=22 nM and B
max2
=9.5 pmol/mg protein. Treatment with dithiothreitol reduced the number of binding sites by > 70%. The rank order of potency for AII analogs was (agent, IC50) [Sar1,Ile8]AII, 0.91 nM > AII, 2.0 nM > AI, 5.3 nM > [Sar1, Ala8]AII, 19 nM > CGP42112A, 1.2 μM ⋙ DuP 753≈PD‐123177, > μM. The relative potencies of these compounds differ markedly from their activities on the two known mammalian AII receptor subtypes, AT1 and AT2. These results indicate that amphibian AII receptors are pharmacologically distinct from both the AT1 and AT2 receptors characterized in mammalian tissues.
A catalytic role for threonine‐12 of E. coli asparaginase II as established by site‐directed mutagenesis
Authors: Harms E. , Wehner A. , Aung H.-P. , Röhm K.H.
Published: July 2002

A threonine‐12 to alanine mutant of E. coli asparaginase II (EC 3.5.1.1) has less than 0.01% of the activity of wild‐type enzyme. Both tertiary and quaternary structure of the enzyme are essentially unaffected by the mutation; thus the activity loss seems to be the result of a direct impairment of catalytic function. As aspartate is still bound by the mutant enzyme, Thr‐12 appears not be involved in substrate binding.
Nanosecond electron transfer kinetics in photosystem I following substitution of quinones for vitamin K1as studied by time resolved EPR
Authors: Sieckman Ina , van der Est Arthur , Bottin Hervé , Sétif Pièrre , Stehlik Dietmar
Published: July 2002

Room temperature transient EPR spectra of photosystem I (PS I) particles fromSynechocystis6803 are presented. Native PS I samples and preparations depleted in the A1‐acceptor site by solvent extraction and then reconstituted with the quinones (Q) vitamin K1(VK1), duroquinone (DQ and DQd12) and naphthoquinone (NQ) have been studied. Sequential electron transfer to P+700A−1(FeS) and P+700A1(FeS)−is recovered only with VK1. With DQ and NQ electron transfer is restored to form the radical pair P+700Q−as specified by a characteristic electron spin polarization (ESP)‐pattern, but further electron transfer is either slowed down or blocked. A qualitative analysis of the K‐band spectrum suggests that the orientation of reconstituted NQ in PS I is different from the native acceptor A1= VK1.
Plasmin cleavage of vitronectin Identification of the site and consequent attenuation in binding plasminogen activator inhibitor‐1
Authors: Chain Daniel , Kreizman Tamar , Shapira Hadar , Shaltiel Shmuel
Published: July 2002

Plasmin is shown to specifically cleave vitronectin at the Arg361‐Ser362 bond, 18 amino acid residues upstream from the site of the endogenous cleavage which gives rise to the two‐chain form of vitronectin in plasma. The cleavage site is established using the exclusive phosphorylation of Ser378 with protein kinase A. As a result of the plasmin cleavage, the affinity between vitronectin and the type‐1 inhibitor of plasminogen activator (PAI‐1) is significantly reduced. This cleavage is stimulated by glycosaminoglycans, which are known to anchor vitronectin to the extracellular matrix. A mechanism is proposed through which plasmin can arrest its own production by feedback signalling, unleashing PAI‐1 from the immobilized vitronectin found in the vascular subendothelium, which becomes exposed at the locus of a hemostatic event.
Determination of the branch location of extra N‐acetyllactosamine units in sialo N‐linked tetraantennary oligosaccharides
Authors: Hokke Cornelis H. , Kamerling Johannis P. , van Dedem Gijs W.K. , Vliegenthart Johannes F.G.
Published: July 2002

An approach is presented for the determination of the branch location of 1 or 2 extra N‐acetyllactosamine units in sialo N‐linked carbohydrate chains from glycoproteins. Tetraantennary oligosaccharides containing extra N‐acetyllactosamine units were digested with endo‐β‐galactosidase, followed by treatment with N‐acetyl‐β‐glucosaminidase, yielding products which could be analysed by 1H‐NMR spectroscopy, thereby giving conclusive data about the location of the extra units in the intact structures.
Effects of the lipase inhibitors, Triton WR‐1339 and tetrahydrolipstatin on the synthesis and secretion of lipids by rat hepatocytes
Authors: Hermier Dominique , Hales Paul , Brindley David N.
Published: July 2002

The lipase inhibitors, Triton WR‐1339 and tetrahydrolipstatin, were incubated with rat hepatocytes. Triton WR‐1339 increased the recovery of triacylglycerol in the hepatocytes and incubation medium by 31% and 38%, respectively. Tetrahydrolipstatin decreased the accumulation of newly synthesized, and of total triacylglycerol in the medium. This compound might be useful in determining mechanisms involved in intracellular triacylglycerol metabolism and the secretion of very low density lipoproteins.
Platelet‐activating factor activates cardiac GK via arachidonic acid metabolites
Authors: Nakajima Toshiaki , Sugimoto Tsuneaki , Kurachi Yoshihisa
Published: July 2002

Platelet‐activating factor (PAF), added to the bathing solution, stimulated the cardiac muscarinic K+ channel (KACh) in the cell‐attached patch (no agonist in the pipette). The PAF‐induced KACh channel activation was blocked by WEB2086, a PAF‐receptor inhibitor, indicating that the PAF‐receptor mediated the response. PAF‐induced activation was prevented by nordihydroguaieretic acid, a lipoxygenase inhibitor, and AA‐861, a 5‐lipoxygenase inhibitor, but was not affected by indomethacin, a cyclo‐oxygenase inhibitor. The PAF‐induced KACh, channel activity disappeared upon formation of inside‐out patch. In this inside‐out patch, intracellular GTP alone induced maximal channel reactivation, which was inhibited by GDP‐βS. These results suggest that 5‐lipoxygenase metabolites of PAF‐released arachidonic acid cause a persistent stimulation of GK but not the KACh channel itself, resulting in a receptor‐independent activation of the KACh channel by GTP.
Arg‐Gly‐Asp constrained within cyclic pentapoptides Strong and selective inhibitors of cell adhesion to vitronectin and laminin fragment P1
Authors: Aumailley Monique , Gurrath Marion , Müller Gerhard , Calvete Juan , Timpl Rupert , Kessler Horst
Published: July 2002

Cyclic Arg‐Gly‐Asp‐Phe‐Val peptides with either D‐Phe or D‐Val residues were 20‐ to more than 100‐fold better inhibitors of cell adhesion to vitronectin and/or laminin fragment P1 when compared to a linear variant or Gly‐Arg‐Gly‐Asp‐Ser. No or only little increase in inhibitory capacity was observed for fibronectin adhesion and for the binding or platelet receptor αIIbβ3 to fibrinogen. NMR studies of the two most active cyclic peptides showed for both an all‐trans conformation with a βII′ and γ turn. Subtle conformational differences, however, exist between both peptides and may contribute to selectivity or inhibition.
A new class of anti‐HIV agents: GAP31, DAPs 30 and 32
Authors: Lee-Huang Sylvia , Kung Hsiang-fu , Huang Paul L. , Huang Philip L. , Li Bao-Qun , Huang Peter , Huang Henry I. , Chen Hao-Chia
Published: July 2002

Three inhibitors of human immunodeficiency virus (HIV) have been isolated and purified to homogeneity from Euphorbiaceae himalaya seeds (Gelonium multiflorum) and carnation leaves (Dianthus caryophytlus). These proteins. GAP 31 (Gelonium Anti‐HIV Protein 31 kDa) and DAPs 30 and 32 (dianthus anti‐HIV proteins, 30 and 32 kDa), inhibit HIV‐1 infection and replication in a dosc‐dependent manner with little toxicity to target cells. The therapeutic indices of these compounds are in the order 104, suggesting that they may be clinically important agents in the treatment of AIDS. The N‐terminal amino acid sequences of these proteins show little homology to those of previously described anti‐HIV proteins. The structure‐function features of these HIV inhibitors, based on the 40–60 amino acid residues of N‐terminal sequences, are examined.
Tunicamycin inhibits the initiation of DNA synthesis stimulated by prostaglandin F2α in Swiss mouse 3T3 cells
Authors: Estevez Alvaro G. , Calamante Gabriela , Goin Mercedes , de Asua Luis Jimenez
Published: July 2002

Tunicamycin, an inhibitor of the asparagine‐linked protein N‐glycosylation, blocks the initiation of DNA synthesis in Swiss 3T3 cells stimulated by prostaglandin F2α alone or with insulin. This effect is exerted only when tunicamycin is added from 0 to 8 h after stimulation and it decreases the rate of entry into S phase. Blocking of labeled sugar incorporation to proteins occurs regardless of the time of PGF2α stimulation. In contrast tunicamicin does not inhibit protein synthesis. These results suggest that N‐glycoprotein synthesis early during the prereplicative phase is an important event controlling the mitogenic action of PGF2α
Construction and characterization of a fusion protein with epidermal growth factor and the cell‐binding domain of fibronectin
Authors: Kawase Yasutoshi , Ohdate Yoichi , Shimojo Tomoko , Taguchi Yuki , Kimizuka Fusao , Kato Ikunoshin
Published: July 2002

An efficient expression system was constructed for C‐EGF, a fusion protein made of a fragment of the cell‐binding domain of human fibronectin (FN) bound with epidermal growth factor (EGF), C‐EGF was produced in Escherichia coli HB101 cells carrying the recombinant plasmid pCE102 as inclusion bodies, which were solubilized and refolded after purification. C‐EGF had both cell‐adhesive and EGF activities, so it might be more effective than EGF in therapeutic applications. This fusion system would be useful for the construction of a recombinant drug delivery system for cells that have fibronectin receptors (integrins).
Molecular link between membrane cholesterol and Na+/H+ exchange within human platelets
Authors: Kochhar N. , Kaul D.
Published: July 2002

Incubation of human platelets with cholesterol‐poor, cholesterol‐normal and cholesterol‐rich liposomes revealed that: (i) acquisition or depletion of platelet membrane cholesterol was highly selective; (ii) variation in membrane cholesterol was highly selective. Variation in membrane cholesterol content (cholesterol‐to‐phospholipid molar ratio from 0.15–1.2) with respect to values found in unmodified normal platelets, was paralleled by the observed changes in amiloride‐sensitive cytoplasmic pH, as well as phospholipase A2 activity. However, a decrease in cytoplasmic pH was accompanied by an increase in phospholipase A2 activity; (iii) membrane cholesterol‐modulated changes in intra‐platelet pH, as well as phospholipase A2 activity, was completely inhibited when platelets were pretreated with quinacrine (a specific phospholipase A2 inhibitor) before exposure to various types of liposomes. Although exposure of platelets (pretreated with amiloride) with various types of liposomes resulted in the inhibition of Na+/H+ exchange it had no noticeable effect upon the observed phospholipase A2 activity. Based upon these results we suggest that membrane cholesterol‐modulated phospholipase A2 activity may be the basic mechanism responsible for the nature of Na+/H+ exchanger activity observed in cholesterol‐enriched platelets, leading these platelets to a hypersensitized state.
ATP and light regulate D1 protein modification and degradation Role of D1* in photoinhibition
Authors: Aro Eva-Mari , Kettunen Reetta , Tyystjärvi Esa
Published: July 2002

We have recently shown that during in vivo photoinhibition the D1 protein is degraded via a modified form, designated D1*. Depending on light conditions, the amount of D1* varies in leaves between 0 and 50% of total D1 content. By isolating thylakoids from leaves acclimated to different light levels, and performing photoinhibition experiments on these thylakoids, the following results on D1 protein degradation were obtained: (1) the protease involved in D1 degradation requires activation by light; (ii) neither acceptor nor donor side photoinhibition of PSII induces formation of D1* in vitro; (iii) in isolated thylakoids, the transformation of D1 to D1* can be induced in low light in the presence of ATP, which suggests that D1* is a phosphorylated form of the D1 protein; (iv) D1*, induced either in vivo or in vitro, is much less susceptible to degradation during illumination of isolated thylakoids than the original D1 protein. We suggest that the modification to D1* is a means to prevent disassembly of photodamaged photosystem II complex in appressed membranes.
Induction of nitric oxide synthase by lipoteichoic acid from Staphylococcus aureus in vascular smooth muscle cells
Authors: Auguet Michel , Lonchampt Marie-Odile , Delaflotte Sylvie , Goulin-Schulz Jocelyne , Chabrier Pierre Etienne , Braquet Pierre
Published: July 2002

Inducible vascular nitric oxide synthase accounts for the contractile impairment observed in ondotoxemia. We provide evidence that lipoteichoic acid (LTA) from Staphylococcus aureus, a micro‐organism without endotoxin, also induces nitric oxide synthase. Our study demonstrates that on endothelium‐free rings of rat aorta. LTA‐like lipopolysaccharide induces a loss of contractility restored by Methylene blue and N
G‐nitro‐l‐arginine‐methyl ester (LNAME). Moreover in cultured vascular smooth muscle cells, LTA produces a dose‐dependent increase in intracellular cyclic GMP which is antagonized by LNAME and prevented by dexamethasone.
Structural determinants of Cys2His2 zinc fingers
Authors: Mortishire-Smith Russell J. , Lee Min S. , Bolinger Lizann , Wright Peter E.
Published: July 2002

Two mutants of the zinc finger peptide Xfin‐31 (Ac‐YKCGLCERSFVEKSALSRHQRVHKN‐CONH2) containing alterations to the conserved hydrophobic core have been constructed and their zinc‐bound structures investigated by 1H NMR techniques. In the first (Xfin‐31B) a double mutation R8F/F10G places the conserved core aromatic residue at position 8 rather than position 10. In the second (Xfin‐31C), Phe‐10 is replaced by Leu. A qualitative analysis of 1H chemical shifts, NOE connectivities and coupling constants indicates that the global folds of both mutants are similar to that of the wild‐type protein. However, amide exchange rates suggest that the F10L mutant is much less stable than either the wild‐type or the R8F/F10G mutant.
Direct expression in Escherichia coli and characterization of bovine adrenodoxins with modified amino‐terminal regions
Authors: Sagara Yasuhiro , Hara Takayuki , Ariyasu Yasuyuki , Ando Fumiko , Tokunaga Naomi , Horiuchi Tadao
Published: July 2002

Four forms of bovine adrenodoxin with modified amino‐termini obtained by direct expression of cDNAs in Escherichia coli are Ad(Met1), Ad(Met−1), Ad(Met−12), and Ad(Met6). The shoulder numbers represent this site of translation initiator Met at the amino‐termini. The adrenodoxins, except for Ad(Met−1), were purified from the cell lysate and the ratios of A
414‐to‐A
276 of the purified proteins were over 0.92. NADPH‐cytochrome c reductase activities of the three forms of adrenodoxin in the presence of adrenodoxin reductase were the same as that of purified bovine adrenocortical adrenodoxin. However, as cytochrome P‐450SCC reduction catalyzed by Ad(Met0) was about 60% or that by Ad(Met1), the contribution of the amino‐terminal region for the electron transfer or binding to cytochrome P‐450SCC would need to be considered.
Irreversible oxidative inactivation of protein kinase C by photosensitive inhibitor calphostin C
Authors: Gopalakrishna Rayudu , Chen Zhen Hai , Gundimeda Usha
Published: July 2002

Isolated protein kinase C (PKC) was irreversibly inactivated by a brief (min) incubation with calphostin C in the presence of light. This inactivation required Ca2+either in a millimolar range in the absence of lipid activators or in a submicromolar range in the presence of lipid activators. In addition, an oxygen atmosphere was required suggesting the involvement of oxidation(s) in this inactivation process. Furthermore, PKC inactivation might involve a site‐specific oxidative modification of the enzyme at the Ca2+‐induced hydrophobic region. Physical quenchers of singlet oxygen such as lycopene, β‐carotene, and α‐tocopherol all reduced the calphostin C‐induced inactivation of PKC. In intact cells treated with calphostin C, the inactivation of PKC was rapid in the membrane fraction compared to cytosol. This intracellular PKC inactivation was also found to be irreversible. Therefore, calphostin C can bring prolonged effects for several hours in cells treated for a short time. Taken together these results suggest that the calphostin C‐mediated inactivation of PKC involves a site‐specific and a ‘cage’ type oxidative modification of PKC.
Involvement of MAP kinase activators in angiotensin II‐induced activation of MAP kinases in cultured vascular smooth muscle cells
Authors: Ishida Yoshihiro , Kawahara Yasuhiro , Tsuda Terutaka , Koide Masanobu , Yokoyama Mitsuhiro
Published: July 2002

In cultured vascular smooth muscle cells (VSMC) angiotensin II (ang II) induces tyrosine and serine/threonine phosphorylation and activation of two mitogen‐activated protein (MAP) kinases, When extracts of ang II‐stimulated VSMC were fractionated by Mono Q anion‐exchange column chromatography, three peaks of the activities which in vitro activate inactive MAP kinases were detected. These MAP kinase activator activities were not detected in extracts of unstimulated VSMC. In vitro activation of MAP kinases by the MAP kinase activators was accompanied by tyrosine and serine/threonine phosphorylation of MAP kinases. These results suggest that the MAP kinase activators are involved in the ang II‐induced phosphorylation and activation of MAP kinases in VSMC.
System A transport activity is stimulated in skeletal muscle in response to diabetes
Authors: Gumà Anna , Mora Conchi , Santalucía Tomàs , Viñals Francesc , Testar Xavier , Palacín Manuel , Zorzano Antonio
Published: July 2002

We have studied the activity of system A transport in skeletal muscle during experimental diabetes. Five days after streptozotocin injection, rats showed a marked hyperglycemia and a substantial decrease in the content of GLUT‐4 protein in skeletal muscle and adipose tissue. Under these conditions, basal uptake of 2‐(methyl)aminoisobutyric acid (MeAIB), an index of system A transport activity, was enhanced in extensor digitorum longus (EDL) muscles from diabetic rats compared to controls. Furthermore, insulin‐stimulated MeAIB uptake by the incubated EDL and soleus muscles was markedly greater in diabetic than in control rats. The derepressive phase of adaptive regulation was partially blocked in the diabetic muscle, and incubation of muscles for 3 h in the absence of amino acids led to a lower stimulation of system A transport activity in muscles from diabetic groups compared to controls. We propose that the activated system A might participle in the enhanced alanine release from muscle cells that occurs in diabetes.
Characterisation of a human bilirubin UDP‐glucuronosyltransferase stably expressed in hamster lung fibroblast cell cultures
Authors: Sutherland Lesley , Senafi Sahidan bin , Ebner Thomas , Clarke Douglas J. , Burchell Brian
Published: July 2002

A cDNA encoding a human bilirubin UDP‐glucuronosyltransferase has been isolated and stably expressed in Chinese hamster V79 lung fibroblast cell line. Western blotting of cell homogenates with anti‐UGT antibody revealed a highly expressed protein of approx. 55.5 kDa in size. The expressed enzyme specifically catalysed the formation of bilirubin mono‐ and diglucuronides, and also catalysed the glucuronidation of two phenolic compounds, which are good substrates for other human UGT isoenzymes, at low rates.
Production and secretion of plasminogen in cultured rat brain microglia
Authors: Nakajima Kazuyuki , Tsuzaki Naoko , Nagata Koichi , Takemoto Nagisa , Kohsaka Shinichi
Published: July 2002

The production and secretion of plasminogen in cultured rat brain microglia was investigated. Urokinase‐dependent cascinolytic activity was detected by zymography in microglial conditioned medium with a molecular weight of about 90 kDa. The 90‐kDa protein was also detected by Western blotting with anti‐rat plasminogen antiserum in the non‐reducing condition. Immunoprecipitation with plasminogen antiserum following [35S]methionine labelling revealed that the plasminogen detected in microglial conditioned medium is synthesized in microglia. The amount of plasminogen in the conditioned medium was increased by stimulation with lipopolysaccharide. These results show that cultured microglia produce plasminogen and secrete it into the culture medium.
Regulation of cardiac insulin receptor function by guanosine nucleotides
Authors: Russ Martina , Reinauer Hans , Eckel Jürgen
Published: July 2002

The present study examined the effect of [35S]GTP on the function of insulin receptors partially purified from adult rat cardiomyocytes by WGA chromatography. [35S]GTP increased receptor autophosphorylation about two times and fully mimicked the stimulatory action of insulin on poly(Glu:Tyr) phosphorylation with no additional effect of the hormone. The effect of [35S]GTP was specific, dose‐dependent, and due to an increase in the V
max of the kinase. In the presence of ATP or AMP‐PNP, insulin significantly enhanced the binding of [35S]GTP to the partially purified insulin receptor. The findings suggest coupling of the insulin receptor to a G‐protein which may be involved in the regulation of tyrosine kinase activity.
A human somatostatin receptor (SSTR3), located on chromosome 22, displays preferential affinity for somatostatin‐14 like peptides
Authors: Corness Jacquie D. , Demchyshyn Lidia L. , Seeman Philip , Van Tol Hubert H.M. , Srikant Coimbatore B. , Kent Gillian , Patel Yogesh C. , Niznik Hyman B.
Published: July 2002

We report here on the cloning of a human intronless gene encoding a member of the G‐protein linked somatostatin (SST) receptor subfamily, termed SSTR3. Based on the deduced amino acid sequence, this gene encodes a 418 amino acid protein displaying sequence similarity, particularly within putative transmembrane domains, with the recently cloned human SSTR1 (62%), SSTR2 (64%) and SSTR4 (58%) receptors. Membranes prepared from COS‐7 cells transiently expressing the human SSTR3 gene bound [125I]Leu8,d‐Trp22,‐Tyr22 SST‐28 in a saturable manner with high affinity (~200 pM) and with rank order of potency (d‐Trp8 SST‐14 > SST‐14 > SMS‐201‐995 > SST‐28) indicative of a somatostatin‐14 selective receptor. The pharmacological profile of the expressed human SSTR3 receptor is similar but not identical to that reported for the rat homolog [(1992) J. Biol. Chem. 267,20422] where the peptide selectivity is SST‐28 ≧ SST‐14 XXX SMS‐201‐995. Northern blot analysis reveals the presence of an SSTR3 mRNA species of ~5 kb in various regions of the monkey brain, including the frontal cortex, cerebellum, medulla, amygdala, with little or no SSTR3 mRNA detectable in brain regions such as the striatum, hippocampus, and olfactory tubercle. The SSTR3 receptor gene maps to human chromosome 22. The existence of at least four distinct human genes encoding somatostatin‐14 selective receptors with diverse pharmacological specificities may help to account for some of the multiple biological actions of somatostatin under normal and pathological conditions.
v‐Ha‐Ras insertion/deletion mutants with reduced protease‐inhibitory activity have no transforming activity
Authors: Sawada Toshie , Sakiyama Shigeru , Hiwasa Takaki
Published: July 2002

We have purified 26 insertion/deletion mutants of v‐Ha‐ras oncogene products produced by Escherichia coli and investigated their protease‐inhibitory activity toward papain and cathepsins B and L. K
i values for papain were relatively similar among the mutants, however, those for cathepsins B and L varied up to 10‐fold. Among them, four mutants, 1–48 LIR 54–189, 1–110 LIS 112–189, 1–130 PDQ 146–189 and 1–155 LIR 166–189, showed significant reduction in the inhibitory activity toward cathepsin L and these four mutants have lost transforming activity toward NIH3T3 mouse fibroblasts. However, some other mutants also showed no transforming activity in spite of possession of the potent protease‐inhibitory activity, suggesting that the protease‐inhibitory activity of Ras might be necessary but not sufficient for its biological activity.
The three‐dimensional structure of the colicin E3 immunity protein by distance geometry calculation
Authors: Yajima Shunsuke , Muto Yutaka , Morikawa Souichi , Nakamura Haruki , Yokoyama Shigeyuki , Masaki Haruhiko , Uozumi Takeshi
Published: July 2002

The three‐dimensional solution structure of the colicin E3 immunity protein (84 residues) was determined by distance geometry calculations. The hydrophilic side of a four‐stranded antiparallel β‐sheet constitutes a part of the surface of the protein, and two loops lie on the hydrophobic side of the sheet. All the three specificity‐determining residues, which are included in the center of the β‐sheet, display their side groups on the protein surface.
Similarity of primary radical pair recombination in photosystem II and bacterial reaction centers
Authors: Volk Martin , Gilbert Matthias , Rousseau Gerhard , Richter Martin , Ogrodnik Alexander , Michel-Beyerle Maria-Elisabeth
Published: July 2002

We report temperature and magnetic field dependent measurements of the recombination dynamics of the radical pair P680+Pheo− in D1D2cytb559 reaction centers of photosystem II and compare the results to those obtained in bacterial reaction centers. In photosystem II the rate of recombination to the groundstate is found to be slower than in the bacterial reaction centers by a factor of at least 50. This difference arises from the different redox potentials of the pigments of plant and bacterial reaction centers. In contrast, the rate of recombination to the triplet state is similar in all reaction centers, indicating a similar electronic coupling which allows us to conclude upon the structural similarity.
Conserved HisVI‐17 of the NK‐1 receptor is involved in binding of non‐peptide antagonists but not substance P
Authors: Zoffmann Sannah , Gether Ulrik , Schwartz Thue W.
Published: July 2002

Residue number 17 in transmembrane segment VI has been shown to be crucial for the binding of agonists in G‐protein‐coupled receptors for the monoamines. In many peptide receptors a histidyl residue has been conserved at this position. We find that replacement of HisVI‐17 in the NK‐1 receptor with either glutamine, phenylalanine, or alanine has no apparent effect on the binding of the natural peptide ligand substance P or on the agonist induced increase in inositolphosphate turnover. However, the binding of certain non‐peptide antagonists was impaired; for example, replacement of HisVI‐17 with alanine decreased the affinity for FK888 and RP67,580 5‐ to 12‐fold, respectively. A glutamine side chain was a good substitute for the imidazole in the binding of all non‐peptide antagonists. It is concluded that the conserved HisVI‐17 in the NK‐1 receptor is involved in the binding of certain non‐peptide antagonists, but is not important for the action of the natural peptide agonist, substance P.
The transforming activity of activated Gα12
Authors: Jiang Huiping , Wu Dianqing , Simon Melvin I.
Published: July 2002

Heterotrimeric GTP‐binding proteins transduce receptor‐mediated extracellular signals to their effectors. Several activated mutations of Gsα and Giα have been shown to be associated with endocrine tumors. In this report, we have evaluated the transforming activity of an putative activated form of Gα12 subunit. We found that transient expression in NIH3T3 cells of the Gα12 mutant with substitution of glutamine‐229 for leucine could lead to focus formation and that stably transfected NIH3T3 cells could form colonies in soft agar and tumors in nude mice.
cDNA cloning and pharmacological characterization of an opioid receptor with high affinities for κ‐subtype‐selective ligands
Authors: Nishi Miyuki , Takeshima Hiroshi , Fukuda Kazuhiko , Kato Shigehisa , Mori Kenjiro
Published: July 2002

The amino acid sequence of a rat opioid receptor, designated as ROR‐D, has been deduced by cloning and sequencing the cDNA. The ROR‐D expressed from the cDNA exhibits high affinities for ligands selective for the opioid receptor κ‐subtype and low affinities for ligands selective for the δ‐ and μ‐subtypes. RNA blot hybridization analysis indicated that ROR‐D mRNA is distributed in cerebrum and brainstem but not in cerebellum.