bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthetic peptide from lipocortin I has no phospholipase A2 inhibitory activity
Authors: van Binsbergen J. , Slotboom A.J. , Aarsman A.J. , de Haas G.H.
Published: July 2002

Two anti‐inflammatory peptides corresponding to a high amino acid similarity region between lipocortins were synthesized and tested on their ability to inhibit porcine pancreatic phospholipase A2. Kinetic assays using monomeric and aggregated phospholipids did not reveal any phospholipase A2 inhibitory activity. The peptides did not inhibit phospholipase A2 activity on monolayers of negatively charged substrate and did not prevent phospholipase A2 action on mixed micelles of 1‐stearoyl‐2‐arachidonoyl‐sn‐glycero‐3‐phosphocholine and sodiumdeoxycholate. Ultraviolet difference spectroscopy did not show binding of the peptides to phospholipase A2. Therefore we conclude that these anti‐inflammatory peptides do not inhibit pancreatic phospholipase A2 in vitro, in contrast to the results recently published [(1988) Nature 335,726–730].
An analysis of the periodicity of conserved residues in sequence alignments of G‐protein coupled receptors
Authors: Donnelly D. , Johnson M.S. , Blundell T.L. , Saunders J.
Published: July 2002

Twenty‐three sequences from the family of G‐protein coupled receptors have been aligned according to the ‘historical alignment’ procedure of Feng and Doolittle. Fourier transform analysis of this reveals that parts of five of the seven putative membrane‐spanning regions exhibit a periodicity of conserved/nonconserved residues which is compatible with the periodicity of the α‐helix. This would place the conserved residues on one side of the helix, which may face the inside of the proposed seven membered helical bundle.
Reductive dehydroxylation of 4‐hydroxybenzoyl‐CoA to benzoyl‐CoA in a denitrifying, phenol‐degrading Pseudomonas species
Authors: Glöckler Rainer , Tschech Andreas , Fuchs Georg
Published: July 2002

The initial reactions in anaerobic degradation of phenol to CO2 have been studied in vitro with a denitrifying Pseudomonas strain grown with phenol and nitrate in the absence of molecular oxygen. Phenol has been proposed to be carboxylated to 4‐hydroxybenzoate [(1987) Arch. Microbiol. 148, 213–217]. 4‐Hydroxybenzoate was activated to 4‐hydroxybenzoyl‐CoA by a coenzyme A ligase. Cell extracts also catalyzed the reductive dehydroxylation of 4‐hydroxybenzoyl‐CoA to benzoyl‐CoA with reduced benzyl viologen as electron donor. This enzyme, benzoyl‐CoA:(acceptor) 4‐oxidoreductase (hydroxylating) (EC 1.3.99.‐), has not been reported before. The data suggest that phenol and 4‐hydroxybenzoate are anaerobically metabolized by this strain via benzoyl‐CoA.
Limited proteolysis of Cl‐inhibitor by chymotrypsin‐like proteinases
Authors: Schoenberger Oeyvind L. , Sprows Jennifer L. , Schechterf Norman M. , Cooperman Barry S. , Rubin Harvey
Published: July 2002

Limited proteolysis of Cl‐inhibitor was observed with human skin chymase, human cathepsin G, and bovine chymotrypsin. In each case, the inhibitor was degraded to one major product migrating slightly faster than the native inhibitor in an SDS‐polyacrylamide gel. The inhibitory activity of Cl‐inhibitor against human plasma kallikrein was not altered by the modification with chymase. Edman degradation of the proteolyzed inhibitor revealed two sequences in a 1:1 ratio: NPNATSSSQ, the N‐terminus of native Cl‐inhibitor, and VEPILEVSSL. This second sequence showed that the Phe33‐Val34 bond was hydrolyzed. Our results provide another example of the susceptibility of the N‐terminal region of Cl‐inhibitor to proteolytic cleavage.
Molecular organization of genes constituting the virulence determinant on the Salmonella typhimurium 96 kilobase pair plasmid
Authors: Taira Suvi , Rhen Mikael
Published: July 2002

The ability of intracellular growth is plasmid‐dependent in Salmonella typhimurium. Only a small portion of this 96 kilobase pair plasmid appears essential for intracellular growth. The genetic organization of this region (the essential virulence determinant) was resolved. Fragments of the virulence determinant were cloned from the 96‐kb plasmid pEX102 and transformed into minicell‐producing E. coli. Plasmid‐directed protein synthesis was investigated in metabolically labeled minicells. This analysis indicated the presence of at least four genes, mkaA, mkaB, mkaC and mkaD, within the virulence determinant encoding proteins of 70, 31, 30 and 29 kDa, respectively. The genes were positioned on the restriction map of the 96‐kb virulence plasmid and the map locations confirmed by nucleotide sequence analysis of two new virulence genes (mkaB and mkaC)
Effects of vasoactive intestinal contractor (VIC) and endothelin on intracellular calcium level in neuroblastoma NG108‐15 cells
Authors: Fu Tao , Chang Wei , Ishida Norio , Saida Kaname , Mitsui Youji , Okano Yukio , Nozawa Yoshinori
Published: July 2002

Effects on [Ca2+]i levels of endothelin‐1 (ET) and vasoactive intestinal contractor peptide (VIC), which is a novel member of the endothelin family, were examined in fura 2‐loaded neuroblastoma NG108‐15 cells. VIC was found to be a very effective stimulus for intracellular Ca2+ mobilization and to be more potent than ET. Intracellular calcium response to sequential addition of two stimulants exhibited the homologous desensitization of either ET or VIC, but no heterologous desensitization between ET and VIC. This indicates evidence suggesting that these two peptides act through distinct receptors.
Additive effects of dexamethasone and calcium on the calcitonin mRNA level in adrenalectomized rats
Authors: Besnard Philippe , Jousset Valérie , Garel Jean-Michel
Published: July 2002

Northern hybridizations were used to evaluate the effects of dexamathasone and calcium on calcitonin mRNA levels in adrenalectomized female rats. Two weeks after adrenalectomy, a 3.6‐fold decrease in the calcitonin mRNA level was observed (28% vs 100% in sham‐operated controls). After 5 days of dexamethasone treatment (1.5 mg/kg b wt), a 2.6‐fold rise in calcitonin mRNA occurred in adrenalectomized rats (73% vs 28%). This increment was higher when dexamethasone treated animals were injected with calcium (100% vs 73%). The effect of calcium on the calcitonin mRNA level of adrenalectomized rats treated or not with dexamethasone was similar, and additive in the former case. Our data suggest that calcium and dexamethasone elevate calcitonin mRNA by two different mechanisms.
Involvement of protein kinase C in the mitogenic and chemotaxis effects of basic fibroblast growth factor on bovine cerebral cortex capillary endothelial cells
Authors: Daviet I. , Herbert J.M. , Maffrand J.P.
Published: July 2002

Basic fibroblast growth factor is increasingly implicated in cellular growth, differentiation, angiogenesis and oncogenesis. In culture, basic fibroblast growth factor greatly improved the growth rate of bovine brain cortex capillary endothelial cells. Down‐regulation of protein kinase C by prolonged treatment with phorbol esters prevented the mitogenic effect of basic fibroblast growth factor on capillary endothelial cells. Furthermore, staurosporine, a potent protein kinase inhibitor, showed strong antiproliferative activity against basic fibroblast growth factor‐induced endothelial cell growth. Similarly, the chemotaxis effect of basic fibroblast growth factor on capillary endothelial cells was abolished by down‐regulation of protein kinase C or by staurosporine treatment. Therefore, it is suggested that protein kinase C could account for part of the angiogenic effect of basic fibroblast growth factor.
Agonist‐sensitive binding of a photoreactive GTP analog to a G‐protein α‐subunit in membranes of HL‐60 cells
Authors: Offermans S. , Schäfer R. , Hoffmann Barbara , Bombien E. , Spicher K. , Hinsch K.-D. , Schultz G. , Rosenthal W.
Published: July 2002

Myeloid‐differentiated HL‐60 cells were used to study the activation of G‐proteins by receptor agonists. Following incubation of membranes with the photoreactive GTP analog. [α‐32P]GTP azidoanilide, and subsequent exposure to ultraviolet light (254 nm), photolabeling of 40 kDa proteins comigrating with the Gi2 α‐subunit was observed. Photolabeling in the absence or presence of the chemoattractant, N‐ionnyl‐methionyl‐leucyi‐phenylalanin (FMLP), absolutely required Mg2+; FMLP stimulated photolabeling at all Mg2+ concentrations employed (up to 30 mM). Addition of GDP (3–50 μM) reduced basal photolabeling to a greater extent than photolabeling stimulated by FMLP. FMLP did not stimulate photolabeling of proteins modified by pertussis toxin. Leukotriene B4 and C5a also stimulated photolabeling of 40 kDa proteins. The results indicate that (i) the major G‐protein in HL‐60 cells, Gi2 requires Mg2+ for basal and receptor‐stimulated activity, (ii) effective receptor‐mediated activation of G‐proteins is observed at mM concentrations of Mg2+, and (iii) receptor agonists apparently reduce the affinity of G‐proteins for GDP.
Mapping of the immunodominant regions of the NAD‐dependent formate dehydrogenase
Authors: Bogdanova Anna V. , Cherednikova Tatyana V. , Egorov Tsezi A. , Harutyunyan Emil G. , Kurochkina Nataleya A. , Lamzin Victor S. , Savitskiy Alexsander P. , Shumilin Igor A. , Popov Vladimir O.
Published: July 2002

A panel of 4 monoclonal antibodies and 7 polyclonal antisera against NAD‐dependent formate dehydrogenase from methylotrophic bacterium Pseudomonas sp. 101 has been obtained. The reactivity of the 37 overlapping proteolytic peptides with the monoclonal antibodies and polyclonal antisera has been studied with ELISA test. The data obtained were interpreted residing on the structural model of the formate dehydrogenase at 3 Å resolution. The immunodominant regions in the formate dehydrogenase molecule and the epitopes for the monoclonal antibodies were elucidated.
aS,7S‐absolute configuration of natural (−)‐colchicine and allocongeners
Authors: Brossi Arnold , Boyé Olivier , Muzaffar Anjum , Yeh Herman J.C. , Toome Voldemar , Wegrzynski Bogda , George Clifford
Published: July 2002

The aS,7S‐absolute configuration of (−)‐colchicine (1) and (−)‐N‐acetylcolchinol methyl ether (3, NCME) suggested on the basis of 1H NMR data and negative Cotton effects at about 260 nm (EtOH) is firmly established by an X‐ray analysis of urea 5, a compound derived from 3. Binding of these compounds to tubulin requires an aS‐configuration of the biaryl system.
Cloning of two additional catecholamine receptors from rat brain
Authors: O'Dowd Brian F. , Nguyen Tuan , Tirpak Anne , Jarvie Keith R. , Israel Yedy , Seeman Philip , Niznik Hyman B.
Published: July 2002

An approach based on the polymerase chain reaction (PCR) was used to isolate additional members of the G‐linked receptor family from a rat striatal λgtII cDNA library. Priming with one degenerate probe corresponding to highly conserved consensus sequences in the third transmembrane (TM) domain of 15 G‐linked receptors and sequences in the phage vector resulted in one clone (G‐13) encoding a dopamine D2 receptor variant with a 29 amino acid insert in the third cytoplasmic loop. In addition, the amino acid sequence encoded by clone G‐36 contained conserved sequences characteristic of the G‐linked class of receptors and displayed sequence homology in TM domains with the β2‐adrenergic receptor (48%). Two conserved serine residues in TM5 postulated to be part of a ligand binding site in the adrenergic receptor, suggests that G‐36 encodes a catecholaminergic receptor. Northern blot analysis confirmed the expression of G‐36 in rat brain, but not in kidney, heart and lung. Several strong hybridizing bands to G‐36 were obtained in both human and rat genomic DNA. The general PCR strategy employed here should prove to be extremely useful for the isolation of other members of the G‐linked receptor family.
Human epidermal growth factor
Authors: Engler David A. , Montelione Gaetano T. , Niyogi Salii K.
Published: July 2002

Site‐directed mutagenesis was employed to examine the function of two highly conserved residues, Tyr‐37 and Arg‐41, of human EGF (hEGF) in receptor binding. Both a conservative change to phenylalanine and a semi‐conservative change to histidine at position 37 yield proteins with receptor affinity similar to wild‐type hEGF. A non‐conservative change to alanine results in a molecule with about 40% of the receptor affinity, indicating that an aromatic residue is not essential at this position. Both conservative (to lysine) and non‐conservative (to alanine) substitutions at position 41 drastically reduced receptor binding to <0.5% of the wild‐type activity. 1D‐NMR data indicate that the replacement of Arg‐41 by lysine does not significantly alter the native protein conformation. Thus, Arg‐41 may be directly involved in ligand‐receptor interaction, whereas the side chain of Tyr‐37, although possibly important structurally, is not essential for receptor binding.
Secondary structure of a mitochondrial signal peptide in lipid bilayer membranes
Authors: Tamm Lukas K. , Bartoldus Ingrid
Published: July 2002

The secondary structure of the synthetic signal peptide of cytochrome c oxidase subunit IV (coxIV‐25) has been measured by circular dichroism spectroscopy in different lipid environments. CoxIV‐25 is polymorphic in membranes. It forms an amphiphilic α‐helix both in negatively charged lipid bilayers (up to 49% helix) and in detergent micelles (up to 42% helix). In associaton with bilayers of the zwitterionic lipid phosphatidylcholine, coxIV‐25 takes an aperiodic, unidentified structure. CoxIV‐25 is also partially α‐helical in bilayers of cardiolipin, mitochondrial lipid extracts and mixtures of synthetic phosphatidylcholine and phosphatidylglycerol.
Expression of soluble guanylyl cyclase
Authors: Harteneck Christian , Koesling Doris , Söling Ariane , Schultz Günter , Böhme Eycke
Published: July 2002

Purified soluble guanylyl cyclase consists of two subunits (70 and 73 kDa) whose primary structures were recently determined. The availability of cDNA clones coding for either subunit allowed to study the question of the functional roles of the two subunits in expression experiments. Enzyme subunits were expressed in COS‐7 cells by transfection with expression vectors containing the coding region for the 70 of 73 kDa subunit of the enzyme. No significant elevation in the activity of soluble guanylyl cyclase was observed in cells transfected with cDNA coding for one of the subunits. In contrast, transfection of cells with cDNAs coding for both subunits resulted in a marked increase in activity of soluble guanylyl cyclase. Enzyme activity was stimulated about 50‐fold by sodium nitroprusside. The results indicate that formation of cyclic GMP by soluble guanylyl cyclase requires'both 70 and 73 kDa subunits.
Monoclonal antibodies to lipocortin‐1 as probes for biological function
Authors: Pepinsky R.Blake , Sinclair Lesley K. , Dougas Irene , Liang Chi-Ming , Lawton Pornsri , Browning Jeffrey L.
Published: July 2002

We have developed two monoclonal antibodies to human lipocortin‐1 (103 and 105) as reagents for quantitating the protein in biological systems and neutralizing its activity. Lipo 105 is a high affinity antibody that is functional in ELISA and Western blot formats. The antibody recognizes a site between amino acids 30 and 55 in the lipocortin‐1 sequence and can be used on native or denatured protein. Lipo 103 is an antibody that neutralizes the phospholipase A2 inhibitory activity of lipocortin‐1 by blocking binding of the protein to phospholipid surfaces. The antibody is specific for native human lipocortin‐1. Lipo 103 was recently shown to block lipocortin‐1‐dependent differentiation of a squamous carcinoma cell line, demonstrating its usefulness as a probe for function.
Low density lipoprotein activates monocytes to express tumor necrosis factor
Authors: Barath Peter , Cao Jin , Forrester James S.
Published: July 2002

We analyzed the effect of acetylated low density lipoprotein (aLDL) incubation on tumor necrosis factor (TNF) mRNA and protein expression in isolated resting human monocytes in serum free DMEM. TNF mRNA expression was about one third that of PMA and was dose dependent. The maximum stimulatory effect on TNF mRNA was at 250 μg/ml, while 500 μg/ml induced downregulation. The maximum stimulatory effect occurred at 6 hours, and by 24 hours TNF mRNA expression returned to the resting state. Acetyl LDL also induced the expression ofimmunoreactive TNF, reaching a sevenfold maximum above control at 12 hours following a 6 hour exposure period. The results suggest that aLDL is a potent stimulator of TNF expression in resting monocytes. This mechanism may be operational in atheroma evolution.
Substrate specificity of two kallikrein family gene products isolated from the rat submaxillary gland
Authors: Elmoujahed A. , Gutman N. , Brillard M. , Gauthier F.
Published: July 2002

Two proteinases which belong to the tissue kallikrein family were purified from rat submaxillary glands. These proteinases correspond to the products of the RSKG‐7 and the rGK8 genes, as shown by the comparison of their partial amino‐acid sequence with that deduced from nucleotide sequences. These two proteinases, kallikrein k7 and kallikrein k8, exhibit a marked preference for cleavage after arginyl residues. However, their overall specificities towards synthetic fluorogenic substrates diner significantly from each other and from that of true tissue kallikrein. Kallikrein k7 is strongly inhibited by soybean trypsin inhibitor, whereas kallikrein k8 is not. These data, demonstrating the individual specificity of these kallikrein‐like proteinases, suggest that they could be involved in the processing of peptides other than kinins.
Immunocytochemical evidence of vertebrate bioactive peptide‐like molecules in the immuno cell types of the freshwater snail Plianorbarius corneus (L.) (Gastropoda, Pulmonata)
Authors: Ottaviani Enzo , Cossarizza Andrea
Published: July 2002

An immunocytochemical investigation was carried out on round and spreading hemocytes of Planorbarius comeus by using 20 antisera to vertebrate bioactive peptides. The immunotests showed the presence of α1antichymotrypsin‐bombesin‐, calcitonin‐, CCK‐8 (INC)‐, CCK‐39‐, gastrin‐, glucagon‐, Met‐enkephalin‐, neurotensin‐, oxytocin‐, somatostatin‐, substance P‐, VIP‐, and vasopressin‐immunoreactive molecules in the spreading hemocytes. The round hemocytes were only positive to anti‐bombesin, anticalcitonin, anti‐CCK‐8 (INC), anti‐CCK‐39, anti‐neurotensin, anti‐oxytocin, anti‐substance P and anti‐vasopressin antibodies. No immunostaining was observed with anti‐CCK‐8 (Peninsula), anti‐insulin, anti‐prolactin, anti‐thyroglobulin and anti‐thyroxin (T4) antibodies. As probably in vertebrates, these bioactive peptides may modulate immuno cell function.
Posttranslational attenuation of peptide gene expression
Authors: Rehfeld Jens F.
Published: July 2002

Studies of the cell‐specific processing of neuroendocrine peptides have shown that neuroendocrine cells occasionally fail to mature the biosynthetic precursors to bioactive peptides, or that they do so to a negligible extent only. Instead, inactive precursors and processing intermediates accumulate in the cells. Thus, the expression of genes encoding hormonal peptides is in certain cells and under certain conditions attenuated at the posttranslational level. The exact molecular mechanisms of posttranslational atenuation are still largely unknown. The review emphasizes that postttranslational attenuation may play a significant role during normal cell differentiation and in the carcinogenic transformation of cells. The existence of post‐translational attenuation has significant biological and clinical implications
The specific inhibitor of protein kinase C, 1‐(5‐isoquinolinylsulfonyl)‐2‐methylpiperazine (H7), induces morphological change and cell differentiation of human neural crest‐derived cell lineages
Authors: Parodi Maria Teresa , Varesio Luigi , Tonini Gian Paolo
Published: July 2002

It has been proved that inhibition of protein kinase C by 1‐(5‐isoquinolinylsulfonyl)‐1‐methylpiperazine (H7) induces morphological differentiation in murine neuroblastoma (nb) cell. Here we report that H7 is also active on human nb cell lines. The human nb cell had originally neuroblast‐like (N) or intermediate (I) morphology. N and I type are thought to represent different stages of neuroblastoma differentiation. Neurite outgrowth was observed in both N and I type morphology treating the cells with 7, 14 or 28 μM of H7. The results confirm previous observations and show that inhibition of PKC by H7 also promotes neuronal differentiation in human cell line variants.
Synergistic and additive induction of metallothionein in Chang liver cells
Authors: Iijima Yoshio , Fukushima Tomihiro , Bhuiyan Lutful A. , Yamada Teruo , Kosaka Futami , Denry Sato J.
Published: July 2002

We have studied metallothionein (MT) induction by three primary inducers; a heavy metal, a glucocorticoid and a cytokine, and by the combinations of these inducers in the cultured cells. MT‐protein was synergistically induced by either a cytokine or a heavy metal with a glucocorticoid hormone and was additively induced by the combination of a cytokine and a heavy metal, but MT‐mRNA levels were not completely correlated with MT‐protein levels. These results suggest that posttranscriptional regulation may be involved in the synergistic induction of MT‐protein. We propose a possible mechanism in which marked MT induction by stress in vivo is dependent on the combined effect of two or more inducers, because marked MT induction is not seen by an injection of a plausible dose of either a glucocorticoid hormone or a cytokine in vivo.
Expression of the cholecystokinin gene in a human (small‐cell) lung carcinoma cell‐line
Authors: Geijer Thomas , Folkesson Ronnie , Rehfeld Jens F. , Monstein Hans-Jürg
Published: July 2002

Expression of the cholecystokinin (CCK), gastrin and enkephalin A genes were studied by Northern blot analysis and a library of sequence‐specific radioimmunoassays in human cell lines. The human small‐cell lung carcinoma line (SCLC) U‐1690 expressed moderate levels of CCK mRNA as compared to the human neuroepithelioma cell line SK‐N‐MC. Neither gastrin nor (pro)enkephalin A mRNAs were detectable in the U‐1690 cell line. In contrast, the SCLC‐line H‐69 expressed Enk A but no CCK mRNA. The radioimmunoassays showed that the CCK mRNA transcript in the SCLC line U‐1690 also is translated, and that preproCCK is processed into bioactive, carboxyamidated CCK peptides. Thus, the human small cell carcinoma cell line U‐1690 is a useful model for studies of cell‐specific CCK gene expression.
Polypeptide components of the apamin receptor associated with a calcium activated potassium channel
Authors: Leveque Christian , Marqueze Beatrice , Couraud Francois , Seagar Michael
Published: July 2002

Photoaflinity labeling of rat brain membraines with [125l]ANPAA‐apamin incorporated radioactivity into polypeptides of 86 and 59 kDa and occasionally a more weakly labeled component of 45 kDa. These polypeptides were immunoprecipitated with anti‐apamin antibodies and treated with glycosidases. Neither the 86 nor the 59 kDa polypeptide appeared to b %glycoxylated. Partial protocolytic mapping of affinity labeled polypeptides with chymotrypsin or V8 protease generated an identical pattern. Thes results suggest that the 59 and 45 kDa components are not additional subunits of an oligomeric protein but results from cleavage of the 86 kDa polypeptide
Identification of a new P1 residue mutation (444Arg→Ser) in a dysfunctional C1 inhibitor protein contained in a type II hereditary angioedema plasma
Authors: Aulak K.S. , Cicardi M. , Harrison R.A.
Published: July 2002

A new reactive‐centre P1 residue mutation (444Arg→Ser), has been identified in a dysfunctional C1 inhibitor protein, C1 inhibitor(Ba), contained in a type II hereditary angioedema plasma. This substitution is compatible with a point mutation of the 444Arg codon (CGC→AGC), and represents the first non‐histidine, non‐cysteine P1 residue mutant described for C1 inhibitor.
Regulation of the catalytic activity and oligomeric composition of enzymes in reversed micelles of surfactants in organic solvents
Authors: Kabanov Alexander V. , Nametkin Sergey N. , Klyachko Nataliya L. , Levashov Andrey V.
Published: July 2002

The Phenomenon of the regulation of the catalytic activity of enzymes via changing their oligomeric composition in the system of reversed micelles of sodium bis(2‐ethylhexy)sulfosuccinate (AOT) in octane was studied using z‐chymotrypsin (CT) from bovine brain and alkaline phosphatase (AP) from calf intestinal mucosa. The dependences of the enzyme catalytic activity on the AOT hydration degree (W=[H2O][AOT]), the parameter determining the radius (r
4) of the inner cavity of micelles, usually represent the bell‐shaped curves. The maximal catalytic activity is observed at such Wo
when r
4 is equal to the size of the enzyme molecule. The position of this maximum strictly correlates with the enzyme oligomeric composition. Thus, in the case of CT this is observed at Wo
=12 when r
1 is equal to the radius (rp
) of the CT globule. In the case of artificially produced conjugate containing six cross‐linked CT molecules, this is observed at W
o=43 when r
4 is equal to the radius of the sphere surrounding the absolute octahedron composed of six CT globules. The dependence of the catalytic activity of AP on Wo represent a curve with two maxima that are observed when r
4 is equal to r
p of either AP monomer (W=17) or AP dimer (W
o=25), Ultracentrifugation experiments revealed that variation of W
o causes a change in the oligomeric composition of AP its transition from monomeric (W
o<20) to demeric form (W
o > 20). Hence, the observed maxima correspond to functioning of different oligomeric forms of AP.
Molecular analysis of tap2, an anther‐specific gene from Antirrhinum majus
Authors: Nacken Wolfgang K.F. , Huijser Peter , Saedler Heinz , Sommer Hans
Published: July 2002

Deficiens is a floral homeotic gene of Antirrhinum majus, mutation of which results in transformation of petals to sepals and stamens to carpels. In a search for putative target genes, controlled by this regulatory locus, cDNA clones representing genes, that are expressed in wild type but not in the deficiens mutant flowers, were isolated by differential screening. The molecular structure and the expression pattern of one of these genes, tap2, is described. Tap2 is transiently and tissue‐specifically expressed in the tapetum of the anthers. It encodes a 131 amino‐acids‐long protein with a hydrophobic N‐terminus, displaying all characteristic features of a signal peptide. This indicates that the TAP2 protein may be secreted.
Voltage‐dependent cationic channels formed by a cytolytic toxin produced by Gardnerella vaginalis
Authors: Moran Oscar , Zegarra-Moran Olga , Virginio Caterina , Rottini Giandomenico
Published: July 2002

A cytolytic toxin produced by G. vaginalis was incorporated in artificial membranes and giant liposomes. The toxin formed ionic channels when incorporated in lipid bilayers. The electrical properties of such channels were studied. Current records revealed a unitary conductance of 126 pS (in symmetrical 150 mM KCl). The open state probability of the cytolysin formed channels was a function of the applied membrane potential. The permeability ratio of cations to anions was estimated to be 6.5.
Sphingosine inhibits attachment of murine Lewis lung carcinoma cells to laminin and type IV collagen
Authors: Inokuchi Jin-ichi , Kumamoto Yukihiro , Jimbo Masayuki , Shimeno Hiroshi , Nagamatsu Atsuo
Published: July 2002

The effect of sphingosine (SPH) on the adhesive properties of Lewis lung carcinoma (3LL) cells was investigated using plastic precoated with the extracellular matrix proteins, laminin, fibronectin, or type IV collagen. Treatment of 3LL cells with SPH (0.5–10 μM) resulted in a dose‐dependent decrease in the ability to bind to laminin and type IV collagen but had little or no effect on atachment to fibronectin. Phorbol 12‐myristate 13‐acetate (PMA) selectively enhanced attachment of 3LL cells to laminin and collagen. The inhibitory effect of SPH on attachment to both proteins was competitively antagonized by PMA. These results suggest that SPH acts as a negative effector for cell attachment to laminin and collagen, and that the cell attachment process to both proteins might be regulated in part by protein kinase C.
Stark effect in P700, the primary electron donor of Photosystem I
Authors: Krawczyk S. , Maksymiec W.
Published: July 2002

Quadratic Stark effect in CP1 pigment‐protein complex was examined at low temperatures in the red spectral region. The Stark spectra of samples containing P700 in reduced form exhibit a strong negative band at 704 nm, which disappears on chemical oxidation of P700. The change in permanent dipole moment, Δμ, of P700 on electronic excitation estimated from these spectra was found to be between 4.7 and 7.7 Debye units. It is suggested to reflect the charge‐transfer contribution to the excited state of P700. For antenna chlorophyll, Δμ≌ 1 D was obtained in accordance with the data for monomeric chlorophyll.
Molecular cloning, nucleotide sequence and expression of the tufB gene encoding elongation factor Tu from Thermus thermophilus HB8
Authors: Satoh Motohide , Tanaka Toshikazu , Kushiro Akira , Hakoshima Toshio , Tomita Ken-ichi
Published: July 2002

The tufB gene encoding elongation factor Tu (EF‐Tu) of Thermus thermophilus HB8 was cloned and expressed. Compared with the known tufA gene of T. thermophilus, nucleotide differences were found at 10 positions out of 1221 nucleotides, and amino acid substitutions were found at 4 positions out of 406 amino acids. The tufB product was 70.9% homologous to the corresponding sequence of the tufB product of E. coli. The G+C content of the third base of the codon in the tufB gene was 84.8% and G was especially preferred in this position.
Magainins affect respiratory control, membrane potential and motility of hamster spermatozoa
Authors: de Waal Anthony , Gomes Ana Vaz , Mensink Anne , Grootegoed J.Anton , Westerhoff Hans V.
Published: July 2002

The hypothesis was tested that the magainin peptides, known to compromise bacterial and mitochondrial energetics, are highly active against spermatozoa. A mixture of magainin A and PGLa (1:1) caused a 50% reduction in motility of hamstet spermatozoa at 4 μg/ml total peptide concentration, All motility was lost at 8 μ/ml. At this concentration, respiratory control was released and respiration in the presence of uncoupler was inhibited. Uptake or the lipophilic cation tetraphenyl phosphonium was largely abolished by addition or magainin A and PGLa at this concentration, indicative of a decrease in the mitochondrial membrane potential. Magainin A and PGLa showed synergism with respect to release of respiratory control.
Nitromethylene actions on in situ and expressed insect nicotinic acetylcholine receptors
Authors: Leech C.A. , Jewess P. , Marshall J. , Sattelle D.B.
Published: July 2002

Single channel recordings from dissociated housefly (Musca domestica) neurons show that a novel type of nitromethylene insecticide, 2(nitromethylene)tetrahydro‐1,3‐thiazine (NMTHT) gates a channel, the conductance and open time histogram of which resemble those obtained when acetylcholine is the agonist. Injection into Xenopus oocytes of a locust (Schistocerca gregaria) α‐subunit mRNA results in the expression of functional nicotinic receptors sensitive to NMTHT. Control oocytes injected with distilled water are insensitive to the same concentration of this compound. Thus NMTHT exhibits agonist actions at both in situ and expressed insect nicotinic receptors, and one site of action of this compound is on an insect nicotinic receptor α‐subunit.
Inactivation of human plasma C1‐inhibitor by human PMN leucocyte matrix metalloproteinases
Authors: Knäuper Vera , Triebel Susanne , Reinke Heinz , Tschesche Harald
Published: July 2002

Highly purified human polymorphonuclear (PMN) leucocyte matrix metalloproteinases, collagenase and gelatinase, cleaved human plasma C1‐inhibitor at the carboxyl site of Ala439 (P6). This led to a concomitant loss of C1‐inhibitor activity. An additional cleavage site at the carboxyl site of Ser441 (P4), was observed during PMN leucocyte gelatinase‐induced inactivation, and a minor fragment of the plasma C1‐inhibitor was generated.
Expression in human lung tumor cells of the proprotein processing enzyme PC1/PC3 Cloning and primary sequence of a 5 kb cDNA
Authors: Creemers John W.M. , Roebroek Anton J.M. , Van de Ven Wim J.M.
Published: July 2002

Northern blot analysis of human lung tumors indicated that the gene, which encodes the subtilisin‐like proprotein processing enzyme PC1/PC3, was highly expressed in almost all carcinoid tumors tested. In small cell lung carcinomas (SCLCs), expression varied. In non‐SCLCs and normal lung, no expression was found. Analysis of SCLC cell lines revealed that expression was restricted preferentially to cell lines of the classical type, to lung tumor cells expressing the PC1/PC3 gene, transcripts of 3 kb and 5 kb were detected, the 5 kb mRNA always being the most abundant species. We isolated a cDNA corresponding to the 5 kb human PC1/PC3 transcript, determined the nucleotide sequence of it and deduced the amino acid sequence of the corresponding protein. Furthermore, we conclude that the two PC1/PC3 transcripts have 3′ non‐coding regions of different size and encode the same protein.
Production of a unique multi‐lamella structure in the nuclei of yeast expressing Drosophila copia gag precursor
Authors: Yoshioka Katsuji , Fujita Atsushi , Kondo Shunzo , Miyake Tadashi , Sakaki Yoshiyuki , Shiba Tadayoshi
Published: July 2002

Drosophila retrotransposon copia produces virus‐like particles (VLPs) in the nuclei of cultured Drosophila cells. The VLPs contain copia RNA and reverse transcriptase activity, and thus, play a major role in copia replication. Here we have expressed the copia gag polyprotein precursor in yeast. The precursor, which includes copia protease itself, showed correct autoprocessing to produce a unique multi‐lamella structure in the nuclei of the yeast cells. This expression system should be useful for the analysis of nuclear localization ot'the major copia VLP protein, and Furthermore, would provide important information concerning the mechanism of copia VLPs formation.
Unusual conformational flexibility in N1‐substituted uncommon purine nucleosides Crystal structure of 1‐allyl‐isoguanosine and 1‐allyl‐xanthosine
Authors: Liaw Yen-Chywan , Chern Ji-Wang , Lin Gwo-Sen , Wang Andrew H.-J.
Published: July 2002

Several new N1‐substituted uncommon purine nucleosides, including doridosine (1‐methyl‐isoguanosine; m‐iG), 1‐allyl‐isoguanosine (a‐iG) and 1‐allyl‐xanthosine (a‐X), have been synthesized and tested as agonists for the adenosine receptors. Some have smooth muscle relaxant or negative chronotropic activities. The X‐ray crystal structure of these compounds has been determined at atomic resolution in order to understand the structure‐activity relationship. The structures were solved by direct methods and refined by full‐matrix least‐squares refinement procedure. The crystallographic parameters are: a‐iG, space group P21, a=10.573 (1) Å, b=21.955 (2) Å, c=14.360 (1) Å, β=110.65 (1)°, no. of 3σ Fo's=4585, R=0.047; a‐X, space group P212121, a=16.015 (2) Å, b=16.239 (1) Å, c=5.3723 (5) Å, no. of 3σ Fo's= 1169, R=0.031. In the a‐iG crystal, there are 4 independent molecules (with different conformation) per asymmetric unit. While all 4 molecules adopt anti
χCN glycosyl torsion angle, their riboses have 3 distinct puckers (C2′‐exo, C2′‐endo and C1′‐exo). In contrast, the a‐X structure adopts a syn
χCN glycosyl torsion angle, which is stabilized by an intramolecular hydrogen bond between the N3 or purine base and the O5′ of the ribose (in C2′‐endo pucker). Both purine bases (a‐iG and a‐X) are mainly in the keto tautomer form. For the isoguanine base, the averaged N1–C2 bond distance (1.42 Å) is significantly longer than that (1.375 Å) of the guanine base. For the xanthine base, N3 nitrogen has an imino proton attached which is unambiguously located in the electron density map. The surprising flexibility in the ribose ring of these N1‐substituted uncommon purine nucleosides suggests flat the ribose moiety may not participate in the binding of nucleoside to the adenosine receptors.
Electrochemical titration of the cytochrome hemes in the Rhodopseudomonas viridis reaction center Cyclic equilibrium titrations yield midpoint potentials without evidence for heme cooperativity
Authors: Fritz F. , Moss D.A. , Mäntele W.
Published: July 2002

The redox and spectral characteristics of the 4‐heme cytochrome c unit of the photochemical reaction center from Rhodopseudomonas viridis were studied by a combination of protein electrochemistry and spectroscopy using an ultra thin‐layer spectroelectrochemical cell. Quantitative and reversible reduction of the high‐potential and the low‐potential hemes was performed in cyclic titrations to record the optical difference spectra in the α‐band region. The titration of the absorbance from the high‐potential hemes can be approximated with a sum of 2 Nernst functions with E
m = 0.113 V and E
m = 0.175 V. The corresponding titration of the absorbance from the low‐potential hemes yielded E
m = −0.257 V and E
m = −0.175 V (all potentials quoted vs. Ag/AgCl/3 M KCl; add 0.208 V for potentials vs. standard hydrogen electrode). The high‐potential hemes equilibrate rapidly and titrate identically for oxidative and reductive titrations. Under identical conditions, the low‐potential hemes exhibit a hysteresis, thus indicating much slower equilibration with the applied potential. Cyclic titrations with increasing equilibration periods, however, indicate the disappearance of the hysteresis for equilibration periods approximately twice as long as for the high‐potential hemes. We take this as evidence for a slower internal equilibration, but against a cooperativity of the low‐potential hemes as observed for other multi‐heme cytochromes.
Order and disorder in 30 nm chromatin fibers
Authors: Subirana Juan A.
Published: July 2002

The tendency of DNA to form fibers upon condensation with counterions is reviewed. It is shown that chromatin fibers may acquire a relatively constant diameter of about 30 nm simply as an optimal size achieved upon neutralization of DNA, without requiring a repetitive internal structure. Thus the size of chromatin fibers would not be determined by any specific spatial interaction between DNA and histones. The driving force for the formation of fibers in chromatin would be similar to that found in proteins when they acquire a compact globular shape.
Crystallization and preliminary X‐ray analysis of recombinant human transforming growth factor β2
Authors: Schlunegger Michael P. , Cerletti Nico , Cox David A. , McMaster Gary K. , Schmitz Albert , Grütter Markus G.
Published: July 2002

Recombinant human transforming growth factor β2 (TGF‐β2) was cloned and expressed in E. coli. The protein was isolated from inclusion bodies, renatured and purified to a single component as judged by reversed‐phase HPLC. The recombinant TGF‐β2 was shown to have a biological activity equal to that of native TGF‐β2 in a fibroblast migration assay. Pure, active recombinant TGF‐β2 has been crystallized from polyethylene glycol 400. The trigonal crystals of spacegroup P3121 or P3221 have unit cell dimensions of a=b=60.6 Å, c=75.2 Å and diffract beyond 2.0 Å.
Structural determination of Saccharomyces cerevisiae rig gene and identification of its product as ribosomal protein S21
Authors: Takasawa Shin , Tohgo Akira , Unno Michiaki , Yonekura Hideto , Okamoto Hiroshi
Published: July 2002

rig was originally isolated from a rat insulinoma‐derived cDNA library. The 145 amino acid sequence of the rig protein is invariant in mammalian cDNAs. In this paper, we have isolated the cDNA and genomic clones for yeast (Saccharomyces cerevisae) rig, determined their nucleotide sequences, and identified the gene product. The gene and the mRNA encode a basic protein of 142 amino acids which has 61.3% amino acid identity with mammalian rig protein. On two‐dimensional gel electrophoresis, the in vitro transcription/translation product of yeast rig cDNA co‐migrated with yeast ribosomal protein S21. These results led to the conclusion that yeast rig encodes ribosomal protein S21 and to the determination of the previously unknown primary structure of yeast S21 protein. Unlike most ribosomal protein genes of S. cerevisiae, the gene exists as a single copy in a haploid set of the yeast genome and has no intron, locating at chromosome VII or XV.
Interaction of plasma gelsolin with tropomyosin
Authors: Koepf Edward K. , Burtnick Leslie D.
Published: July 2002

Horse plasma gelsolin labelled with benzophenone‐4‐isothiocyanate can be photochemically cross‐linked to rabbit cardiac tropomyosin. The cross‐linking proceeds with greater efficiency in calcium‐containing buffers. Further evidence for interaction between these proteins is provided by retention of fluorescently labelled gelsolin on tropomyosin‐agarose affinity columns and by the ability of tropomyosin to cause an increase in the fluorescence intensity of gelsolin labelled with fluorescein‐5‐isothiocyanate. Both of these effects require the presence of calcium ions.
Serine residues responsible for tetracycline transport are on a vertical stripe including Asp‐84 on one side of transmembrane helix 3 in transposon Tn10‐encoded tetracycline/H+ antiporter of Escherichia coli
Authors: Yamaguchi Akihito , Ono Nobukazu , Akasaka Takaaki , Sawai Tetsuo
Published: July 2002

Putative transmembrane helix 3 of the tetracycline/H+ antiporter encoded by a transposon, Tn10, contains four serine residues, Ser‐77, Ser‐82, Ser‐91 and Ser‐92. Each of these serine residues was replaced by site‐directed mutagenesis. Of these four serine residues, Ser‐77 was important for the transport function, and a bulky side chain at position 91 hindered substrate translocation, whereas Ser‐82 and Ser‐92 did not play any role. Ser‐77 and Ser‐91 are on the same vertical stripe, that includes the essential Asp‐84, on the hydrophilic side of putative helix 3. These observations suggest that helix 3 is part of the tetracycline translocation channel across the membrane. Tetracycline; Antiporter; Serine; Tetracycline/H+ antiporter; Site‐directed mutagenesis; Antibiotic resistance
Solubilization of active GLP‐1 (7–36)amide receptors from RINm5F plasma membranes
Authors: Göke Rüdiger , Oltmer Britta , Sheikh Søren P. , Göke Burkhard
Published: July 2002

Glucagon‐like peptide‐1 (7–36)amide (GLP‐1 (7–36)amide) represents a physiologically important incretin in mammals including man. Receptors for GLP‐1 (7–36)amide have been described in RINm5F cells. We have solubilized active GLP‐1 (7–36)amide receptors from RINm5F cell membranes utilizing the detergents octyl‐β‐glucoside and CHAPS; Triton X‐100 and Lubrol PX were ineffective. Binding of radiolabeled GLP‐1(7–36)amide to the solubilized receptor was inhibited conentration‐dependently by addition of unlabeled peptide. Scatchard analysis of binding data revealed a single class of binding sites with K
a= 0.26 ± 0.03 nM and B
max= 65.4 ± 21.24 fmol/mg of protein for the membrane‐bound receptor and K
a= 22.54 ± 4.42 μM and B
max= 3.9 ± 0.79 pmol/mg of protein for the solubilized receptor. The binding of the radiolabel to the solubilized receptor was dependent both on the concentrations of mono‐ and divalent cations and the protein/detergent ratio in the incubation buffer. The membrane bound receptor is sensitive to guanine‐nucleotides, however neither GTP‐γ‐S nor GDP‐β‐S affected binding or labeled peptide to solubilized receptor. These data indicate that the solubilized receptor may have lost association with its G‐protein. In conclusion, the here presented protocol allows solubilization of the GLP‐1(7–36)amide receptor in a functional state and opens up the possibility for further molecular characterization of the receptor protein.
Is the cytosolic catalase induced by peroxisome proliferators in mouse liver on its way to the peroxisomes?
Authors: Eriksson Anna Messing , Lundgren Bo , Andersson Karin , DePierre Joseph W.
Published: July 2002

Dietary treatment of male C57B1/6 mice with clolibrate, nafenopin or WY‐14.643 resulted in a modest (at most 2‐fold) increase in the total catalase activity in the whole homogenate and mitochondrial fraction prepared from the livers of these animals. On the other hand, the catalase activity recovered in the cytosolic fraction was increased 12‐ to 18‐fold, i.e. 30–35% of the total catalase activity in the hepatic homogenate was present in the high‐speed supernatant fraction after treatment with these peroxisome proliferators. A study of the time course of the changes in peroxisomal and cytosolic catalase activities demonstrated that the peroxisomal activity both increased upon initiation of exposure and decreased after termination of treatment several days after the increase and decrease, respectively, in the corresponding cytosolic activity. This finding suggests that the cytosolic catalase may be on its way to incorporation into peroxisomes.
Construction of dystrophin fusion proteins to raise targeted antibodies to different epitopes
Authors: Ginjaar H.B. , van Paassen H.B.M. , den Dunnen J.T. , thi Man Nguyen , Morris G.E. , Moorman A.F.M. , van Ommen G.J.B.
Published: July 2002

For the study of the structure and function relationship of dystrophin, defective in DMD, and for diagnostic purposes it is important to dispose of untibodies against different parts of the protein. We have made five different constructs for the expression of fusion proteins containing parts of the four domains of dystrophin. Two different recombinant expression vectors, pATH2 and pEX1, were used. Rabbits were immunized with the fusion products and several polyclonal antibodies were raised. At a later stage, monoclonal antibodies were also raised to some of the fusion proteins. One polyclonal antibody, named P20 AB, is directed against the region covering amino acid sequence 1749–2248 or the nucleotide sequence 5456–6953 of the mRNA, which corresponds to the major deletion‐prone region of the DMD gene. We show the particular value, sensitivity and specificity of the P20 AB in dystrophin analysis.
A new crystal form of the complex between seryl‐tRNA synthetase and tRNASer from Thermus thermophilus that diffracts to 2.8 Å resolution
Authors: Yaremchuk A.D. , Tukalo M.A. , Krikliviy I. , Malchenko N. , Biou V. , Berthet-Colominas C. , Cusack S.
Published: July 2002

Two distinct complexes between seryl‐tRNA synthetase and tRNA Ser from Thermus themophilus have been crystallized using ammonium sulphate as a precipitant. Form III crystals grow from solutions containing a 1:2.5 stoichiometry of synthetase dimer to tRNA. They are of monoclinic space group C2 with unit cell dimensions a = 211.6 Å, b = 126.8 Å, c = 197.1 Å, β = 132.4° and diffract to about 3.5 Å. Preliminary crystallographic results show that the crystallographic asymmetric unit contains two synthetase dimers. Form IV crystals grow from solutions containing a 1:1.5 stoichiometry of synthetase dimer to tRNA. They are of orthorhombic space group P212121 with unit cell dimensions a = 124.5 Å, b = 128.9 Å, c = 121.2 Å and diffract to 2.8 Å resolution. Preliminary crystallographic results show that these crystals contain only one tRNA molecule bound to a synthetase dimer.
A mutant α‐amylase with enhanced activity specific for short substrates
Authors: Matsui Ikuo , Ishikawa Kazuhiko , Miyairi Sachio , Fukui Sakuzo , Honda Koichi
Published: July 2002

The 210th lysine (K210) at the active site in Saccharomycopsis fibuligera α‐amylase was altered to arginine (R) or asparagine (N) by site‐directed mutagenesis. Replacement of K210 by R strengthened the 7th and weakened the 8th subsite affinities. K210 was found to contribute to both the 8th and the 7th subsites. The catalytic activity of the K210R enzyme for the hydrolysis of maltose (G2) was three‐times higher than that of the native enzyme due to an increase in the affinity of the 7th subsite adjacent to the catalytic site, whereas the activity of the K210N enzyme for G2 was decreased to 1% of that of the native enzyme by a reduction in the 7th subsite affinity.