bluuebunny/crossref_metadata_2025 huggingface dataset:
Co‐operation between metastatic tumor cells and macrophages in the degradation of basement membrane (type IV) collagen
Authors: Henry Nicole , Eeckhout Yves , van Lamsweerde Anne-Louise , Vaes Gilbert
Published: July 2002

The co‐culture of mouse peritoneal macrophages and Lewis lung carcinoma cells induces the relase of a metal‐dependent type IV collagen‐degrading proteinase which is not produced in detactable amounts by either cell type cultivated alone. Conditioned media of the co‐cultures degrade both pepsin‐extracted type IV collagen from human placenta and mouse type IV procollagen. Thus macrophages can interact with tumor cells to degrade basement membrane type IV collagen: this might be of importance to allow cancer invasion and metastasis.
Decreased rate of fatty acid synthesis in brown adipose tissue of hypothalamic obese rats
Authors: Saito Masayuki , Shimazu Takashi
Published: July 2002

Obese rats with lesions of the ventromedial hypothalamus (VMH) exhibited a 3‐fold increase in the wet weight of interscapular brown adipose tissue (IBAT), due to an incresed triglyceride content, compared with lean controls. The rate of lipogenesis in IBAT was much higher than that in white adipose tissues in control rats, but decreased greatly in rats with VMH lesions and approximated the value in white adipose tissues. It was suggested that thermogenesis in BAT was impaired after VMH lesions by decreasing triglyceride turnover in BAT.
Purification of a novel pancreatic secretory factor (PSF) and a novel peptide with VIP‐ and secretin‐like properties (helodermin) from Gila monster venom
Authors: Vandermeers André , Vandermeers-Piret Marie-Claire , Robberecht Patrick , Waelbroeck Magali , Dehaye Jean-Paul , Winand Jaques , Christophe Jean
Published: July 2002

A combination of three HPLC procedures applied to the venom of Gila monster (Heloderma suspectum) has led to the purification to homogeneity of two bioactive components: (i) a 17.5 kDa protein, isolated on the basis of its potent secretory effect on dispersed rat pancreatic acini, was accordingly designated PSF (pancreatic secretory factor); (ii) a 5.9‐kDa peptide, designated helodermin, was purified on the basis of its ability to stimulate adenylate cyclase in rat pancreatic membranes. PSF was unable to activate adenylate cyclase and, conversely, helodermin was devoid of secretory action.
Ligand‐specific state transitions of the membrane‐bound acetylcholine receptor
Authors: Covarrubias Manuel , Prinz Heino , Maelicke Alfred
Published: July 2002

We have developed a simple, direct and time‐resolved method to monitor ligand‐induced changes in agonist affinity of the membrane‐bound acetylcholine receptor. The assay is based on the quenching of fluorescence of NBD‐5‐acylcholine observed upon binding of this cholinergic agonist to the receptor. Under conditions of partial saturation with the fluorescent agonist, agonists and local anesthetics but not antagonists can induce an increase in affinity of the receptor for NBD‐5‐acylcholine. The effect is not observed with receptor fully saturated with the fluorescent agonist. The half‐life of the observed change in affinity is independent of the nature of the agonist or local anesthetic applied (t
½ ∼60 s at 22°C). We conclude that the same state transition of the receptor can be induced by two groups of cholinergic ligands that are assumed to be non‐competitive with each other and to have distinctly different modes of action. The time course of the transition is reminiscent of the slow process of desensitization observed in vivo.
Characterization of Leydig cell protein kinase
Authors: Winters Christine A. , Dufau Maria L.
Published: July 2002

Leydig cell cAMP‐dependent protein kinase has been characterized using rapid fractionation and optimal conditions to minimize proteolysis. DEAE‐cellulose analysis showed a single Type I peak of cAMP binding and enzyme activity that eluted at 0.1 M KCl. Photoaffinity labelling with 8‐azido[32P]cAMP followed by SDS‐PAGE showed a doublet with M

r
54000 and 51000 for the peak fraction, while the original extract exhibited only the smaller form. Autophosphorylation revealed a doublet of M

t
, 54000 + 573 and M

r
51 000 ± 710. To titrate the occupancy of regulatory subunits during hCG action, free cAMP receptors were measured by 8‐azido[3H]cAMP binding under non‐exchange conditions followed by photolysis. hCG treatment caused a dose‐related decrease of free receptors and SDS‐PAGE analysis of the 8‐azido[32P]cAMP regulatory subunit from control and hCG treated cells also showed a hormone dependent decrease in a single band of M

r
, 50000. These results have shown that the Leydig cell protein kinase behaves as a Type I enzyme on DEAE analysis, but has the physical characteristics of the Type II enzyme. The dose‐dependent fall in available receptor sites during hCG stimulation further indicates the central role of cAMP in hormone action in the Leydig cell.
Structural differences in the cell binding region of human fibronectin molecules isolated from cultured normal and tumor‐derived human cells
Authors: Borsi Laura , Allemanni Giorgio , Castellani Patrizia , Rosellini Claudio , Di Vinci Angela , Zardi Luciano
Published: July 2002

Fibronectins isolated from human plasma (pFN) and from the conditioned media of normal (N‐cFN) and tumor (T‐cFN) human cells were compared by cathepsin D digestion followed by immunostaining of released fragments with the monoclonal antibody 3E3, specific for the cell binding site. Two different staining patterns were obtained, one specific for pFN and N‐cFN, the second common to fibronectins from the 3 different kinds of tumors studied. This indicates structural differences between N‐cFN and T‐cFN in the cell binding region of the fibronectin molecule.
Direct measurements of increased free cytoplasmic Ca2+ in mouse pancreatic β‐cells following stimulation by hypoglycemie sulfonylureas
Authors: Abrahamsson Hakan , Berggren Per-Olof , Rorsman Patrik
Published: July 2002

The effects of the hypoglycemic sulfonylureas tolbutamide and glibenclamide on free cytoplasmic Ca2+, [Ca2+]i, were compared with that of a depolarizing concentration of K+ in dispered and cultured pancreatic β‐cells from ob/ob mice. [Ca2+]i was measured with the fluorescent Ca2+‐indicator quin2. The basal level corresponded to 150 nM and increased to 600 nM after exposure to 30.9 mM K+. The corresponding levels after stimulation with 1 μM glibenclamide and 100 μM tolbutamide were 390 and 270 nM respectively. K+ depolarization increased [Ca2+]i more rapidly than either of the sulfonylureas. It is suggested that the increased [Ca2+]i obtained after stimulation by sulfonylureas is due to depolarization of the β‐cells with subsequent entry of Ca2+ through voltage‐dependent channels.
The encephalitogenic protein of myelin forms hexamers in which the polypeptides have a pleated‐sheet structure
Authors: Smith Ross
Published: July 2002

Sedimentation equilibrium data are shown to be consistent with the existence in solution of an equilibrium between monomers and hexamers of bovine myelin basic protein, without significant accumulation of intermediates. At low concentrations circular dichroism spectra were indicative of an aperiodic coiled secondary structure. At higher concentrations, where the protein self‐associates, they showed formation of a β‐pleated sheet conformation. At low molar ratios myristoyllysophosphatidylcholine promotes protein self‐association and the concomitant conformational transition. The data are consistent with the existence of an equilibrium mixture of relatively unstructured monomers and hexamers in which the polypeptides have a welldefined three‐dimensional structure.
Identification of the peptide bond cleaved during activation of human Clr
Authors: Arlaud Gerard J. , Gagnon Jean
Published: July 2002

CNBr cleavage of unreduced proenzyme Clr yielded fragment CP2b, isolated by gel filtration and highpressure gel permeation chromatography. This fragment (~ M
τ 55000) comprised at least 4 disulphidelinked peptides, which were separated by gel filtration after reduction and alkylation. Peptide CP2bRA4, overlapping the A‐ and B‐chain regions in proenzyme Clr was digested by V8 staphylococcal protease, and the digest separated by reversed‐phase HPLC. N‐terminal sequence analysis of peptide CP2bRA4SP9 established that Clr activation involves the cleavage of a single Arg‐Ile bond, located in the sequence:⋯ Gln‐Arg‐Gln‐Arg‐Ile‐Ile‐Gly‐Gly⋯
Targeting of enzyme immobilized on erythrocyte membrane to collagen‐coated surface
Authors: Muzykantov V.R. , Sakharov D.V. , Smirnov M.D. , Domogatsky S.P. , Samokhin G.P.
Published: July 2002

Drug targeting
Collagen
Immobilized enzyme
Antibody attachment
Peroxidase
Specific binding
Mechanisms of poly(ADP‐ribose) polymerase catalysis; mono‐ADP‐ribosylation of poly(ADP‐ribose) polymerase at nanomolar concentrations of NAD
Authors: Bauer Pal I. , Hakam Alaeddin , Kun Ernest
Published: July 2002

Calf thymus and rat liver poly(ADP‐ribose) polymerase enzymes, and the polymerase present in extracts of rat liver nuclei synthesize unstable mono‐ADP‐ribose protein adducts at 100 nM or lower NAD concentrations. The isolated enzyme‐mono‐ADP‐ribose adduct hydrolyses to ADP‐ribose and enzyme protein at pH values slightly above 7.0 indicating a continuous release of ADP‐ribose from NAD through this enzyme‐bound intermediate under physiological conditions. NH2OH at pH 7.0 hydrolyses the mono‐ADP‐ribose enzyme adduct. Desamino NAD and some other homologs at nanomolar concentrations act as ‘forward’ activators of the initiating mono‐ADP‐ribosylation reaction. These NAD analogs at micromolar concentrations do not affect polymer formation that takes place at micromolar NAD concentrations. Benzamides at nanomolar concentrations also activate mono‐ADP‐ribosylation of the enzyme, but at higher concentrations inhibit elongation at micromolar NAD as substrate. In nuclei, the enzyme molecule extensively auto‐ADP‐ribosylates itself, whereas histones are trans‐ADP‐ribosylated to a much lower extent. The unstable mono‐ADP‐ribose enzyme adduct represents an initiator intermediate in poly ADP‐ribosylation.
Cooperativity of the αβ ‐protomer structure in Na+, K+‐ATPase functioning
Authors: Chetverin A.B. , Khechinashvili N.N. , Filimonov V.V.
Published: July 2002

Heat denaturation of the free and ligand‐bound forms of purified Na+,K+‐ATPase from pig kidney is studied with the scanning microcalorimetry technique. A single two‐state transition is observed during denaturation of the free enzyme, the molar concentration of the cooperatively melting units being equal to the concentration of αβ‐protomers (M
r≈140000). Upon interaction of the enzyme with phosphate, Mg2+, and strophanthidin, but not with Na+, the cooperativity of the protomer unfolding is lost, and the protein stabilization enthalpy becomes ≈ 230 higher. The data suggest that (i) in a functionally active enzyme form, the αβ‐protomers possess a rigid structure with tight association of their subunits and domains, (ii) this structural rigidity is essential for the Na+,K+‐ATPase functioning and (iii) there is a unique non‐active conformation of the enzyme which may play an important role in its in vivo regulation.
Palindromic units from E. coli as binding sites for a chromoid‐associated protein
Authors: Gilson Eric , Perrin David , Clement Jean-Marie , Szmelcman Sevec , Dassa Elie , Hofnung Maurice
Published: July 2002

Several hundred copies of a highly conserved extragenic palindromic sequence, 20–40 nucleotides long, exist along the chromosome of E. coli and S. typhimurium. These have been defined as palindromic units (PU) or repetitive extragenic palindromes (REP). No general function for PUs has been identified. In the present work, we provide data showing that a protein associated with a chromoid extract of E. coli protects PU DNA against exonuclease III digestion. This provides the first experimental evidence that PU constitutes binding sites for a chromoid‐associated protein. This result supports the hypothesis that PUs could play a role in the structure of the bacterial chromoid.
The acute GH action in rat adipocytes is associated with enhanced phosphorylation of a 46 kDa plasma membrane protein enriched by GH‐Sepharose
Authors: Donnér Jakob , Eriksson Hans , Belfrage Per
Published: July 2002

The acute anti‐lipolytic effect of human growth hormone (hGH) in maximally noradrenaline‐stimulated intact rat adipocytes was selectively associated with increased phosphorylation of a 46 kDa plasma membrane protein which was highly enriched by hGH‐Sepharose chromatography. The same protein was also phosphorylated by an endogenous protein kinase in isolated plasma membranes, although then no hGH effect could be demonstrated. About 14% of the phosphate incorporated into the protein in isolated plasma membranes was found in tyrosine residues and the remainder in serine and threonine. The possible relation of the 46 kDa protein with the hGH plasma membrane receptor is discussed.
Rearrangements to the JP1, JP and JP2 segments in the human T‐cell rearranging gamma gene (TRGγ) locus
Authors: Huck Sylvie , Lefranc Marie-Paule
Published: July 2002

In the human T‐cell rearranging gamma (TRGγ) locus, five joining (J) segments have been identified: J1, J2 and three additional segments JP, JP1 and JP2. We report the sequence of the germline JP1 segment and compare it with the other human and mouse Jγ segments. We also demonstrate that rearrangements to the three additional Jγ segments can be identified by hybridization of the KpnI digests to the Jγ1 probe pH60. Since rearrangements to J1 or J2 can be assigned, using the same pH60 probe, to one of the nine variable (V) γ genes known to rearrange [(1987) EMBO J. 6, 1945–1950], our results show that a unique probe can detect all the TRGγ rearrangements and be particularly useful for assessing the preferential usage of Vγ and Jγ segments in the TRG γ‐expressing cells.
The ATP,Mg‐dependent protein phosphatase: Regulation by casein kinase‐1
Authors: Agostinis Patrizia , Vandenheede Jackie R. , Goris Jozef , Meggio Flavio , Pinna Lorenzo A. , Merlevede Wilfried
Published: July 2002

The free modulator subunit of the ATP,Mg‐dependent phosphatase is phosphorylated up to 1 mol per mol by casein kinase‐1, up to 1.85 mol per mol after dephosphorylation by the PCSH1 phosphatase, but 10‐fold less when purified in the presence of NaF, suggesting an in vivo phosphorylation of the casein kinase‐1 sites. Peptide mapping of 32P‐modulator labeled by casein kinase‐1 or ‐2 shows a different phosphorylation pattern. Phosphorylation of the inactive phosphatase by casein kinase‐1 prevents the subsequent kinase FA‐mediated activation, while it does not impair the activated phosphatase.
On the molecular size of thymosins
Authors: Haritos A.A. , Yialouris P.P. , Heimer E.P. , Felix A.M. , Rosemeyer M.A.
Published: July 2002

The immunoregulatory polypeptide prothymosin α and its biologically active N‐terminal fragment thymosin α1m, with relative molecular masses of 12 500 and 3108 respectively, were found to behave as oligomers (trimers to hexamers) in gel‐filtration measurements. This phenomenon of an apparent association of polypeptides has been reported for other thymosins — parathymosin α, thymosin β4 and thymosin β10. In contrast, sedimentation equilibrium ultracentrifugation shows that thymosin α1 is a monomer with a relative molecular mass of 3000±200. Measurement of the diffusion coefficient as 221 μm2/s suggests that the molecule is approximately spherical. The implications for the molecular species of prothymosin α, parathymosin α, and β‐thymosins are discussed.
Cause of the blue shift of the absorption spectrum of tetranitromethane‐treated bacteriorhodopsin
Authors: Higashiyama Hiroyuki , Tokunaga Fumio
Published: July 2002

The reaction of tetranitromethane with bacteriorhodopsin under irradiation was separated into two stages by selecting wavelengths of the irradiation light. The first stage of the reaction was characterized by a blue shift of the absorption spectrum and the second stage was characterized by decrease of the absorption of the visible region. The blue shift at the first stage was not caused by nitration of tyrosine residues but a modification of the chromophore. This conclusion was confirmed by reconstitution experiments and chromophore analysis. Nitration of tyrosine residues progressed at the second stage of the reaction.
Only water‐exposed carboxyl groups are protonated during the transition to the cation‐free blue bacteriorhodopsin
Authors: Gerwert Klaus , Ganter Ulrich M. , Siebert Friedrich , Hess Benno
Published: July 2002

FTIR (Fourier‐transform‐infrared) difference spectra between cation‐free blue and purple bacteriorhodopsin were recorded. The results indicate that during the blue to purple transition no isomerization of the chromophore takes place. It is further observed that approx. 14 water‐exposed carboxyl groups ofamino acids are protonated in blue bacteriorhodopsin. The groups were deprotonated during the blue to purple transition. Protonation of carboxyl groups in the interior of the protein, which are not accessible to water, can be excluded.
Humoral mediator‐dependent activation of the Sarcophaga lectin gene
Authors: Shiraishi Akihiro , Natori Shunji
Published: July 2002

Sarcophaga lectin is a defence protein synthesized by the fat body and secreted into the hemolymph in response to injury of the body of third instar larvae of Sarcophaga peregrina (flesh‐fly). In this paper, we demonstrate that the stimulus of body injury is first transmitted to a certain tissue present in the anterior part of the body, and from there a mediator molecule that interacts directly with fat body cells is secreted into the hemolymph. On interaction with this mediator molecule, the fat body begins to synthesize mRNA for Sarcophaga lectin.
Cysteine proteases: The S2P2 hydrogen bond is more important for catalysis than is the analogous S1P1 bond
Authors: Asbo´th B. , Majer Z. , Polga´r L.
Published: July 2002

High hydrophobicity of the second amino acid N‐terminal to the scissile bond (P2 residue) is generally considered to be the major factor in the specificity of the substrates for cysteine proteases of the papain family. To examine the catalytic contribution of the S2P2 hydrogen bond apparent from X‐ray crystallographic studies, the kinetics of Z‐Phe‐Gly‐OEt and its thiono derivative were compared. The thiono compound contains a sulfur atom in place of the carbonyl oxygen of the phenylalanine residue. It was found that the specificity rate constants for the reactions of the thiono substrate with various cysteine proteases are lower by 2–3 orders of magnitude as compared to the corresponding rate constants for the oxo substrate. This remarkable effect is not expected in the light of previous studies indicating that the change from oxygen to sulfur in the P1 residue was without an appreciable effect. The results are interpreted in terms of a distorted binding of the thiono substrate.
Effect of hydrocortisone on sialyltransferase activity in the rat small intestine during maturation Changes along the villus‐crypt axis and in fetal organ culture
Authors: Kolínská Jiřina , Ivanov Svetoslav , Chelibonova-Lorer Heny
Published: July 2002

Sialyltransferase activity was assayed in rat intestinal cells isolated as fractions reflecting the villus‐crypt axis of differentiation. In 13‐day‐old rats both endo‐ and exogenous sialyltransferase activity reached their maximum in undifferentiated crypt cells and their peaks overlapped. In contrast, sialyltransferase of the adult intestine was 4‐fold lower than that of sucklings in the crypts, with slight tendency to be transferred to the villus cells. Hydrocortisone applied to 10‐day‐old rats caused three days later a precocious drop of sialyltransferase activity in the crypt cells. Unlike in vivo, glucocorticoid responsiveness was accompanied by increased sialyltransferase activity in fetal small intestine cultivated for 17 days.
Location of a region of the muscarinic acetylcholine receptor involved in selective effector coupling
Authors: Kubo Tai , Bujo Hideaki , Akiba Isamu , Nakai Junichi , Mishina Masayoshi , Numa Shosaku
Published: July 2002

Chimaeric muscarinic acetylcholine receptors (mAChR) in which corresponding portions of mAChR I and mAChR II are replaced with each other have been produced in Xenopus oocytes by expression of cDNA constructs encoding them. Functional analysis of the chimaeric mAChRs indicates that a region mostly comprising the putative cytoplasmic portion between the proposed transmembrane segments V and VI is involved in selective coupling of mAChR I and mAChR II with different effector systems. In contrast, the exchange of this region between mAChR I and mAChR II does not significantly affect the antagonist binding properties of the two mAChR subtypes.
The amino‐terminal sequence of the Xenopus laevis mitochondrial SSB is homologous to that of the Escherichia coli protein
Authors: Mahoungou C. , Ghrir R. , Lecaer J.P. , Mignotte B. , Barat-Gueride M.
Published: July 2002

Two closely related forms of the single‐stranded DNA binding protein purified from Xenopus laevis oocytes mitochondria have been identified. Their amino terminal sequences exhibit homology with the Escherichia coli SSB protein.
Phosphatidylinositol turnover in human monocyte‐derived macrophages by native and acetyl LDL
Authors: Ishikawa Yuichi , Asaoka Yoshinori , Taniguchi Takahiro , Tsunemitsu Masahiko , Fukuzaki Hisashi
Published: July 2002

Monocyte‐derived macrophages play a key role in pathogenesis of atherosclerosis. However, the mechanism of activating macrophages in atheromatous lesions has not been fully investigated. This report describes the contribution of phosphatidylinositol turnover to the uptake of low density lipoproteins (LDL) by macrophages. Both native and acetyl LDL stimulated inositol 1,4,5‐triphosphate (IP3) formation in a dose‐dependent manner at concentrations of 0–70 μg/ml. The potency of IP3 formation by acetyl LDL (0.44 nmol/mg protein) was 2‐fold higher than that by native LDL (0.21 nmol/mg protein). Time course studies showed that a maximal effect of IP3 formation by acetyl LDL at concentrations of 30 μg/ml was observed at 3 min. Longer incubation diminished IP3 formation. Oxidized LDL also stimulated IP3 formation with a similar efficiency to acetyl LDL. It was indicated that chemically modified LDL which were taken up through the scavenger receptor pathway activated the macrophages by mediating the phosphatidylinositol hydrolysis and IP3 formation.
n‐Butyrate and dexamethasone synergistically modulate the surface expression of epidermal growth factor receptors in cultured rat hepatocytes
Authors: Gladhaug Ivar P. , Christoffersen Thoralf
Published: July 2002

n‐Butyrate was previously found to increase the epidermal growth factor (EGF) receptor binding in primary cultures of rat hepatocytes. We show here that butyrate and dexamethasone synergistically modulate the surface expression of the EGF receptors. The butyrate‐induced enhancement of high‐affinity EGF binding was only slight in the absence of glucocorticoid, but was strongly and dose‐dependently amplified by dexamethasone. Butyrate counteracted the inhibition by insulin of the dexamethasone‐induced increase in EGF binding. The results indicate that the glucocorticoid has a permissive effect on a butyrate‐sensitive process that determines the surface expression of the high‐affinity class of EGF receptors.
P‐700 photooxidation in state 1 and state 2 in cyanobacteria upon flash illumination with phycobilin‐ and chlorophyll‐absorbed light
Authors: Tsinoremas Nicholas F. , Hubbard Julia A.M. , Evans Michael C.W. , Allen John F.
Published: July 2002

Photosynthesis; Light harvesting; Photosystem I; State transition; Phycobilisome; Excitation energy distribution; Cyanobacteria; (Nostoc MAC; Synechococcus 6301)
Effect of sialidase on blood group specificity of hog submaxillary glycoproteins
Authors: Aminoff David , Morrow Marianne P.
Published: July 2002

Hogs can be divided into three phenotypes A, H and In, depending on the ability of extracts of their submaxillary glands to inhibit the agglutination of human A cells with human anti‐A, and human O cells with eel‐anti‐H serum. Evidence is here provided that the inactive glycoprotein, In, represents H active glycoprotein containing additional N‐glycolyl neuraminic acid, which stereospecifically masks the H determinant, α (1 → 2)‐L‐fucosyl residue.
Heterogeneity of metabolic response to muscular exercise in humans
Authors: Bendahan David , Confort-Gouny Sylviane , Kozak-Reiss Geneviève , Cozzone Patrick J.
Published: July 2002

31P NMR spectroscopy at 4.7 T has been used in vivo to follow metabolic changes associated with exercise and subsequent recovery in the forearm flexor digitorum superficial muscle of 14 healthy volunteers. The muscle content in phosphomonoesters at rest provides an index of glycogenolytic activity. Quantitative linear correlations have been shown to link end‐of‐exercise acidosis to recovery kinetics of phosphocreatine and phosphocreatine/organic phosphate ratio. These linear relationships constitute new metabolic invariants to be used in the study of myopathies and muscle adaptation to exercise.
Mitogen‐induced oscillations of membrane potential and Ca2+ in human fibroblasts
Authors: Peres Antonio , Giovannardi Stefano
Published: July 2002

Using the whole‐cell technique, we have measured recurring hyperpolarizations induced by fetal calf serum and bradykinin in human fibroblasts. By coupling fura‐2 microfluorimetry to electrophysiology, we have also measured directly cytosolic Ca2+ and found that Ca2+ oscillations occur in synchrony with membrane currents. Mitogen stimulation of cells in which intracellular K+ had been replaced with Cs+ resulted in the abolishment of the outward current. We conclude then that the mitogen‐induced recurring hyperpolarizations in human fibroblasts are due to the opening of Ca2+‐activated K+ channels.
Complement components C1r/C1s, bone morphogenic protein 1 and Xenopus laevis developmentally regulated protein UVS.2 share common repeats
Authors: Bork Peer
Published: July 2002

Property patterns were constructed, based on an alignment of related domains in human complement subcomponents C1r and C1s as well as in the sea urchin protein uEGF. This kind of consensus pattern was able to identify similar domains in a human bone morphogenic protein, in a Xenopus laevis embryonal protein involved in dorsoanterior development and in a calcium‐dependent serine protease secreted from malignant hamster embryo fibroblast cells. Because of the high level of overall sequence homology this protease may be the hamsters' equivalent of the human complement subcomponent C1s. The resulting multiple alignment of all studied domains suggests functionally and structurally important regions.
Cloning and nucleotide sequence of the fabD gene encoding malonyl coenzyme A‐acyl carrier protein transacylase of Escherichia coli
Authors: Magnuson Kelly , Oh Won , Larson Timothy J. , Cronan John E.
Published: July 2002

We report the cloning and nucleotide sequence of the gene encoding malonyl coenzyme A‐acyl carrier protein transacylase of Escherichia coli, Malonyl transacylase has been overexpressed 155‐fold compared to a wild‐type strain, Overexpression of this enzyme alters the fatty acid composition of a wild‐type E. coli strain; increased amounts of cis‐vaccenate are incorporated into the membrane phospholipids.
A molecular model for the tumour‐associated antigen, p97, suggests a Zn‐binding function
Authors: Garratt Richard C. , Jhoti Harren
Published: July 2002

The primary structure of p97 (melanotransferrin) has been compared with other members of the transferrin superfamily. A molecular structure of p97 has been modelled based on the crystal structure of diferric rabbit serum transferrin. The most significant amino acid substitutions in p97 are almost exclusively limited to only two regions; the C‐lobe iron‐binding cleft and the interlobe contact region. The latter includes within the N‐terminal lobe a Zn‐binding consensus sequence found in metallopeptidases, and in the C‐terminal lobe a glutamic acid residue (Glu‐394) capable of completing a potential thermolysin‐like Zn‐binding site. Thus, p97 may have a Zn‐binding potential, unique amongst the transferrin superfamily.
Cross‐linking of apolipoproteins is involved in a loss of the ligand activity of high density lipoprotein upon Cu2+‐mediated oxidation
Authors: Sakai Masakazu , Miyazaki Akira , Sakamoto Yu-Ichiro , Shichiri Motoaki , Horiuchi Seikoh
Published: July 2002

A recent study demonstrated that Cu2+‐mediated oxidation of high density lipoprotein (HDL) resulted in a loss of the capacity to reduce cholesterol from macrophage foam cells [(1991) Proc. Natl. Acad. Sci. USA 88, 6457–6461]. In the present study we characterized the physicochemical properties of oxidized HDL and correlated them with the ligand activity toward the HDL receptor. Among them, the cross‐linking of apolipoproteins and an increase in lipid peroxides were characteristic and closely similar to those of tetranitromethane‐treated HDL, an abortive ligand for the HDL receptor. Cellular experiments with murine peritoneal macrophages revealed that both the cellular binding activity of HDL and its capacity to enhance cholesterol efflux from macrophage foam cells were markedly reduced upon oxidation. These results suggest that cross‐linking of HDL apolipoproteins is involved in the loss of the ligand activity of oxidized HDL.
Growth hormone acutely decreases type III iodothyronine deiodinase in chicken liver
Authors: Darras V.M. , Berghman L.R. , Vanderpooten A. , Kühn E.R.
Published: July 2002

Growth hormone (GH) increases plasma T3 and decreases plasma T4 in 18‐day old chicken embryos, in newly hatched chicks and in adult chickens within 2 h after injection. The in vivo increase in T3 can be linked to an increased in vitro T3 recovery from liver homogenates incubated with T4. Specific type I and type III deiodinase tests (5′D‐I and 5D‐III), however, show that GH has no effect at all on the amount of hepatic type I enzyme (catalyzing T4 deiodination to T3) but acutely decreases the amount of type III enzyme (catalyzing T3 deiodination). This suggests that the GH‐induced increase in plasma T3 is not due to an increased T3 production, but is the result of a decreased T3 breakdown. The lack of a stimulatory effect of GH injection in 3‐day‐old fed chicks might be the combined result of a low hepatic type III enzyme level and a low GH receptor availability at that stage.
Proto‐oncogene c‐jun and c‐fos messenger RNAs increase in the liver of carnitine‐deficient juvenile visceral steatosis (jvs) mice
Authors: Tomomura Mineko , Nakagawa Kyoko , Saheki Takeyori
Published: July 2002

We determined the mRNA levels of c‐jun and c‐fos in the liver of C3H‐H‐2° jvs mice. Both were higher in jvs mice than in normal mice. The level of c‐jun mRNA increased gradually after birth, but in the control mice there was almost no change. In addition, α‐fetoprotein and aldolase A mRNA levels were also higher than in normal littermates. These results suggest that the pattern of the gene expression in jvs mice partly resembles the one that occurs in undifferentiated hepatocytes and/or hepatocellular carcinoma.
Synthesis and characterization of photoactivatable peptide agonists of the human thrombin receptor
Authors: Bischoff Rainer , Cordier Yves , Rasmussen Ulla B. , Schlesinger Yasmin , Gachet Christian , Jaquinod Michel , Tripet Brian , Chong Pele C.S. , Pavirani Andrea
Published: July 2002

Chemical synthesis and biochemical analysis of modified agonist peptides of the human thrombin receptor derived from the sequence SFLLRNP containing photoactivatable azido groups and biotin for sensitive detection is described. Substitution of leucine in position three with p‐azidophenylalanine and extension of the C‐terminus with a KGGK spacer containing biotin covalently linked to the side chain of the C‐terminal lysine residue resulted in an active receptor agonist as determined by intracellular Ca2+ mobilization in human erythroleukemia (HEL) cells. In contrast, substitution of phenylalanine in position two with p‐azidophenylalanine reduced agonist activity significantly.
Carbohydrate isoforms of antithrombin variant N135Q with different heparin affinities
Authors: Turko Illarion V. , Fan Bingqi , Gettins Peter G.W.
Published: July 2002

We have changed one of the carbohydrate‐bearing asparagine residues of human antithrombin to glutamine by site‐directed mutagenesis and expressed the variant antithrombin, N 135Q, in baby hamster kidney cells. Two isoforms were secreted, both of which had higher affinity for heparin than human plasma α antithrombin. Both forms had normal inhibitory activity toward factor Xa and showed normal heparin acceleration of proteinase inhibition. The mutation resulted in a higher production of the very high affinity form from about 30% to 60% of the total secreted antithrombin. This form should be the most useful for comparison of the effects of other mutations on heparin binding and proteinase inhibition.
Preparations of Rp‐cyclic adenosine 3',5'‐phosphorothioate (Rp‐cAMPS) can contain biologically active amounts of adenosine
Authors: Musgrave I.F. , Genieser H.G. , Maronde E. , Seifert R.
Published: July 2002

Superoxide anion (O2
.−) production from human neutrophils stimulated by N‐formyl‐l‐methionyl‐l‐leucyl‐l‐phenylalanine (fMLP, 1 μM) was inhibited by preparations of the inhibitor of cAMP‐dependent protein kinase, Rp‐cyclic adenosine 3',5'‐phosphorothioate (Rp‐cAMPS, 100 μM). This effect of Rp‐cAMPS was reversed by xanthine amine congener (0.1 μM), an adenosine receptor antagonist, and by low concentrations of adenosine desaminase (0.02 mg/ml). HPLC analysis shows that these preparations of Rp‐cAMPS contained concentrations of adenosine which could produce significant inhibition of fMLP‐induced O2
.− production. These results suggest that Rp‐cAMPS should be used with caution in cells or tissues containing adenosine receptors, and that preparations of Rp‐cAMPS should be treated with adenosine desaminase before use to avoid activation of adenosine receptors.
Purification and antipathogenic activity of lipid transfer proteins (LTPs) from the leaves of Arabidopsis and spinach
Authors: Segura Ana , Moreno Manuel , García-Olmedo Francisco
Published: July 2002

Two homogeneous proteins active in vitro against the bacterial pathogen Clavibacter michiganensis subsp. sepedonicus were obtained from a crude cell‐wall preparation from the leaves of Columbia wild‐type Arabidopsis. The N‐terminal amino acid sequences of these proteins allowed their identification as lipid transfer proteins (LTP‐a1, LTP‐a2); the LTP1‐a1 sequence was identical to that deduced from a previously described cDNA (EMBL M80566) and LTP‐a2 was quite divergent (44% identical positions). These proteins were not detected in the cytoplasmic fraction by Western‐blot analysis. Proteins LTP‐s1 and LTP‐s2 were similarly obtained from spinach leaves; LTP‐s1 was 91% identical to a previously purified spinach LTP (Swiss Prot P10976), and LTP‐s2 was moderately divergent (71% identical positions). About 1/3 of the total LTPs were detected in the cytoplasmic fraction from spinach by Westem‐blot analysis. Concentrations of these proteins causing 50% inhibition (EC‐50) were in the 0.1–1 μM range for the bacterial pathogens C. michiganensis and Pseudomonas solanacearum and close to 10 μM for the fungal pathogen Fusarium solani.
Compositional compartmentalization of the nuclear genomes of Trypanosoma brucei and trypanosoma equiperdum
Authors: Isacchi Antonella , Bernardi Giacomo , Bernardi Giorgio
Published: July 2002

High molecular weight DNA preparations from Trypanosoma brucei and Trypanosoma equiperdum were fractionated by preparative centrifugation in a Cs2SO4 density gradient in the presence of BAMD, bis(acetatomercurimethyl)dioxane, a sequence‐specific DNA ligand. Analytical centrifugation in CsCl of the DNA fractions so obtained showed that both DNAs had a bimodal distribution with two major peaks banding at 1.702–1.703 and 1.708
3 and representing and of total DNA, respectively. Several minor components were also detected. These results indicate that a compositional compartmentalization is not only found in the genome of vertebrates and plants, as already described, but also in those of protozoa such as Trypanosomes.
Photosynthesis modulates the sign of phototaxis of wild‐type Chlamydomonas reinhardtii
Authors: Takahashi Tetsuo , Watanabe Masakatsu
Published: July 2002

We have found that the sign of phototaxis, i.e. the direction of phototactic migration either towards or away from the light source, of wild‐type Chlamydomonas reinhardtii depends on its photosynthetic activity. This accounts for the frequently reported transient positive phototaxis that precedes a negative phototaxis of wild‐type cells, as well as the earlier observations that preillumination affects the sign of phototaxis. The bases for our conclusion are as follows. (1) The transient nature of phototaxis was preferentially observable with blue‐green actinic light rather than with green actinic light. (2) Red background light induces negative phototaxis under the actinic‐light conditions in which, without background light, Chlamydomonas cells show exclusively positive phototaxis. (3) Both the effect of red background light and the transient change in the sign of phototaxis were inhibited by 3‐(3',4'‐dichlorophenyl)‐1,1‐dimethylurea, a specific inhibitor of photosynthesis. The conclusion modifies the accepted view that photosynthesis does not link with the phototaxis of this microorganism, thus constituting a necessary part in elucidating mechanisms of algal phototaxis.
Cloning and characterization of the elongation factor EF‐1β homologue of Saccharomyces cerevisiae
Authors: Hiraga Kazumi , Suzuki Katsuhito , Tsuchiya Eiko , Miyakawa Tokichi
Published: July 2002

A Saccharomyces cerevisiae cDNA homologue of the elongation factor EF‐1β was found among the clones obtained by immunoscreening of a yeast cDNA expression library with an antibody against calmodulin affinity‐purified proteins. The cDNA encoded a protein of 206 amino acids which was highly homologous (about 70% homology) with Artemia salina and human EF‐1β. A protein with an apparent molecular mass of 33,000, significantly larger than that expected from the gene, was identified by Western blotting. Gene disruption experiments indicated that EF‐1β is essential for growth.
Endothelin‐1, phorbol esters and phenylephrine stimulate MAP kinase activities in ventricular cardiomyocytes
Authors: Bogoyevitch Marie A. , Glennon Peter E. , Sugden Peter H.
Published: July 2002

ET‐1 stimulated MBP kinase activity in cultured cardiomyocytes. Maximal activation (3.5‐fold) was at 5 min. EC50 was 0.2 nM. PMA or PE also increased MBP kinase (4‐ or 2.5‐fold, respectively). Pre‐treatment with PMA down‐regulated the subsequent response to ET‐1 or PMA. ET‐1‐ or PMA‐stimulated MBP kinase was resolved into 2 major (peaks II and IV) and 2 minor peaks by FPLC on Mono Q. Peaks II and IV were inactivated by either LAR or PP2A. Renatured MBP kinase activities following SDS‐PAGE in MBP‐containing gels and immunoblot analysis showed that peak II was a p42 MAP kinase and peak IV was a p44 MAP kinase.
The humoral antibacterial response of Drosophila
Authors: Hoffmann Jules A. , Hetru Charles , Reichhart Jean-Marc
Published: July 2002

Drosophila, like other insects, responds to the injection of bacteria by the rapid and transient synthesis of a battery of potent antibacterial peptides. Only a few of these peptides have been fully characterized to date. We review our recent data on the control of the expression of a gene encoding one of the induced peptides, i.e. diptericin. Our data highlight the role of proximal cis‐regulatory motifs similar to regulatory elements binding nf‐κb and NF‐IL6 in promoters of some immune genes of mammals. We argue that the Drosophila host defense is homologous to the mammalian acute phase response.
High level expression of the large subunit of mouse ribonucleotide reductase in a baculovirus system
Authors: Salem Jerome S. , Scott Charles P. , Li Lin-Sheng , Cooperman Barry S. , Rubin Harvey
Published: July 2002

The large subunit of ribonucleotide reductase from mouse has been overexpressed in Spodoptera frugiperda cells infected with recombinant baculovirus. The expressed protein was purified by affinity chromatography to apparent homogeneity as determined by SDS‐PAGE. The homogeneous protein is recognized in Western blot analysis by a monoclonal antibody raised to the large subunit of ribonucleotide reductase from calf thymus, has the correct N‐terminal sequence, and, in the presence of the small subunit of mouse ribonucleotide reductase and nucleoside triphosphate effectors, catalyzes the reduction of both purine and pyrimidine nucleoside diphosphates.
The 3′‐terminal untranslated region of alfalfa mosaic virus RNA 4 facilitates the RNA entry into translation in a cell‐free system
Authors: Ryabova Lyubov A. , Torgashov Aleksey F. , Kurnasov Oleg V. , Bubunenko Michail G. , Spirin Alexander S.
Published: July 2002

In order to understand the role of the 3′‐terminal untranslated region (3′‐UTR) of alfalfa mosaic virus (A1MV) RNA 4 in viral RNA translation we have constructed the RNA derivatives differing in the length of their 3′‐terminal portions and expressed them in a wheat germ extract. The result shows that the removal of the 3′‐UTR from A1MV RNA 4 causes a lagged RNA translation in the cell‐free system as compared with the translation of the full length RNA 4, thus suggesting the involvement of the 3′‐UTR in the translation initiation pathway.
The covalent maleimidobenzoyl‐actin‐myosin head complex
Authors: Bertrand Raoul , Derancourt Jean , Kassab Rhida
Published: July 2002

We have identified the region of actin involved in the covalent coupling of maleimidobenzoyl‐G‐actin to the central 50 kDa segment of the myosin‐S‐1 heavy chain by analyzing the structure of the maleimidobenzoyl‐G‐actin‐S‐1 conjugate using selective proteolytic digestions, amino acid sequence determinations and novel cross‐linking reactions between S‐1 and different maleimidobenzoyl‐G‐actin derivatives. The cross‐linking is shown to occur only on the stretch of residues 48–67 in actin subdomain‐2 with Lys‐50, residing on the outer part of the DNase‐I‐binding loop, as the most likely site of cross‐linking. Because the maleimidobenzoyl‐F‐actin‐S‐1 complex undergoes the same coupling process, the data provide experimental evidence in favor of the recent model of the rigor F‐actin‐S‐1 complex suggesting a close contact between structural elements of the lower domain of the 50 kDa fragment and the top of actin subdomain‐2.
The occupancy of two distinct conformations by active‐site histidine‐119 in crystals of ribonuclease is modulated by pH
Authors: J. de Mel V.Srini , Doscher Marilynn S. , Martin Philip D. , Edwards Brian F.P.
Published: July 2002

Structures of a semisynthetic RNase have been obtained to a resolution of 2.0 Å at pH values of 5.2, 6.5, 7.5, and 8.8, respectively. The principle structural transformation occurring over this pH range is the conversion of the side chain of active site residue His‐119 from one conformation (X
1 = −43° to −57°) at low pH to another (X
1 = + 159° to + 168°) at higher pH values. On the basis of this observation, a model is proposed that reconciles the disparate pK values for His‐119 in the enzyme‐substrate complex that have been deduced from kinetic studies and from proton NMR measurements in the presence of pseudosubstrates.
Changes in the NAD(P)H concentration caused by addition of nitrogenase ‘switch‐off’ effectors in Rhodospirillum rubrum G‐9, as measured by fluorescence
Authors: Norén Agneta , Nordlund Stefan
Published: July 2002

The effect of nitrogenase ‘switch‐off’ effectors on the concentration of NAD(P)H in Rhodospirillum rubrum G‐9 was investigated by fluorescence. A rapid decrease in fluorescence was observed when cells, either N2‐grown or nitrogen‐starved, were subjected to the effectors, but not when sodium chloride or Tris buffer was added. No effects on the fluorescence were observed in non‐nitrogen fixing cultures except when NAD+ was added. The results strongly indicate that the redox state of the pyridine nucleotide pool affects the control of the regulation of nitrogenase activity in R. rubrum.
Identification of in vivo phosphorylation sites of SET, a nuclear phosphoprotein encoded by the translocation breakpoint in acute undifferentiated leukemia
Authors: Adachr Yoshifumi , Pavlakis George N. , Copeland Terry D.
Published: July 2002

SET, the translocation breakpoint‐encoded protein in acute undifferentiated leukemia (AUL), is identified as a 39‐kDa phosphoprotein found predominantly in the cell nuclei [1994, J. Biol. Chem. 269,2258‐2262]. SET is fused to a putative oncoprotein, CAN, in AUL and is thought to regulate the transformation potential of SET‐CAN by its nuclear localization and phosphorylation. We investigated in detail the in vivo phosphorylation of SET. Phosphorylation of SET occurred in all human cell lines examined in vivo, primarily on serine residues. Endoproteinase Glu‐C digestion of phosphorylated SET yielded two phosphopeptides. By radiosequencing, we identified the in vivo phosphorylation sites of SET as Ser9 and Ser24. The surrounding sequences of Ser9 and Ser24 contained an apparent consensus site sequence for protein kinase C.
Membrane interactions and surface hydrophobicity of Bacillus thuringiensis δ‐endotoxin CryIC
Authors: Butko Peter , Cournoyer Micheline , Pusztai-Carey Marianne , Surewicz Witold K.
Published: July 2002

The interaction between Bacillus thuringiensis insecticidal 5‐endotoxin CryIC and phospholipid vesicles was studied by fluorescence spectroscopy. The toxin dissipates the diffusion potential across vesicular membranes, presumably by creating ion permeable channels or pores. This effect is pH‐dependent and strongly increases under acidic conditions. The enhanced membrane‐perturbing activity of CryIC at low pH correlates with the increased surface hydrophobicity of the toxin molecule. The membrane permeabilizing effect of the toxin is further increased by the presence of acidic phospholipids. These findings are discussed in relation to the mode of insecticidal action of the toxin.
The origin of human immunodeficiency virus type‐1 rev gene
Authors: Kubota Satoshi , Oroszlan Stephen , Hatanaka Masakazu
Published: July 2002

The Rev protein of human immunodeficiency virus type‐1 is an RNA‐binding posttranscriptional transregulator encoded by an accessory gene that is distinct from retroviral oncogenes and whose origin is unclear. We hypothesize that the rev gene was generated by duplication of a viral RNA segment having a secondary‐structure that evolved into the Rev‐responsive element (RRE). This hypothesis is based on the following findings. First, accumulated data on functional mapping of Rev, Tat, and the transmembrane protein of Env suggested that the major coding exon of rev should have been inserted into the transmembrane region of env during the course of its evolution. Experiments with equine infectious anemia virus, another complex retrovirus, also indicate that a large portion of rev is located within the dispensable transmembrane region of env. Second, base usage analysis suggests the same origin for rev and RRE. Our hypothesis may provide a new insight into the evolutionary aspect of RNA0binding transactivators.