bluuebunny/crossref_metadata_2025 huggingface dataset:
Low‐cytopathic infectious clone of human immunodeficiency virus type I (HIV‐I)
Authors: Sakai Koji , Ma Xiaoyue , Volsky David J.
Published: July 2002

Single genotypic variants of HIV‐I, contained in a parental cytopathic HIV‐I isolate, were isolated by molecular cloning and propagated in susceptible cells. Two such HIV‐I clones, designated N1T‐E and N1T‐A, exhibited similar restriction endonuclease maps but strikingly different biological activities. Infection of T lymphocytes or monocytes by clone N1T‐E was characterized by slow kinetics and lack of significant cytopathic effects, but high reverse transcriptase activity levels in culture supernatants of chronically‐infected cells. Clone N1T‐A, like the parental HIV‐I isolate, exhibited fast kinetics of infection in T cells and monocytes and strong cytopathicity in these cells. Full characterization of the low‐cytopathic virus in comparison to the structurally similar cytopathic clone may facilitate the elucidation of the molecular basis of HIV cytopathogenicity.
Predictions of linear T‐cell and B‐cell epitopes in proteins encoded by HIV‐1, HIV‐2 and SIVMAC and the conservation of these sites between strains
Authors: Zvelebil Markéta J.J.M. , Sternberg Michael J.E. , Cookson John , Coatest Anthony R.M.
Published: July 2002

An important consideration in the design of vaccines to prevent HIV‐1 infection effective against dilterent strains is the amino acid sequence conservation of antigenic determinants. Even one amino acid change can destroy the antigenicity of a site for the antibody or T‐cell receptor. The comparisons of predicted T‐ and B‐cell epitopes between human HIV‐1, HIV‐2 and monkey SIVMAC AIDS viruses are presented. The three major gene products (env,gag and pol) were examined. A number of epitopes were identical between strains of HIV‐1. Our analysis highlights the problem of designing an effective HIV‐1 and HIV‐2 vaccine and also the problem of testing human vaccines in monkey models.
Na+‐driven ATP synthesis in Methanobacterium thermoautotrophicum can be modulated with sodium ion concentrations in the growth medium
Authors: Šmigáň Peter , Horovská Ľubica , Greksák Miloslav
Published: July 2002

Methane formation by nongrowing cells of Methanobacterium thermoautotrophicum ΔH can be enhanced with sodium ions only when the cells were originally grown in the presence of a low (5 mM) concentration of NaCl. ATP synthesis in the cells driven by an artificially imposed Na+ gradient can be modulated by changing the sodium ion concentration in the growth medium while H+ gradient‐driven ATP synthesis does not depend on it. Stimulation of the entire ATPase activity with Na+ in permeabilized cells depends also on the Na+ concentration in the growth medium. The results obtained might indicate the presence of an inducible Na+‐translocating ATPase in Methanobacterium thermoautotrophicum.
Solubilization and partial purification of a thylakoidal enzyme of spinach involved in the processing of D1 protein
Authors: Inagaki N. , Fujita S. , Satoh K.
Published: July 2002

The enzyme involved in the processing of D1 precursor protein was solubilized from spinach thylakoids by Triton X‐100 treatment and then partially purified in the presence of the detergent by Sephadex G‐75 gel‐filtration chromatography. The apparent molecular mass of the enzyme was estimated via this procedure to be about 34 kDa. The D1 precursor protein translated from the extracted spinach chloroplast RNA by a wheat germ cell‐free system was used here as a substrate in measurements of the activity.
S‐100a0 protein stimulates the basal (Mg2+‐activated) adenylate cyclase activity associated with skeletal muscle membranes
Authors: Fanò G. , Angelella P. , Mariggiò D. , Aisa M.C. , Giambanco I. , Donato R.
Published: July 2002

S‐100a0, protein, the αα isoform of the S‐100 family, stimulates basal (Mg2+‐activated) adenylate cyclase (AC) activity associated with the sarcolemma, longitudinal tubules and terminal cisternae of rat skeletal muscle cells. The stimulatory effect of S‐100a0 on AC activity is maximal around 5μM S‐100a0 and half‐maximal around 0.2 μM S‐100a0. Also, the stimulatory effect is greatest on the AC activity associated with the terminal cisternae than on the other membrane fractions studied. These data are discussed in relation to the subcellular localization of S‐100a0 in muscle cells.
Immunological evidence for the role of phosphoprotein p68/pI=7.3 in premessenger RNA splicing
Authors: Liew C.C. , Smith H.C.
Published: July 2002

A 68 kDa pI=7.3 nuclear phosphoprotein has been previously characterized as a component of transcriptionally active chromatin. Two‐dimensional PAGE Western blotting radioimmunoassay with monoclonal antibodies have identified this protein in nuclear extracts used for in vitro RNA splicing. In vitro splicing activity could be quantitatively inhibited by preincubating nuclear extracts with the antibodies, but the assembly of 60 S spliceosomes could not.
XANES spectroscopy sensitivity to small electronic changes Case of carp azidomethemoglobin
Authors: Pin Serge , Le Tilly Véronique , Alpert Bernard , Cortes Robert
Published: July 2002

Spin states equilibrium of hemoglobin‐iron varies with external conditions: pH, allosteric effectors, temperature. The small electronic reorganization of the iron caused by the spin state changes has been detected by X‐ray absorption near edge structure (XANES) spectroscopy at room temperature. The iron K‐edge region which is sensitive to spin state is located in 7110–7130 eV. Here are presented the 100% high spin and 100% low spin XANES spectra of carp azido ferric hemoglobin.
Detection of a proteolytic activity in the micrococcal nuclease used for preparation of messenger‐dependent reticulocyte lysates
Authors: Hemmer Odile , Demangeat Gérard , Greif Charles , Fritsch Christiane
Published: July 2002

Some, but not all, commercial preparations of micrococcal nuclease used to remove endogenous mRNA from reticulocyte lysates were found to contain proteolytic activity. The protease(s) caused a time‐dependent cleavage of the polyprotein primary translation products of genome RNA of several plant viruses, but did not affect the translation products of some other virus mRNA. The activity resulted in the production of smaller proteins and was inhibited by zinc ions. Thus the protease(s) mimicked virus‐coded proteases and represents a potential artifact in studies of translation products of virus RNA.
Muscarinic agents modify kinetics properties of membrane‐bound guanylyl cyclase activity
Authors: de Becemberg I.Lippo , de Aguilar A.E.Peña , Camarillo I. , de Alfonzo R.Gonzalez , Alfonzo M.
Published: July 2002

Plasma membranes from bovine tracheal smooth muscle show guanylyl cyclase activity, which can be stimulated by muscarinic agonists such carbamylcholine and oxotremorine and blocked by atropine. This stimulation was observed in the presence of 150 mM NaCl. In the absence of this salt, guanylyl cyclase activity was considerably higher but was not affected by muscarinic agonists. Carbamylcholine decreased the apparent K
m but did not change the V
max of this enzyme. When plasma membrane fractions were extracted with 1% octylglucoside, guanylyl cyclase activity was preserved, however the muscarinic activation was abolished, despite a muscarinic receptor capable of [3H]quinuclidinylbenzilate binding being present in the extract. The detergent extraction changed the affinity of guanylyl cyclase for GTP but the Mn2+ kinetics was unaltered. Based on these findings and on current information in the literature, we propose that another component is required to restore the link between the muscarinic receptor and guanylyl cyclase, however the nature of this component remains to be established.
Fluorescence of the reaction centre of photosystem II in cells of green alga Chlamydomonas reinhardtii
Authors: Lebedev N.N. , Barskaya Irina V.
Published: July 2002

Cells of green alga Chlamydomonas reinhardtii, mutant ASS66, which only exhibit the spectra and polypeptide composition of photosystem II, possess a weak fluorescence at about 673 nm along with two main emission bands at 686 and 698 nm, after cultivation in the dark. The 673 nm fluorescence exhibits an excitation spectrum similar to the absorbance of the isolated reaction centre of photosystem II (D1/D2 protein complex). It contains chlorophyll a, pheophytin a, and carotenoid bands with the chlorophyll:pheophytin ratio of about 3. Under illumination, the intensity of the 673 nm emission band decreases with concomitant increase in the emission band at 698 nm, which is thought to belong to the core antenna of photosystem II. Under illumination, in the presence of dithionite (strong reducing conditions), the relative intensity of 673 to 686 nm emission does not change, but the intensities of the pheophytin bands in its excitation spectrum were reduced two‐fold. That the relative intensity of the 673 nm band in the emission spectrum under illumination in the presence of dithionite does not change indicates that it originates from an emitter other than pheophytin (possibly reaction centre chlorophyll a).
Location of enzymatic and DNA‐binding domains on E. coli protease La
Authors: Baker Michael E.
Published: July 2002

Escherichia coli protease La is an ATP‐dependent enzyme that has a DNA‐binding site. The locations of the enzymatic and DNA‐binding sites are not known. We report that a 75‐residue segment at the carboxy‐terminus of the protease La is similar to part of Bacillus licheniformis β‐lactamase, a serine enzyme. The comparison score is 8.2 standard deviations higher than that obtained with 10 000 comparisons of randomized sequences of these segments. The probability of obtaining such a score by chance is 1.2 × 10−16. We also find that a 107‐residue segment in the amino‐terminus half of protease La is similar to part of the sopB protein, a DNA‐binding protein of the plasmid F of E. coli. The comparison score for these segments is 8 standard deviations (P = 6 × 10−16). These strong amino acid sequence similarities suggest the locations of the catalytic serine and the DNA‐binding domains of protease La.
Interaction of the pore forming‐peptide antibiotics Pep 5, nisin and subtilin with non‐energized liposomes
Authors: Kordel Marianne , Schüller Friederike , Sahl Hans-G.
Published: July 2002

The cationic peptide antibiotics Pep 5, nisin and subtilin depolarize bacterial and artificial membranes by formation of voltage‐dependent multi‐state pores. Studies with non‐energized liposomes indicated that the peptides do not span the membrane in the absence of a membrane potential. The effects of Pep 5 and nisin on neutral membranes, as studied by membrane fluidity, phase transition points and carboxyfluorescein efflux, were small compared to melittin. Acidic liposomes were affected more strongly, indicative of primarily electrostatic interactions with phospholipid head groups. Subtilin may slightly enter the hydrophobic core as suggested by tryptophan fluorescence quenching and liposome fusion experiments.
Synthesis of paf‐acether from exogenous precursors by the prokaryote Escherichia coli
Authors: Denizot Yves , Dassa Elie , Kim Hee-Yong , Bossant Marie-Jeanne , Salem Norman , Thomas Yolène , Benveniste Jacques
Published: July 2002

Paf‐acether (paf) is a potent mediator of inflammatory diseases and septic shock. Using normal‐phase HPLC, a paf‐like activity was found in culture supernatants from E. coli. Prokaryotic paf exhibited the same biological and physico‐chemical properties as eukaryotic cells and synthetic paf. Further, reverse‐phase HPLC indicates that paf generated by bacteria is predominantly of the hexadecyl and octadecyl species. When cultures were supplemented with lyso‐paf, a dramatic increase in paf production was observed. The purity and molecular structure of bacterial paf were further characterized by mass spectral analysis. These results could be of importance considering the pathogenetic role of enterobacteria. Further, it appears that the competence to form and release paf is an early phylogenetic development.
Identification of S‐100 proteins and S‐ 100‐binding proteins in a detergent‐resistant EDTA/KCl‐extractable fraction from bovine brain membranes
Authors: Donato Rosario , Giambanco lleana , Aisa Maria Cristina , Ceccarelli Paolo
Published: July 2002

The Triton X‐100‐resistant residue of brain membranes contains appreciable amounts of S‐100 proteins. This fraction of S‐100 can be solubilized by high concentrations of EDTA plus or minus high concentrations of KCl. Whereas KCl (0.6 M) extracts the detergent‐resistant S‐100, NACl (1 M) does not. Endogenous Ca2+ is required and is sufficient for S‐100 to remain associated with the detergent‐resistant residue. However, 0.6 M KCl extracts a further fraction of Triton X‐100‐resistant S‐100. In contrast, the Triton X‐100‐extractable fraction of S‐100 resists the action of EDTA. These data suggest that Ca2+ regulates the extent of association of S‐100 with Triton X‐100‐resistant components in brain membranes, whereas the association of S‐100 with the lipid bilayer of brain membranes and/or with some intrinsic membrane proteins is less Ca2+‐regulated. Several S‐100‐binding proteins are identified in the detergent‐resistant residue of brain membranes by an overlay procedure.
Isolation and N‐terminal amino acid sequence of an octopamine ligand binding protein
Authors: Nathanson James A. , Kantham Lakshmi , Hunnicutt Edward J.
Published: July 2002

An octopamine receptor photoaffinity probe was used to label membranes from the light organs of Photinus pyralis, a tissue highly enriched in octopamine receptors. Labeling was concentrated in a glycoprotein of 75 ± 2 kDa with lesser labeling of a 79 ± 2 kDa component. Labeling could be displaced by prior incubation with octopamine, mianserin, cyproheptadine, phentolamine or propranolol, with a relative potency that correlated with the ability of these same agents to modulate light organ octopamine‐sensitive adenylate cyclase. The 75 kDa binding protein was isolated and its N‐terminal amino acid sequence was determined.
Association of higher molecular weight ribonucleoproteins of 7 S particle preparations with multimers of transcription factor IIIA
Authors: Coyer James , Andersen Janet , Delihas Nicholas
Published: July 2002

Protein dimer; Nucleic acid‐binding protein; TFIIIA; RNP, 7 S
Increased calcium permeability is not responsible for the rapid lethal effects of amphotericin B on Leishmania sp
Authors: Cohen B.Eleazar , Benaim Gustavo , Ruiz Marie-Christine , Michelangeli Fabián
Published: July 2002

The mode of action of the polyene antibiotic amphotericin B (AmB), the drug of choice for the treatment of systemic fungal infections and visceral leishmaniasis, is still unclear. An increase in intracellular Ca2+ concentration ([Ca2+]i), toxic in many cases, has been postulated as a possible lethal mechanism for AmB. Cell permeabilization to ethidium bromide (EB) was used as a criterion of viability. Kinetics of the DNA‐EB fluorescent complex formation was studied in ergosterol‐containing Leishmania promastigotes. Intracellular Ca2+ concentration was measured using quin‐2 fluorescence in parallel aliquots. It is shown in this work that AmB can act as an efficient Ca2+ ionophore. However, the rapid permeabilization effect induced by AmB on these cells was not dependent on an increase in [Ca2+]i. On the contrary, it was found that leishmanicidal effect of AmB was enhanced in the absence of external calcium. Furthermore, A23187 a Ca2+ ionophore did not provoke cell penneabilization to EB.
Cloning and sequencing of the gene encoding cytochrome c‐551 from Pseudomonas aeruginosa
Authors: Arai Hiroyuki , Sanbongi Yoshihiro , Igarashi Yasuo , Kodama Tohru
Published: July 2002

The cytochrome c‐551 gene from Pseudomonas aeruginosa was cloned by using two oligonucleotide probes, which had been synthesized based on the known primary structure of the protein. The restriction map of the cloned DNA and sequence analysis showed that the cytochrome c‐551 gene is located 50 bp downstream of the nitrite reductase gene, which has recently been cloned and sequenced. DNA sequence analysis also indicated that cytochrome c‐551 is synthesized in vivo as a precursor having an amino‐terminal signal sequence consisting of 22 amino acid residues.
Human HLA‐DRα gene: a rare oligonucleotide (GTATA) identifies an upstream sequence required for nuclear protein binding
Authors: Barbieri Rafaella , Giacomini Patrizio , Volinia Stefano , Nastruzzi Claudio , Mileo Anna Maria , Ferrini Umberto , Soria Marco , Barrai Italo , Natali Pier Giorgio , Gambari Roberto
Published: July 2002

Synthetic oligonucleotides containing putative regulatory sequences are currently employed to identify and isolate genes coding for nuclear binding factors. Upstream DNA sequences of eukaryotic genes required for transcriptional activity and tissue specificity can be identified by means of biochemical techniques as well as computer analysis using homology searching. An alternative approach has been recently proposed by our research group. Scanning DNA sequences 1.8 megabases in length from a Genetic Sequence Data Bank, we have identified rare oligonucleotides 5 base pairs (bp) long, which are localized within or close to regulatory segments in mammalian promoters. In this paper we demonstrate that the rare GTATA sequence identifies an upstream region of the HLA‐DRα gene which operates in conjunction with the sequence AGAAGTCAG, homologous to a box found in many interferon‐inducible genes, in binding nuclear proteins.
The primary structure of the Lotus tetragonolobus seed lectin
Authors: Konami Yukiko , Yamamoto Kazuo , Osawa Toshiaki
Published: July 2002

The complete amino acid sequence of the Lotus tetragonolobus lectin was determined by a protein sequencer after digestion with endoproteinases of Lys‐C and Asp‐N, and compared with those of other leguminous plant lectins.
Norepinephrine‐induced synthesis of the uncoupling protein thermogenin (UCP) and its mitochondrial targeting in brown adipocytes differentiated in culture
Authors: Herron David , Rehnmark Stefan , Néchad Myriam , Lončar Dragutin , Cannon Barbara , Nedergaard Jan
Published: July 2002

Synthesis of the brown adipocyte‐specifie mitochondrial uncoupling protein thermogenin (UCP) is demonstrated here in brown adipocytes differentiated in culture from precursor cells. By immunoblotting, no UCP was detectable in untreated multilocular adipocytes. The synthesis of UCP was stimulated by norepinephrine at physiological concentrations and was observable already after 2 h. It was evident from immunoelectron microscopy that the newly synthesised protein was targeted to the mitochondrial inner membrane, demonstrating the functional competence of these cultured cells.
Sequence homology between purple acid phosphatases and phosphoprotein phosphatases
Authors: Vincent John B. , Averill Bruce A.
Published: July 2002

The amino acid sequences of mammalian purple acid phosphatases and phosphoprotein phosphatases are shown to possess regions of significant homology. The conserved residues contain a high percentage of possible metal‐binding residues. The phosphoprotein phosphatases 1, 2A and 2B are proposed to be iron‐zinc metalloenzymes with active sites isostructural (or nearly so) with those of the purple phosphatases.
DNA polymerase activity of tomato fruit chromoplasts
Authors: Serra Esteban C. , Carrillo Nestor
Published: July 2002

DNA polymerase activity was measured in chromoplasts of ripening tomato fruits. Plastids isolated from young leaves or mature red fruits showed similar DNA polymerase activities. The same enzyme species was present in either chloroplasts or chromoplasts as judged by PH and temperature profiles, sensitivities towards different inhibitors and relative molecular mass (M
1 88 kDa). The activities analyzed showed the typical behaviour of plastid‐type polymerases. The results presented here suggest that chromoplasts maintain their DNA synthesis potential in fruit tissue at chloroplast levels. Consequently, the sharp decrease of the plastid chromosome transcription observed at the onset of fruit ripening could not be due to limitations in the availability of template molecules. Other mechanisms must be involved in the inhibition of chromoplast RNA synthesis.
Disruption of the gene encoding subunit VI of yeast cytochrome bc1 complex causes respiratory deficiency of cells with reduced cytochrome c levels
Authors: Kim Chong H. , Zitomer Richard S.
Published: July 2002

A double mutant of Saccharomyces cerevisiae, in which CYCl gene is deleted and the chromosomal copy of the 17 kDa protein gene is disrupted, has been constructed. This mutant cannot grow on nonfermentable carbon sources, but normal growth can be restored by complementation of either mutation with a yeast vector containing either the wild‐type 17 kDa protein gene or the CYCl gene. These results show that although the 17 kDa protein, subunit VI of yeast cytochrome bc
1 complex is dispensable for yeast mitochondrial respiration in cells with the wild‐type levels of cytochrome c, the 17 kDa protein is essential for respiration when the level of cytochrome c is limited, indicating that is plays a role in electron transport. This glycerol− phenotype of the double mutant can serve as the basis for further genetic studies on the function of the 17 kDa protein in yeast mitochondria and may provide insight into the physiological function of the hinge protein, the counterpart of the yeast 17 kDa protein, in beef heart mitochondria.
An upstream regulatory element of the NCAM promoter contains a binding site for homeodomains
Authors: Hirsch Marie-Rose , Valarché Isabelle , Deagostini-Bazin Hermine , Pernelle Christine , Joliot Alain , Goridis Christo
Published: July 2002

In the present study, we have analyzed an upstream regulatory element of the neural cell adhesion molecule (NCAM) promoter which is required for full promoter activity. It contains an ATTATTA motif that resembles the core recognition sequence of homeodomain (HD) proteins of the Antennapedia (Antp) and related types. Electrophoretic mobility shift (EMSA) and DNase I footprinting analyses revealed that the Drosophila HDs coded by the Antp and the zerknüllt (zen) genes bind this site in vitro. In contrast, the engrailed (en) protein did not produce a detectable footprint. The functional relevance of the ATTATTA motif was demonstrated by showing that a two‐nucleotide exchange curtailed stimulation of an heterologous promotor. An oligonucleotide known to be recognized with high affinity by Antp‐like HDs efficiently competed for endogenous factor binding. These results suggest that the NCAM gene may be a target for HD proteins.
A heptapeptide repeat contributes to the unusual length of chloroplast ribosomal protein S18 Nucleotide sequence and map position of the rpl33‐rps18 gene cluster in maize
Authors: Weglöhner Wolfgang , Subramanian Alap Raman
Published: July 2002

The rpl33‐rps18 gene cluster of the maize chloroplast genome has been mapped and sequenced. The derived amino acid sequence of the S18 protein shows a 7‐fold repeat of a hydrophilic heptapeptide domain. S K Q P F R K, in the N‐terminal region. Such a sequence is absent in the E. coli S18 and in the chloroplast S18 of the lower plant liverwort. In tobacco and rice chloroplast S18 it is present 2 and 6 times, respectively. Thus a long N‐terminal repeat (resembling in composition the large C‐terminal heptapeptide repeat in the eukaryotic pol II) appears to be characteristic of monocot cereal S18.
Deletion of exon 8 causes glycosylasparaginase deficiency in an African American aspartylglucosaminuria (AGU) patient
Authors: Fisher Krishna J. , Aronson Nathan N.
Published: July 2002

We have indentified a T‐to‐T transversion at the splice donor site of intron 8 in the glycosylasparaginase gene from an African American aspartylglucosaminuria (AGU) patient, This mutation causes abnormal splicing of glycosylasparaginase pre‐mRNA by joining exon 7 to 9 and excluding 134 bp exon 8. The effect of the mutation is compounded by a frame shift that occurs after the deletion site resulting in premature translational termination. The truncated AGU protein was neither catalytically active nor processed into mature α and β subunits. Both this and a previously characterized Finnish AGU mutation appear to affect folding of the single‐chain precursor of glycosylasparaginase and thereby prevent transport of the enzyme to lysosomes.
Interactions between the antimicrobial peptide, magainin 2, and Salmonella typhimurium lipopolysaccharides
Authors: Rana Fazale R. , Blazyk Jack
Published: July 2002

Using FT‐IR spectroscopy, the effects of magainin 2 on the thermotropic behavior of LPS isolated from wild‐type (SL3770) and LPS‐mutant strains of Salmonella typhimurium are characterized and compared. The mutant strains include Ra (SL3749), polymyxin‐sensitive Rb2(s) (SH5014), polymyxin‐resistant Rb2(r) (SH5357) and Rc (HN202) LPS chemotypes, whose polysaccharide chains differ in length but possess an identical number of phosphorylation sites. In all cases, magainin 2 causes a concentration‐dependent disordering of the LPS fatty acyl chains. Differences in disordering of LPS correlate more closely with the charge on the LPS molecule (determined by high‐resolution 31P NMR) rather than with the length of the LPS sugar side chain, contradicting the currently accepted model for the interaction of cationic antibiotics with the Gram‐negative cell envelope.
Effective detergent/lipid ratios in the solubilization of phosphatidylcholine vesicles by Triton X‐100
Authors: Partearroyo M.Aránzazu , Urbaneja M.Angeles , Goñi Félix M.
Published: July 2002

Effective detergent:lipid ratios (i.e. molar ratios in the mixed aggregates, vesicles or micelles) have been estimated for the solubilization of phosphatidylcholine vesicles by Triton X‐100. Effective molar ratios are given for both the onset and the completion of bilayer solubilization; small unilamellar, large unilamellar and multilamellar vesicles have been used. Effective detergent:lipid ratios are independent of phospholipid concentration, and their use allows a deeper understanding of membrane‐surfactant interactions.
β‐Turns induced in bradykinin by (S)‐α‐methylproline
Authors: Welsh John H. , Zerbe Oliver , von Philipsborn Wolfgang , Robinson John A.
Published: July 2002

The ability or (S)‐α‐methylproline(α‐MePro) to stabilise reverse‐turn conformations in the peptide hormone bradykinin (BK = Arg1‐Pro2‐Pro3‐Gly4I‐Phe5‐Ser6‐Pro7‐Phe8‐Arg9) has been investigated. Two BK analogues containing α‐MePro at position 3 or position 7 were synthesised and their conformations in aqueous solution investigated by NMR spectroscopy. Whereas BK is largely disordered on the NMR time scale both analogues showed ROE connectivities in 2D‐ROESY spectra indicative of reverse‐turn conformations at both Pro2‐Phe5 and Ser6‐Arg9, whose formation appears to be cooperative. Some potential applications of α‐MePro as a reverse‐turn mimetic in the construction of synthetic peptide libraries is discussed.
Zwitterionic amphiphiles that raise the bilayer to hexagonal phase transition temperature inhibit protein kinase C The exception that proves the rule
Authors: Epand Richard M. , Stafford Alan , Wang Jian , Epand Raquel F.
Published: July 2002

We synthesized the zwitterionic amphiphile cholesterylphosphorylethylpyridinium. This substance activated protein kinase C (PKC) in a micelle‐based assay, but in a vesicle assay it was inhibitory. An analog of this compound, in which the pyridine ring is saturated and the nitrogen methylated, showed similar behaviour with PKC. Replacing cholesterol by an aliphatic alcohol lowered the extent of activation in the micelle assay. These results demonstrate that, with some membrane additives, the vesicle and micelle assays give opposite results. Results from the membrane‐based vesicle assay for PKC are in accord with the generalization that zwitterionic amphiphiles that raise the bilayer to hexagonal phase transition temperature in model membranes are inhibitors of PKC
Effects of apocynin, a drug isolated from the roots of Picrorhiza kurroa, on arachidonic acid metabolism
Authors: Engels Ferdi , Renirie Bastien F. , 't Hart Bert A. , Labadie Rudi P. , Nijkamp Frans P.
Published: July 2002

Apocynin is a constituent of root extracts of the medicinal herb Picrorhiza kurroa and has been shown to possess anti‐inflammatory properties. We investigated the effects of apocynin on the production of arachidonic acid‐derived inflammatory mediators by guinea pig pulmonary macrophages. Apocynin concentration‐dependently inhibited the formation of thromboxane A2, whereas the release of prostaglandins E2 and F2α was stimulated. Apocynin potently inhibited arachidonic acid‐induced aggregation of bovine platelets, possibly through inhibition of thromboxane formation. The present results suggest that apocynin might, beside its therapeutic effects in inflammatory conditions when given in a root extract of P. kurroa, also be a valuable tool in the development of new anti‐inflammatory or anti‐thrombic drugs.
Monitor peptide gene expression is increased by exogenous CCK in the rat pancreas and in a rat pancreatic acinar cell line (AR4‐2J)
Authors: Tsuzuki Satoshi , Kondo Atsuko , Fushiki Tohru , Sugimoto Etsuro
Published: July 2002

Monitor peptide (CCK‐releasing peptide) mRNA increased on the administration of CCK in rat pancreas and the AR4‐2J pancreatic cell line. Subcutaneous injection of CCK into rats at 8 h intervals increased the level of monitor peptide mRNA in the pancreas. Concomitant injection of CCK antagonist CR‐1409 strongly decreased it. The monitor peptide mRNA was also increased by CCK in AR4‐2J cells and was decreased by the antagonist. These findings suggest that the plasma CCK induced by prolonged intake of a high protein diet may be responsible for the adaptive increase in the monitor peptide as well as exocrine proteases in the pancreas.
The inhibitory effects of okadaic acid on platelet function
Authors: Higashihara Masaaki , Takahata Kyoya , Kurokawa Kiyoshi , Ikebe Mitsuo
Published: July 2002

Okadaic acid (OA), a potent inhibitor of protein phosphatases type 1 and type 2A, inhibited thrombin‐induced platelet aggregation (IC50 = 0.8 μM), [14C]serotonin release and increase in intracellular Ca2+ ([Ca2+]i) in the same dose dependence. In the absence of thrombin OA increased the phosphorylation of 50‐kDa protein and 20‐kDa myosin light chain (MLC20). The 50‐kDa protein phosphorylation was accomplished within a shorter time period and at a lower concentration than was the MLC20, OA decreased the thrombin‐induced phosphorylation of 47‐kDa protein and MLC20, although phosphorylation of MLC20 reincreased at higher concentrations of OA (5−10 μM). Since type 2A phosphatase is more sensitive to OA than type 1, these results suggest that type 2A phosphatases are involved in the regulation of Ca2+ signaling in thrombin‐induced platelet activation.
Insulin receptor and glucose transporter mRNA expression in skeletal muscle of genetically obese Zucker rats
Authors: Knott Rachel M. , Hesketh John E.
Published: July 2002

Insulin resistance and defective glucose transport are associated with muscle tissue in the genetically obese Zucker rat and are accompanied by changes in the number of insulin receptors and the availability of glucose transporters, The present study was carried out to assess whether. in male Zucker rats at 10 weeks of age. these defects were reflected by changes in the levels of mRNAs for the insulin receptor and for the insulin responsive glucose transporter (GLUT‐4). Total RNA was extracted from plantaris and soleus muscles and the levels of insulin receptor and GLUT‐4 mRNAs and 18 S rRNA were determined by Northern hybridization and quantified by image analysis of the autoradiographs. A 50% increase in the level of insulin receptor mRNA was detected in both the plantaris and the soleus muscle from the obese rats compared to the lean rats, No change in the level of GLUT‐4 mRNA was detected in the plantaris muscle although increases were observed in the soleus muscle from the obese rats.
Atomic force microscopy of DNA molecules
Authors: Yang Jie , Takeyasu Kunio , Shao Zhifeng
Published: July 2002

DNA‐cytochrome complexes adsorbed on carbon‐coated micasurfaces were directly imaged by atomic force microscopy in air using commercially available cantilevers, with a routine resolution of 6 nm. Images of M13 phage DNA and M13‐DNA polymerase complex are also shown.
Photoaffinity labeling of the phylloquinone‐binding polypeptides by 2‐azidoanthraquinone in photosystem I particles
Authors: Iwaki Masayo , Takahashi Masaaki , Shimada Keizo , Takahashi Yuichiro , Itoh Shigeru
Published: July 2002

A photoaffinity label, 2‐azido‐9,10‐anthraquinone, binds at the quinone‐binding (Qφ) site with high affinity and can substitute for the secondary acceptor, phylloquinone, in photosystem I reaction center of spinach. Phylloquinone‐depleted photosystem I particles reconstituted with azido‐[3H]anthraquinone were illuminated with UV light and analyzed by sodium dodecylsulfate‐polyacrylamide gel electrophoresis. The large core polypeptides (psaA and/or psaB) were selectively labeled. The labeling was competitively inhibited in the presence of anthraquinone. These results indicate that the Qφ site is located on psaA or psaB polypeptides.
Codon—anticodon pairing A model for interacting codon—anticodon duplexes located at the ribosomal A‐ and P‐sites
Authors: Lim Valery I. , Venclovas Česlovas
Published: July 2002

The interaction between two codon—anticodon duplexes of the ribosomal A‐ and P‐site‐bound tRNAs is the key feature of the proposed model. This interaction prohibits non‐canonical base pairing at the first and second positions of the codon and controls base pairing at the third position (wobbling rules ensuing from the model are in good accord with those generated from experiments). The model is capable of predicting codon context effects. It follows from the model that modifications of the first anticodon residue of the P‐site tRNA can affect the stability of the A‐site duplex, and that the translation of a DNA single chain analogue of mRNA should be accompanied by non‐canonical base pairing at all three positions of the codon. These predictions of the model can be subjected to experimental tests.
Imidazole antimycotics inhibitors of cytochrome P450 increase phosphatidylserine synthesis similarly to K+‐channel blockers in Jurkat T cells
Authors: Aussel Claude , Breittmayer Jean Philippe
Published: July 2002

The imidazole antimycotics, miconazole, econazole and triclomazole as well as α‐naphtoflavone, known as powerful inhibitors of cytochrome P450 and previously recognized as K+ channel blockers are shown to be potent activators of the base exchange enzyme system responsible for the biosynthesis of phosphatidylserine in Jurkat T cells. The inhibition of CD3‐induced Ca2+ influx by antimycotics but not by K+ channel blockers, demonstrated that the rise in phosphatidylserine synthesis caused by the two classes of drugs, was independent of Ca2+ influx in the cells. In addition, we show that the action of these drugs on phosphatidylserine synthesis was not mimicked by modifications of membrane potential. The regulation of both K+ channels and the base exchange enzyme system thus occurs through a similar (or common) pathway that is independent of Ca2+‐influx and membrane potential.
Picosecond dynamics of excitations in light‐harvesting complex B800‐850 from Chromatium minutissimum studied using fluorescence spectrochronography
Authors: Godik Valentina , Timpmann Kou , Freiberg Arvi , Moskalenko Andrey A.
Published: July 2002

The picosecond dynamics of excitations in the isolated B800‐850 light‐harvesting complex of the purple sulfur bacterium Chromatium minutissimum has been studied using picosecond fluorescence spectrochronography. A short‐lived component of about 20 ps lifetime has been found at 77K at the short wavelength part of the B850 fluorescence spectrum similar to that previously described for the core antenna bacteriochlorophyll band B880 of Rhodospirillum rubrum. Evidence has been presented indicating that this component is likely to reflect excitation energy relaxation step(s) involving both photoexcited bacteriochlorophyll and the protein environment. A new kinetic scheme of excitation transfer from the peripheral antenna to the photoreceptor units in purple bacteria is suggested which takes into account these findings.
Identification of a nuclear protein binding element within the rat brain protein kinase C γ promoter that is related to the developmental control of this gene
Authors: Chen Kuang-Hua , Widen Steven G. , Wilson Samuel H. , Huang Kuo-Ping
Published: July 2002

Protein kinase C γ (PKC γ) is a brain‐specific isozyme expressed at a high level in the adult but not in the fetal or newborn rat. At least seventeen nuclear protein binding sites within the 5'‐flanking region extending from −1612 to +243 had been identified by DNase I footprinting analysis and gel mobility shift assays. Among them, one site, GAATTAATAGG, at −669 to −679 is protected from DNase I digestion by nuclear protein from newborn but not from the adult rat brain. The levels of this binding protein, as determined by gel mobility shift assay, were found inversely related to the levels of PKC γ in rat brain at different stages of development. These results suggest that this particular binding site may participate in the developmental regulation of PKC γ gene.
Human liver microsomal glutathione transferase
Authors: Mosialou E. , Andersson C. , Lundqvist G. , Andersson G. , Bergman T. , Jörnvall H. , Morgenstern R.
Published: July 2002

Human liver microsomal glutathione transferase displays the following glutathione peroxidase/transferase activities: dilinoleoylphosphatidylcholine hydroperoxide (0.03 and 0.17 · mg, unactivated and N‐ethylmaleimide‐activated enzyme, respectively), linoleic acid hydroperoxide (0.09 and 0.15 · mg), cumene hydroperoxide (0.04 and 3 · mg), methyl linoleate ozonide (0.02 and 1.2 · mg) and 1‐chloro‐2,4‐dinitrobenzene (1.9 and 24 · mg). The activation of glutathione peroxidase activities are much higher than previously observed. The activity towards a phospholipid hydroperoxide is noteworthy since protection against lipid peroxidation has been implied. Methyl linoleate ozonide has not previously been characterised as substrate for any microsomal glutathione transferase. Human liver microsomal glutathione transferase displays an isoelectric point of 9.4 and a structure in agreement with that deduced from the cDNA sequence. Gel electrophoretic analysis shows that proteolytic activation of the human enzyme corresponds to cleavage at Lys‐41, thus denning the critical activation site.
Membrane phospholipid asymmetry in DMPC‐induced human red cell vesicles
Authors: de Jong Kitty , Ott Peter
Published: July 2002

Vesicles that do not contain spectrin were released from human erythrocytes by incubation with dimyristoylphosphatidylcholine. The transbilayer orientation of membrane phospholipids was subsequently determined by two independent methods. Incubation with phospholipase A2 revealed that the phospholipid asymmetry observed in red blood cells was essentially preserved in vesicles. By use of the prothrombinase assay a still highly asymmetric distribution of phosphatidylserine could be demonstrated in spite of its slightly increased exposure on the vesicle surface. These results show that membrane phospholipid asymmetry can be maintained in a system that does not contain an intact membrane skeleton or spectrin.
Identification of an aprotinin antiviral domain
Authors: Pellegrini Antonio , Thomas Ursula , Franchini Marco , Stöckli Martina , Klauser Stefan , Hunziker Peter , von Fellenberg Roland
Published: July 2002

Digestion of the proteinase inhibitor aprotinin, by clostripain, a cysteine proteinase, yielded five oligopeptide fragments. Two fragments exhibited both antiviral and antibacterial activities, two fragments only antiviral activity, and one fragment showed no antimicrobial activity. One of the former oligopeptides showed antiviral activity against human herpes simplex virus type 1 and bovine parainfluenza virus type 3. It consisted of the hexapeptide Y‐F‐Y‐N‐A‐K corresponding to amino acids 21–26 of intact aprotinin. An identical synthetic peptide had the same antiviral spectrum as the natural hexapeptide, exhibited no antibacterial activity, and was also devoid of trypsin inhibiting activity. Intact aprotinin, in contrast, is ineffective against human herpes simplex virus 1 and bovine parainfluenza virus 3 but possesses antibacterial properties against several bacterial species [(1992) J. Appl. Bact. 72, 180–187].
A 66‐kDa protein associated with epidermal growth factor receptor is a proteolytic fragment of phosphoinositide‐specific phospholipase C
Authors: Medvedeva N.D. , Alexeyev V.Yu. , Tsupkina N.V. , Nikolsky N.N.
Published: July 2002

It is shown that in the A431 cells, EGFR is co‐immunoprecipitated with a group of proteins recognized by antibodies to phospholipase Cγ. These are 145‐ and 47‐kDa proteins corresponding to phospholipase Cγ and Nck, respectively, and an unidentified 66‐kDa protein. The association of phosphoinositide‐specific phospholipase Cγ and 66‐kDa protein to EGFR was observed in the A431 cells with or without the EGF treatment. Trypsin peptide maps of these two proteins are similar so it is assumed that the 66‐kDa protein is related to phospholipase Cγ.
The interaction of hydrophobic bile acids with the α1‐proteinase inhibitor
Authors: Janciauskiene Sabina , Eriksson Sten
Published: July 2002

An in vitro complex formation between cholesterol and human α1‐proteinase inhibitor (α1‐antitrypsin, α1‐Pi) has been described. Hydrophobie bile acids were studied for a similar interaction using lithocholic acid (LC) as a prototype of a hydrophobic acid. At a molar ratio of 5:1, LC induced conformational changes of α1‐Pi reflected in an abnormal gel‐electrophoretic appearance, loss of anodal immunoreactivity on crossed immunoelectrophoresis, exposition of new antigenic determinant(s) on immunodiffusion, and loss of antiproteinase activity. After 6 h incubation, LC and α1‐Pi form a complex of approximately 200 kDa molecular mass seen following gel‐filtration. After prolonged (24 h) interaction a series of large α1‐Pi polymers were seen on SDS‐PAGE under reducing conditions followed by Western blotting. Glycolitho‐, sulfolitho‐, deoxycholic and 3‐β‐hydroxy‐5‐cholenoic acids induced similar but less pronounced changes of α1‐Pi, whereas transferrin remained unaffected. Hydrophilic acids lacked effect on α1‐Pi. The results are compatible with a specific, irreversible interaction of α1‐Pi with hydrophobic bile acids affecting its physical and proteinase inhibitory properties. The cholestatic potency of the hydrophobic acids studied and their ability to induce α1‐Pi polymerization may be important in cholestatic conditions.
Changing root system architecture through inhibition of putrescine and feruloyl putrescine accumulation
Authors: Ben-Hayyim Gozal , Damon Jean-Pierre , Martin-Tanguy Josette , Tepfer D.
Published: July 2002

Plant roots provide anchorage and absorb the water and minerals necessary for photosynthesis in the aerial parts of the plant. Since plants are sessile organisms, their root systems must forage for resources in heterogeneous soils through differential branching and elongation [(1988) Funct. Ecol. 2, 345‐351; (1991) Plant Roots: The Hidden Half, pp. 3‐25, Marcel Dekker, NY]. Adaptation to drought, for instance, can be facilitated by increased root growth and penetration. Root systems thus develop as a function of environmental variables and the needs of the plant [(1988) Funct. Ecol. 2, 345‐351; (1986) Bot. Gaz. 147, 137‐147; (1991) Plant Roots: The Hidden Half, pp. 309‐330, Marcel Dekker, NY], We show, in a model system consisting of excised tobacco roots, that both α‐dl‐difluoromethylornithine (an inhibitor of putrescine biosynthesis) and the rolA gene (from the root‐inducing transferred DNA of Agrobacterium rhizogenes) stimulate overall root growth and cause a conversion in the pattern of root system formation, producing a dominant or ‘tap’ root. These morphological changes are correlated with a depression in the accumulation of polyamines and their conjugates.
The primary structure of two chlorosome proteins from Chloroflexus aurantiacus
Authors: Niedermeier Georg , Shiozawa Judith A. , Lottspeich Friedrich , Feick Reiner G.
Published: July 2002

The complete nucleotide sequence of two chlorosome proteins with apparent molecular weights of M
r 18,000 and M
r 11,000 from Chloroflexus aurantiacus have been determined. The two polypeptides were 145 and 97 amino acids long and possessed true molecular masses of 15,545 and 10,820 Da, respectively. Protein chemical sequencing was done in parallel to confirm the primary structure deduced from nucleotide sequencing. By Northern blot analysis of RNA isolated from phototrophically grown cells a transcript of 0.95 kb was detected which is the expected length for a mRNA encoding both genes.
The vitamin D3 analogue, calcipotriol, induces sphingomyelin hydrolysis in human keratinocytes
Authors: Geilen Christoph C. , Bektas Meryem , Wieder Thomas , Orfanos Constantin E.
Published: July 2002

The possible role of sphingomyelin cycle for the regulation of cell proliferation was investigated in human keratinocytes. The time‐dependent breakdown of sphingomyelin was observed in the immortalized human keratinocyte cell line HaCaT as well as in primary human keratinocytes thereby providing evidence that the sphingomyelin cycle might be of importance in the epidermis. Peak levels of 20–30% sphingomyelin hydrolysis were measured 3 h after treatment of the cells with 1α,25‐dihydroxyvitamin D3 or with the vitamin D3 analogue, calcipotriol. The decrease of sphingomyelin upon addition of vitamin D3 or calcipotriol was accompanied by an approximately 70% increase of ceramide in the cells. The effects of vitamin D3 and calcipotriol on sphingomyelin breakdown were paralleled by their antiproliferative potency. Furthermore, the cell‐permeable ceramide, N‐acetylsphingosine, and natural ceramide inhibited cell proliferation of human keratinocytes. The results presented suggest that induction of the sphingomyelin cycle represents one mechanism mediating the therapeutic effect of calcipotriol in treatment of psoriasis.
A monoclonal antibody that causes the heterotrimeric G‐protein Go to release its βγ subunits
Authors: Yilla Mamadi , Oleinick Jonathan , Ploegh Hidde L.
Published: July 2002

Heterotrimeric (αβγ) guanine nucleotide binding proteins (G‐proteins) dissociate into their constituent subunits in the course of signal transduction. Exposure of the G‐protein Go to the α
o‐specific monoclonal antibody 3E7 results in recovery of α
o alone. We identified the 3E7 epitope as ERSKAIEKNL (positions 14–23) using synthetic peptides and phage display. Go isolated with α
o‐specific monoclonal antibodies MONO and 3C2 dissociates and releases its βγ subunits when exposed to 3E7. Exposure to 3E7, but not MONO or 3C2, results in the displacement of βγ from trimers, in the absence of added activators of G‐proteins (GTPγS, Mg2+AIF4−). We propose that stable binding of 3E7 to α
o requires displacement of γγ and occurs in the absence of guanine nucleotide exchange.
Peroxisome proliferator‐activated receptors are expressed in mouse bone marrow‐derived mast cells
Authors: Sugiyama Hiroyuki , Nonaka Takashi , Kishimoto Tadashi , Komoriya Keiji , Tsuji Kohichiro , Nakahata Tatsutoshi
Published: July 2002

We examined the expression of peroxisome proliferator‐activated receptors (PPARs) and the role of PPARs in cytokine production in mouse bone marrow‐derived mast cells (mBMMCs). mBMMCs expressed PPARβ strongly and γ slightly, but not α. Activation of mBMMCs with antigen or calcium ionophore resulted in the increased expression of PPARγ mRNA specifically. 15‐Deoxy‐Δ12,14‐prostaglandin J2 (15d‐PGJ2) and troglitazone, all PPARγ ligands, attenuated the antigen‐induced cytokine production by mBMMCs. Carbaprostacyclin, a PPARβ ligand, also inhibited cytokine production, whereas PPARα ligands did not. These results suggest that PPARβ and γ might be included in the negative regulation of mast cell activation.