bluuebunny/crossref_metadata_2025 huggingface dataset:
Complete amino acid sequence of the nucleic acid‐binding protein of bovine leukemia virus
Authors: Copeland Terry D. , Morgan M.Alice , Oroszlan Stephen
Published: July 2002

The complete amino acid sequence of the nucleic acid‐binding protein p12 of bovine leukemia virus (BLV) has been determined. Peptides were generated by enzymatic digestion and formic acid cleavage, purified by reversed‐phase liquid chromatography and subjected to automated Edman degradation. BLV p12 is a proline‐rich linear polypeptide composed of 69 amino acids with M
r 7558. A comparison of the p12 structure to that of the avian and murine type C retroviral nucleic acid‐binding proteins shows significant homology only in the putative binding domain. This conserved region is duplicated in BLV p12 as in the avian homolog.
Polymorphism of human pituitary lutropin (LH)
Authors: Weise H.C. , Graesslin D. , Lichtenberg V. , Rinne G.
Published: July 2002

The complete microheterogeneous system of human pituitary lutropin was demonstrated by gel isoelectric focusing. In addition 7 LH isohormones could be isolated by preparative column focusing and characterized with respect to physicochemical, biological and immunological properties. The in vivo biopotencies ranged from 4.50–11.50 IU/mg, the in vitro bioactivity was from 1.20–10.10 IU/mg, and the immunological activity was from 3.10–7.55 IU/mg. The sialic acid content was found to be 1.8–3.2%. Treatment with neuraminidase resulted in a shift of all bands to the alkaline region, however the 7 LH forms were still present.
Effect of codon shortening and the antibiotics viomycin and sparsomycin upon the behaviour of bound aminoacyl‐tRNA
Authors: Hornig Horst , Woolley Paul , Lührmann Reinhard
Published: July 2002

70 S ribosomes were programmed with initiator tRNA and messenger oligonucleotides AUG(U)
n
and AUG(C)
n
, where n = 1, 2 or 3. The binding of the ternary complexes [Phe‐tRNA·EF‐Tu·GTP] and [Pro‐tRNA·EF‐Tu·GTP] to the programmed ribosomes was studied. If codon—anticodon interaction is restricted to only one basepair, the ternary complex leaves the ribosome before GTP hydrolysis. Two basepairs allow hydrolysis of GTP, but the aminoacyl‐tRNA dissociates and is recycled, resulting in wastage of GTP. Three basepairs result in apparently stable binding of aminoacyl‐tRNA to the ribosome. The antibiotic sparsomycin weakens the binding by an amount roughly equivalent to one messenger base, while viomycin has the reverse effect.
Genes for elongation factor EF‐1α in the brine shrimp Artemia
Authors: van Hemert Formijn J. , Lenstra Johannes A. , Möller Wim
Published: July 2002

A plasmid carrying a cDNA sequence coding for elongation factor EF‐1α from Artemia was used to probe blots of mRNA and chromosomal DNA from Artemia. A messenger length for EF‐1α corresponding to 1850 nucleotides was found. Southern blots pointed to a limited number (1–4) genes, coding for EF‐1α. From an Artemia gene library a recombinant phage was isolated, which contains genomic sequences of EF‐1α. S1‐nuclease mapping indicated the presence of intervening sequences within this cloned gene.
Effect of succinylacetone on heme and cytochrome P450 synthesis in hepatocyte culture
Authors: Giger Urs , Meyer Urs A.
Published: July 2002

The effects of succinylacetone, a tyrosine metabolite, on the hepatic biosynthesis of heme and cytochrome P450 were studied in primary culture of chick embryo hepatocytes. Succinylacetone potentiated the phenobarbital‐mediated induction of δ‐aminolevulinate synthase, strongly inhibited porphobilinogen synthase activity, reduced cellular heme concentration and impaired induction of cytochrome P450. Enhanced induction of δ‐aminolevulinate synthase and decreased cytochrome P450 induction may be explained by the succinylacetone‐mediated inhibition of porphobilinogen synthase and the subsequent depletion of intracellular heme, since these effects of succinylacetone were reversed by addition of heme. These results suggest clinical implications for patients with tyrosinemia, who accumulate succinylacetone.
The presence of LHRH‐like receptors in Dunning R3327H prostate tumors
Authors: Hierowski Marion T. , Perla Altamirano , Redding Tommie W. , Schally Andrew V.
Published: July 2002

Quantitative analyses of LH‐RH‐like membrane receptors were performed in five tumors from the transplantable Dunning R3372H rat prostatic adenocarcinoma. The binding of D‐Trp6‐LH‐RH, an agonist of LH‐RH, was observed in all 5 tumors. The antagonist [Ac‐Dp‐Cl‐Phe1,2,D‐Trp3,D‐Lys6,D‐Ala10]‐LH‐RH was bound to 4 tumors. The apparent equilibrium dissociation constant (K
d) for D‐Trp6‐LH‐RH receptor was from 2.6–3.9 × 10−10 M. The apparent equilibrium B
max values (maximum number of binding sites) were from 17.2–86.0 fmol/mg membrane protein for D‐Trp6‐LH‐RH receptor. The K
d for the antagonist was from 2.4–2.7 × 10−10 M and the B
max values were from 35.5–66.0 fmol/mg membrane protein. Similar binding studies performed in 6 normal rat prostates showed no binding capacities.
Fucosyl transferase activity and fucose incorporation in vivo as markers for subfractionating cucumber microsomes
Authors: Sturm A. , Kindl H.
Published: July 2002

Golgi apparatus only rarely seen in electromicroscopic examinations of post‐germinative storage tissues such as cucumber cotyledons could be detected by biochemical means. Enzyme activity transferring the L‐fucosyl moiety from GDP fucose to membrane‐glycoproteins was almost exclusively confined to golgi vesicles. This was demonstrated by comparing highly purified preparations of the ER, golgi and other organelles. The findings in vitro were supplemented by feeding experiments in vivo, establishing vesicles of golgi and plasma membrane as the by far dominating intracellular sites characterizable by fucose‐containing glycoproteins. Detailed analysis including the single peptides within the fractions revealed that the ER can be easily distinguished from golgi apparatus on the basis of glycoproteins.
Gossypol binds to a high‐affinity binding site on human serum albumin
Authors: Royer Robert E. , Vander Jagt David L.
Published: July 2002

The triterpene gossypol competes with bilirubin for a high‐affinity binding site on human serum albumin. Similar competition between bilirubin and gossypol occurs in the binding of these ligands to the glutathione S‐transferases from human liver and placenta. In each case, gossypol and bilirubin exhibit similar binding constants. The binding properties of gossypol may generally mimic those of bilirubin.
Processing of the propeptide form of rat renal γ‐glutamyltranspeptidase
Authors: Capraro Michael A. , Hughey Rebecca P.
Published: July 2002

The biosynthesis of rat renal γ‐glutamyltranspeptidase (EC 2.3.2.2) was studied by sodium dodecyl sulfate gel electrophoresis and fluorography of specific immunoprecipitates obtained at varying times' postinjection with [35S]methionine. At 20 min postinjection 3 endo‐β‐N‐acetylglucosaminidase H‐sensitive bands were observed representing the propeptide (M
r 75 000) large subunit (M
r 49 500) and small subunit (M
r 29 000) of transpeptidase. The alterations in M
r are consistent with removal of 6 N‐linked coreoligosaccharides from the propeptide; 4 from the large subunit and 2 from the small subunit. All 3 bands became more diffuse and less endoglycosidase H‐sensitive by 40 min and completely resistant by 60 min postinjection. At 20 h postinjection no propeptide remained. Thus, the primary propeptide cleavage reaction occurs prior to the loss of endoglycosidase H sensitivity while about 30% of the propeptide is processed along with the heterodimer and cleaved at a later time.
Comparative study of the fluid‐phase proteolytic cleavage of human complement subcomponents C4 and C2 by Cs and Cr2‐Cs2
Authors: Thielens Nicole M. , Villiers Christian L. , Villiers Marie-Bernadette , Colomb Maurice G.
Published: July 2002

The C3 convertase of the classical pathway of complement is composed of fragments C4b and C2a resulting from cleavage of C4 and C2 by activated C. The limited proteolysis of these two different substrates by the same protease, Cs, has been studied in the fluid phase using purified proteins. The turnover numbers of C2 and C4 cleavage by Cs were affected to different extents, depending on whether Cs was alone or associated with Cr or with monoclonal antibodies to Cs. The binding of C2 to C4 favours the proteolysis of C2 by Cs, as revealed by the use of I2‐treated C2.
Xenopus oocytes can synthesise but do not secrete the Z variant of human α1‐antitrypsin
Authors: Foreman R.C. , Judah J.D. , Colman A.
Published: July 2002

Human liver mRNA was prepared from a patient homozygous for α1‐antitrypsin deficiency (PiZZ) and from a normal subject (PiMM). Both liver RNAs were microinjected into Xenopus oocytes and α1‐antitrypsin identified by immunoprecipitation. The normal M variant of α1‐antitrypsin is synthesised and secreted by Xenopus oocytes, the abnormal Z protein is not secreted and an intracellular form accumulates in the oocytes. In the presence of tunicamycin an unglycosylated form of M α1‐antitrypsin appears in the incubation medium but no corresponding unglycosylated version of the Z protein is secreted.
Evidence for solvent‐induced conformational changes of the soluble Dunaliella chloroplast coupling factor 1 (CF1)
Authors: Selman-Reimer Susanne , Selman Bruce R.
Published: July 2002

The ATPase activity of the chloroplast coupling factor 1 (CF1) isolated from the green alga Dunaliella is completely latent. A brief heat treatment irreversibly induces a Ca2+ ‐dependent activity. The Ca2+ dependent ATPase activity can be reversibly inhibited by ethanol, which changes the divalent cation dependency from Ca2+ to Mg2+. Both the Ca2+ ‐dependent and Mg2+ ‐dependent ATPase activities of heat‐treated Dunaliella CF1 are inhibited by monospecific antisera directed against Chlamydomonas reinhardi CF1. However, when assayed under identical conditions, the Ca2+ ‐dependent ATPase activity is significantly more sensitive to inhibition by the antisera than is the Mg2+ ‐dependent activity. These data are interpreted as indicating that soluble Dunaliella CF1 can exist in a variety of conformations, at least one of which catalyzes a Ca2+ ‐dependent ATPase and two or more of which catalyze an Mg2+ ‐dependent ATPase.
The bond in the bacteriophage øX174 gene A protein‐DNA complex is a tyrosyl‐5'‐phosphate ester
Authors: van Mansfeld A.D.M. , van Teeffelen H.A.A.M. , Baas P.D. , Veeneman G.H. , van Boom J.H. , Jansz H.S.
Published: July 2002

Bacteriophage øX174
Gene A protein
DNA replication
Protein‐DNA complex
Synthetic oligonucleotide
Tyrosyl‐5'‐phosphate ester
Identification of a potential protease‐coding gene in the genomes of bovine leukemia and human T‐cell leukemia viruses
Authors: Sagata Noriyuki , Yasunaga Teruo , Ikawa Yoji
Published: July 2002

The genomes of bovine leukemia and human T‐cell leukemia viruses both contain an unidentified region between the gag and pol genes. These regions harbor an open reading frame that is in a different phase from the reading frames of the gag and pol genes. Based on the deduced amino acid sequences, we show here that they potentially encode a gag precursor‐cleaving protease, which is known to be fused to the gag and pol products of avian and murine retroviruses, respectively. This finding raises the interesting question of the expression and evolution of retroviral genes.
Symmetrical structure of the L7 protein dimer
Authors: Bushuev V.N. , Sepetov N.F. , Gudkov A.T.
Published: July 2002

500 MHz NMR studies of the L7 monomer (oxidized protein) and of the dimer (intact protein) show that its N‐terminal sequence takes part in the dimer formation. The identical environment of equivalent amino acid residues in different polypeptide chains in the dimer is evidence of the symmetrical structure of the L7 dimer.
The formation of products containing a conjugated tetraenoic system by pure reticulocyte lipoxygenase
Authors: Kühn H. , Wiesner R. , Stender H.
Published: July 2002

Lipoxygenase product
Reticulocyte lipoxygenase
Conjugated tetraene
Reaction mechanism
Isolation and characterization of proSS1–32, a peptide derived from the N‐terminal region of porcine preprosomatostatin
Authors: Schmidt Wolfgang E. , Mutt Viktor , Kratzin Hartmut , Carlquist Mats , Conlon J.Michael , Creutzfeldt Werner
Published: July 2002

A peptide derived from the N‐terminal region of porcine prosomatostatin, proSS1–32, has been purified to homogeneity from extracts of porcine upper intestine. Amino acid analysis revealed that the peptide consists of 32 residues. The complete primary structure was determined as: A P S D P R L R Q F L Q K S L A A A A G K Q E L A K Y F L A E L This sequence obviously comprises residues 1–32 of porcine prosomatostatin since it is identical to the corresponding sequence in human preprosomatostatin. The postulated cleavage site in porcine prosomatostatin is a Leu‐Leu bond between residues 32 and 33, thus confirming previous studies of the processing of the somatostatin precursor in the rat and transgenic mouse.
Islet‐activating protein discriminates between different inhibitors of thyroidal cyclic AMP system
Authors: Cochaux P. , Van Sande J. , Dumont J.E.
Published: July 2002

TSH‐induced cyclic AMP accumulation in dog thyroid slices is inhibited by norepinephrine through an α2‐adrenergic receptor, by carbamylcholine through a muscarinic cholinergic receptor, and by iodide. The inhibitory effect of iodide bears on the adenylate cyclase, but the exact mechanism of its action is still unknown. It is known that norepinephrine acts through activation of the N1 subunit of the cyclase, and that carbamylcholine, activating a phosphodiesterase, acts independently of Ni IAP (islet‐activating protein) has been shown to inactivate the Ni subunit. We studied the effect of IAP on the inhibitory action of iodide, norepinephrine, and carbamylcholine on cyclic AMP accumulation in TSH‐stimulated thyroid slices. Incubations of 15 or 22 h, and relatively high concentrations of IAP (250 ) were necessary to demonstrate an effect of IAP on thyroid slices. We report here that, under those conditions, inhibition of cyclic AMP accumulation by norepinephrine, but not by carbamylcholine or iodide, was suppressed by IAP treatment. These results indicate that the cyclase inhibition by iodide, is either not mediated by Ni or if mediated by Ni involves a mode of regulation of this coupling protein that is different from that by which the other 'Ni‐mediated' inhibitory hormones act on the enzyme.
Phosphoprotein phosphatase inhibitor‐2 is phosphorylated at both serine and threonine residues in mouse diaphragm
Authors: DePaoli-Roach Anna A. , Lee Fook-Thean
Published: July 2002

Phosphoprotein phosphatase inhibitor‐2 (i‐2) was rapidly isolated from mouse diaphragm extracts by the use of specific antibodies. The i‐2 so obtained was associated with ATP‐Mg and F
a
/GSK‐3 dependent phosphatase activity, supporting the idea that i‐2 is in fact a component of this form of phosphatase. Inhibitor‐2 isolated from diaphragms incubated with [32P]phosphate contained both phosphoserine (~ 90%) and phosphothreonine (~ 10%). Therefore, i‐2 is multiply phosphorylated in mouse diaphragm and the potential exists for control of the ATP‐Mg‐dependent phosphatase via multiple phosphorylation sites in vivo.
Intersubunit RNA‐protein contacts in pre‐ and post‐translocated E.coli ribosome
Authors: Abdurashidova G.G. , Tsvetkova E.A. , Chernyi A.A. , Kaminir L.B. , Budowsky E.I.
Published: July 2002

E. coli ribosome
Pre‐translocated state
Post‐translocated state
Intersubunit RNA‐protein contact
Ultraviolet‐induced cross‐link
Activation of protein kinase C in neutrophil cytoplasts
Authors: Gennaro Renato , Florio Chiara , Romeo Domenico
Published: July 2002

Treatment of enucleated, granule‐free neutrophil cytoplasts with the protein kinase C activator phorbol 12O‐myristate‐13‐acetate (PMA) causes an increased ‐32P‐incorporation into a variety of polypeptides. Permeabilization of PMA‐stimulated, 32P‐labeled cytoplasts by 0.01% digitonin fully releases the majority of these phosphorylated proteins. A statistically significant correlation is found between the extent of PMA‐induced activation of generation of Superoxide anion (O−
2) and the phosphorylation of a cytosolic polypeptide with an apparent M
r, of 46000, whose ‐32P‐labeling is also enhanced by the treatment of cytoplasts with 1‐oleyl‐2‐acetylglycerol, the Ca2+ ionophore ionomycin or latex beads. Furthermore, treatment of cytoplasts with the protein kinase C inhibitor trifluoperazine markedly inhibits the 32P‐labeling of proteins in the 40000 M
r range, including the 46 kDa polypeptide, and almost totally abolishes the activation of O−
2 production by PMA.
Structural homology between mouse liver and horse spleen ferritins
Authors: Rice D.W. , Dean B. , Smith J.M.A. , White J.L. , Ford G.C. , Harrison P.M.
Published: July 2002

Mouse liver ferritin is composed almost exclusively of polypeptide chains similar in molecular mass (22 kDa) to that characteristic of the major chain (H) found in heart ferritin isolated from human, horse or rat. In these species the predominant polypeptide of liver (L) is smaller (about 20 kDa). Here we show that mouse liver and horse spleen ferritins and apoferritins exhibit extensive structural homology as judged by the similarity in the diffraction patterns of their crystals grown from cadmium sulphate solutions. Implications of this finding are discussed.
Chemical characterization and opioid activity of an exorphin isolated from in vivo digests of casein
Authors: Meisel Hans
Published: July 2002

The in vivo formation of an opioid peptide (exorphin) derived from β‐casein has been proved for the first time. It was isolated from duodenal chyme of minipigs after feeding with the milk protein casein. The exorphin has been identified as a β‐casein fragment by end‐group determinations and qualitative amino acid analysis of the purified peptide. This peptide, named β‐casomorphin‐11, displayed substantial opioid activity in an opiate receptor‐binding assay.
Synthesis and maturation of recombinant human tumor necrosis factor in eukaryotic systems
Authors: Müller Rita , Marmenout Anne , Fiers Walter
Published: July 2002

The biosynthesis of human tumor necrosis factor (hTNF) was studied. The amino‐terminal extension of the hTNF precursor (26 kDa polypeptide) was not cleaved off in a cell‐free system supplemented with dog pancreas microsomes. Correct maturation of pre‐hTNF was nevertheless not restricted to the macrophage system: in the medium of a TNF‐producing, transformed CHO cell line, a (weak) ~20 kDa, an ~18.5 kDa (doublet) and a 17 kDa TNF polyeptide, the latter corresponding to mature hTNF, were revealed by specific immunoprecipitation. Similar results were obtained with Xenopus laevis oocytes, injected with hTNF mRNA, except that the 20 kDa band was lacking. The results are discussed in relation to the secretion mechanism of hTNF.
Protein that induces cell differentiation causes nicks in double‐stranded DNA
Authors: Weisinger Gary , Korn Alex P. , Sachs Leo
Published: July 2002

The growth and differentiation of myeloid hematopoietic cells are regulated by different macrophage and granulocyte inducing proteins, those that induce growth and others that induce differentiation. The proteins that induce differentiation but not those that induce growth bind to double‐stranded DNA. We now report that purified myeloid cell differentiation‐inducing protein causes single strand breaks (nicks) in double‐stranded DNA. This DNA nicking may initiate the changes in gene expression that are required for differentiation.
α‐Subunits of Ns are released from the plasma membrane following cholera toxin activation
Authors: Lynch Christopher J. , Morbach Laura , Blackmore Peter F. , Exton John H.
Published: July 2002

Cholera toxin (CT) and islet‐activating protein (IAP, a Bordetella pertussis toxin) were employed to test the hypothesis that GTP‐binding regulatory proteins are released from plasma membranes to a greater extent when ‘activated’ than when ‘inactivated’. CT, which activates Ns (the stimulatory GTP‐binding regulatory protein of the adenylate cyclase system), catalyzed the incorporation of radioactivity from [32P]NAD into 45 and 47.5 kDa peptides associated with rat liver plasma membranes. Following ADP‐ribosylation and centrifugation at 100000 × g for 1 h, approx. 30–35% of these CT‐labelled peptides were no longer associated with the plasma membranes, but were recovered from the supernatant fraction. IAP, which inactivates Ni (the inhibitory GTP‐binding regulatory protein of the adenylate cyclase system) catalyzed the incorporation of radioactivity from [32P]NAD into a 41 kDa peptide associated with the membranes. However, in contrast to the CT‐labelled peptides, typically less than 5% of the lAP‐labelled peptide was found in the 100000 × g supernatant fraction, but rather was almost exclusively associated with the membrane pellet. The data indicate that the α‐subunits of Ns are released from the plasma membrane following activation, and support the hypothesis that the βγ‐subunits act to anchor the α‐subunits to the plasma membrane. Cholera toxin
Islet‐activating protein
GTP‐binding protein
12‐O‐Tetradecanoyl phorbol 13‐acetate stimulates the myristylation of an ∼ 82 kDa protein in HL‐60 cells
Authors: Malvoisin Etienne , Wild Fabian , Zwingelstein Georges
Published: July 2002

We have studied protein acylation using [3H]myristate in the two leukemia cell lines HL‐60 and HL‐60 Blast II. The latter is a variant which does not differentiate after treatment with 12‐O‐tetradecanoyl phorbol 13‐acetate (TPA). The acylation profiles of the two cell lines as examined by SDS‐PAGE differed. TPA induced the myristylation of an ∼ 82 kDa protein in the sensitive cells, but not in the resistant cells. Myristic acid was shown to be covalently linked to these proteins. Analysis of the cell lipids labelled with [3H]myristate showed that in contrast to observations with the proteins, the changes induced by TPA were observed in both TPA‐sensitive and TPA‐resistant cells. We conclude that the induction of myristylation may be an important step in the mechanism of differentiation.
On the role of microsomal aldehyde dehydrogenase in metabolism of aldehydic products of lipid peroxidation
Authors: Antonenkov Vasily D. , Pirozhkov Sergej V. , Panchenko Leonid F.
Published: July 2002

To elucidate a possible role of membrane‐bound aldehyde dehydrogenase in the detoxication of aldehydic products of lipd peroxidation, the substrate specificity of the highly purified microsomal enzyme was investigated. The aldehyde dehydrogenase was active with different aliphatic aldehydes including 4‐hydroxyalkenals, but did not react with malonic dialdehyde. When Fe/ADP‐ascorbate‐induced lipid peroxidation of arachidonic acid was carried out in an in vitro system, the formation of products which react with microsomal aldehyde dehydrogenase was observed parallel with malonic dialdehyde accumulation.
Isolation and sequence analysis of a cDNA clone encoding the entire catalytic subunit of phosphorylase kinase
Authors: da Cruz e Silva Edgar F. , Cohen Patricia T.W.
Published: July 2002

Synthetic oligonucleotides have been used to isolate a 1.85 kb clone containing the full length coding sequence for the catalytic subunit of rabbit skeletal muscle phosphorylase kinase from a cDNA library constructed in λgt10. Sequence analysis of the clone predicted an amino acid sequence in agreement with a published primary structure. Inspection of the codon usage revealed a strong preference for G or C nucleotides at the third codon position as found for several other skeletal muscle proteins. This cDNA clone should facilitate identification of functional domains, including the calmodulin‐binding site, and investigation of the molecular basis of X‐linked phosphorylase kinase deficiencies.
Some characteristics of hormone (pheromone) processing enzymes in yeast
Authors: Wagner Jean-Claude , Escher Claudia , Wolf Dieter H.
Published: July 2002

The KEX2 gene‐encoded, membrane‐bound Ca2+‐dependent thiol endoproteinase, proteinase yscF, responsible for processing of the precursor protein of the sex pheromone α‐factor of the yeast Saccharomyces cerevisiae was solubilized from the membraneous fraction and partially purified. Gel filtration revealed an apparent Mr
of the native protein of around 150 000. Ca2+ concentration for half‐maximal activity was in the micromolar range and concentration of the substrate Cbz‐Tyr‐Lys‐Arg‐4‐nitroanilide for half‐maximal velocity was 0.05 mM. The enzyme is able to cleave basic amino acids from the carboxy‐terminus of peptides and probably involved in final maturation of the α‐factor peptides generated by proteinase yscF is membrane‐associated, active at neutral pH and responds strongly to the serine proteinase inhibitor phenylmethylsulfonyl fluoride as well as to ‐SH group blocking agents.
The interaction of amino‐deuteromethylated melittin with phospholipid membranes studied by deuterium NMR
Authors: Dempsey Christopher E. , Cryer Geoff D. , Watts Anthony
Published: July 2002

Melittin, deuteromethylated on each of the four amino groups (Gly‐1 Nα and Lys‐7, 21, and 23 Nε), was prepared by reductive methylation using deuteroformaldehyde and NaBD3CN. Deuterium NMR spectra were obtained for the modified peptide (D‐melittin) bound to phospholipid bilayers and erythrocyte ghosts. D‐Melittin at 4 mol% (peptide:lipid) induced reversible transitions between extended bilayers and micelles at the phase‐transition temperature in dimyristoylphosphatidylcholine (DMPC) bilayers. These changes in lipid morphology did not occur at 1 mol% D‐melittin: DMPC and the peptide was highly motionally restricted in gel‐phase lipid.
Mechanism of decreased insulinogenesis in manganese‐deficient rats Decreased insulin mRNA levels
Authors: Baly Deborah L. , Lee Inho , Doshi Rupa
Published: July 2002

Manganese‐deficient rats exhibited seven‐fold lower preproinsulin mRNA levels compared to control, as detected by dot blot hybridization of both total and poly(A)+ RNA using a preproinsulin cDNA probe. No differences in the size of the insulin mRNA were observed. Thus, decreased mRNA levels may be a major contributing factor to the decreased insulinogenesis observed in manganese‐deficient rats.
Genetic fusion of a non‐toxic heat‐stable enterotoxin‐related decapeptide antigen to cholera toxin B‐subunit
Authors: Sanchez J. , Svennerholm A.-M. , Holmgren J.
Published: July 2002

A decapeptide highly homologous to the STa Escherichia coli heat‐stable enterotoxin and to several other heat‐stable enterotoxins was fused genetically to the amino‐end of the B‐subunit of cholera toxin (CTB) and the hybrid protein gene expressed from a tacP overexpression system. The STa‐related decapeptide used, which was encoded by a synthetic oligodeoxynucleotide, contained a single mutation which substituted a disulfide‐linked cysteine by alanine. After its fusion to CTB the decapeptide was able to both react with and to give rise to anti‐STa antibodies. Expression of the decapeptide‐CTB hybrid by non‐toxigenic Vibrio cholerae resulted in its full secretion into the extracellular milieu from where it could then be readily purified by single‐step affinity chromatography using immobilized GM1 ganglioside. Bacteria producing this non‐toxic, immunogenic decapeptide‐CTB toxoid might be useful for the development of oral vaccines against diarrhea caused by E.coli and other bacteria producing immunologically related heat‐stable enterotoxins, and as a source of immunoreagents for methods used to diagnose disease caused by these bacteria.
Transformation of mouse mammary epithelial cells with the Ha‐ras but not with the neu oncogene results in a gene dosage‐dependent increase in transforming growth factor‐α production
Authors: Ciardiello Fortunato , Hynes Nancy , Kim Nancy , Valverius Eva M. , Lippman Marc E. , Salomon David S.
Published: July 2002

An enhanced expression of transforming growth factor‐α (TGFα) was demonstrated in two clones of NOG‐8 mouse mammary epithelial cells, NOG‐8 SR1 and NOG‐8 SR2, that have been transformed by a v‐Ha‐ras oncogene. The amount of TGFα production in NOG‐8 SR1 and NOG‐8 SR2 cells was dependent on the level of p21
ras
expression in these clones, which directly correlated with their cloning efficiency in soft agar. There was also a decrease in the number of epidermal growth factor (EGF) receptors on the NOG‐8 SR1 and NOG‐8 SR2 cells that is proportional to the amount of TGFα secreted. These effects were specific for ras because neu‐transformed NOG‐8 cells grew in soft agar at a comparable level to NOG‐8 SR2 cells yet did not show any increase in TGFα production or change in EGF receptor expression.
Conformational change in beef‐heart mitochondrial F1 ATPase to ATP synthesis mode induced by dimethylsulfoxide and ATP revealed by sulfhydryl group labeling
Authors: Beharry Seelochan , Bragg Philip D.
Published: July 2002

Treatment of beef‐heart mitochondrial F1 ATPase with 5,5′‐dithiobis(2‐nitrobenzoic acid) (DTNB) results in the incorporation of 1 mol DTNB/mol F1 without loss of ATPase activity. Incorporation is not prevented by ATP. Labeling occurs predominantly on an α‐subunit, but also with a significant degree of modification of γ‐ and ε‐subunits. It is suggested that the modified sulfhydryl groups of the α‐, γ‐ and ε‐subunits are in proximity so that only one can be modified by the reagent. Guanidine hydrochloride (0.3 M) dissociates F1 into its subunits. Eight sulfhydryl groups/mol F1 can be modified under these conditions. Guanidine hydrochloride does not cause dissociation of F1 in the presence of 30% (v/v) dimethylsulfoxide (Me2SO) and 2 mM ATP. Under these conditions a second molecule of DTNB is incorporated into F1 with nearly equal modification of the ε‐subunit and an α‐subunit. It is proposed that Me2SO and ATP induce a more stable conformation of F1, which is resistant to dissociation by guanidine hydrochloride, but in which the site of reaction with DTNB is made more accessible by the guanidine hydrochloride to permit the simultaneous modification of an α‐subunit and the ε‐subunit. Ths conformation is probably that which occurs during ATP synthesis by F1 in the presence of Me2SO.
Orientation dependence of the EPR signal from the reduced iron‐quinone complex in a single crystal of the reaction center protein from Rhodopseudomonas viridis
Authors: Gast P. , Gottschalk A. , Norris J.R. , Closs G.L.
Published: July 2002

In the light‐induced charge separation that takes place in the bacterial photosynthetic reaction center protein a quinone molecule (Q) is reduced. The EPR spectrum of this reduced quinone is strongly broadened which is assumed to arise from interaction with the high spin (S=2) Fe2+, which is located near Q. Computer simulations of this EPR spectrum have been somewhat limited by the lack of structure in the EPR spectrum. The availability of single crystals of the reaction center protein offers the possibility to analyze the spectrum of the reduced iron‐quinone complex (FeQ−) in much greater detail than ever before. We have therefore measured the EPR spectrum of FeQ− using single crystals from the reaction center protein from the photosynthetic bacterium Rhodopseudomonas viridis. Transitions were obtained in the g value range of 1.7–1.9 for both crystal orientations, i.e. for rotation along and rotation perpendicular to the crystal's long axis. Strong line broadening was observed at many orientations, which could indicate a heterogeneity in the zerofield parameters of Fe2+.
Substrate structural requirements of Schizosaccharomyces pombe RNase P
Authors: Drainas Denis , Zimmerly Steven , Willis Ian , Söll Dieter
Published: July 2002

RNase P from Schizosaccharomyces pombe has been purified over 2000‐fold. The apparent K
m for two S. pombe tRNA precursors derived from the supS1 and sup3‐e tRNASer genes is 20 nM; the apparent V
max is 2.5 nM/min (supS1) and 1.1 nM/min (sup3‐e). Processing studies with precursors of other mutants show that the structures of the acceptor stem and anticodon/intron loop of tRNA are crucial for S. pombe RNase P action.
Involvement of cytochrome bc1 complex in the electron transfer pathway for N2O reduction in a photodenitrifier, Rhodobacter sphaeroides f. s. denitrificans
Authors: Itoh Masahiko , Matsuura Katsumi , Satoh Toshio
Published: July 2002

The electron transfer pathway for N2O reduction in Rhodobacter sphaeroides f. s. denitrificans was studied. Myxothiazol and antimycin showed inhibiting effects on the consumption of N2O in whole cells grown under photo‐denitrifying conditions. This indicated the involvement of the cytochrome bc
1 complex in the process. Reduced redox dyes that donate electrons directly to cytochrome c
2 overcame this inhibition. Similar results were obtained with Rhodobacter capsulatus, in contrast to earlier studies by McEwan et al. [(1985) J. Bacteriol. 164, 823–830] that suggested the lack of involvement of the cytochrome bc
1 complex in this process. Effects of myxothiazol and antimycin were also observed on the N2O‐induced oxidation of cytochromes c
1 and c
2 and on the simultaneous generation of membrane potential measured by the carotenoid band shift. Thus, reducing equivalents for N2O reduction from substrates pass through the cytochrome bc
1 complex coupling to the generation of an electrochemical gradient of protons.
A rapid burst preceding the steady‐state rate of H+‐transhydrogenase during illumination of chromatophores of Rhodobacter capsulatus
Authors: Palmer Tracy , Jackson J.Baz
Published: July 2002

At the onset of illumination of chromatophores there was a burst (t
approx. 5 ms) in the rate of the H+‐transhydrogenase reaction before establishment of the steady‐state rate. The burst was suppressed at high pH with a pK
a, of approx. 8.5. The burst and the steady‐state rate were inhibited by either (i) a combination of myxothiazol and carbonylcyanide‐p‐trifluoromethoxyphenylhydrazone, or (ii) NAD+ or (iii) dicyclohexylcarbodiimide. The results support a model in which substrate binding to H+‐transhydrogenase is relatively fast. A subsequent slow step is accelerated by the protonmotive force and a third step, possibly product release, is rate‐limiting in steady‐state turnover during illumination.
Two phosphorylation sites on eIF‐2α
Authors: Kramer Gisela
Published: July 2002

Protein synthesis in mammalian cells can be regulated through phosphorylation/dephosphorylation of the α subunit of initiation factor 2, eIF‐2. Two specific kinases have been identified that apparently phosphorylate the same site(s). Controversy exists as to whether serine‐48 is a phosphorylation site in addition to serine‐51. A recent publication is discussed that, in this author's view, answers the question of the phosphorylation sites. It is suggested that phosphorylation procedes sequentially with serine‐51 being the first and serine‐48 the second phosphorylation site. Phosphorylation of both sites is required for inhibition of protein synthesis.
A protein with sequence identity to Skp (FirA) supports protein translocation into plasma membrane vesicles of Escherichia coli
Authors: Thome Birgit M. , Hoffschulte Hedda K. , Schiltz Emile , Müller Matthias
Published: July 2002

We have purified to homogeneity a 15 kDa‐protein from a ribosomal salt extract of Escherichia coli that compensates in vitro a defect of SecA but not of SecB. Removal of this protein from a cell‐free transcription/translation system impairs translocation into plasma membrane vesicles of the precursors of LamB and to a lesser degree also of OmpA. These results suggest a role of the 15 kDa‐protein in bacterial protein export. The NH2‐terminal 35 amino acids were found to be identical to those of the skp (firA) gene product, to which several putative functions have previously been attributed.
Characterization of muscarinic acetylcholine receptors on the rat pancreatic gastrin‐producing cell line B6 RIN
Authors: Abello Jacques , Roche Colette , Cuber Jean-Claude , Bernard Christine , Philippe Jacques , Chayvialle Jean-Alain
Published: July 2002

The mechanisms of cholinergic stimulation of gastrin cells were studied in the rat pancreatic cell line B6 RIN. Carbachol induced an increase in intracellular Ca2+ and stimulated gastrin release in a dose‐dependent manner over the range 10−5‐10−3 M. These effects were completely abolished by atropine, suggesting the implication of muscarinic cholinergic receptors. The binding properties of these receptors were investigated. [N‐Methyl‐3H]scopolamine ([3h]nms) binding on cell homogenates was time‐dependent, saturable and consistent with a single high‐affinity binding class (K
d = 39.5 pM, and B
max = 7.9 DNA). Carbachol competitively inhibited [3H]NMS binding. The potency of inhibition of [3H]NMS binding by subtype selective antagonists was hexahydrodifenidol > pirenzepine > AF‐DX 116. These results suggest the M3, muscarinic receptors may be involved in the carbachol‐induced gastrin release from B6 RIN cells.
Interaction of benzoquinones with mitochondria interferes with oxidative phosphorylation characteristics
Authors: Makawiti Dominic W. , Konji Victor N. , Olowookere Julius O.
Published: July 2002

Studies with four benzoquinones, viz. juglone, embelin, maesaquinone and maesanin, on rat liver mitochondria oxidative phosphorylation have been carried out. Three of the benzoquinones are uncouplers in the order juglone > maesoquinone > embelin, while maesanin is an inhibitor of electron transport and oxidative phosphorylation.
High‐velocity mechanical DNA transfer of the chloramphenicolacetyl transferase gene into rodent liver, kidney and mammary gland cells in organ explants and in vivo
Authors: Zelenin Alexander V. , Alimov Andrey A. , Titomirov Alexander V. , Kazansky Alexander V. , Gorodetsky Stanislav I. , Kolesnikov Viktor A.
Published: July 2002

Mouse and rat liver, kidney and mammary gland explants were bombarded with high‐velocity microprojectiles carrying a chloramphenicolacetyl transferase gene under different promoters (pTAT‐cat, pχ‐Cusein‐cat, pβ‐Cusein‐cat). The expression of a CAT gene was revealed in all organ explants 24 h after transfection. The most pronounced expression was found when a TAT‐CAT construction was used. In experiments in vivo rat liver was bombarded in situ with microprojectiles carrying a pTAT‐cat DNA. A marked activity of the CAT gene was detected 24 h after the bombardment.
Characterization of PC2, a mammalian Kex2 homologue, following expression of the cDNA in microinjected Xenopus oocytes
Authors: Shennan Kathleen I.J. , Smeekens Steven P. , Steiner Donald F. , Docherty Kevin
Published: July 2002

A human insulinoma cDNA (PC2) that encodes a protein homologous to the Kex2/subtilisin‐like proteinases has recently been described [1990, J. Biol. Chem. 265, 2997–3000]. In order to characterise the associated proteinase activity, mRNA encoding PC2 was synthesised in vitro and micro‐injected into Xenopus oocytes. The proteinase activity released into the media from oocytes microinjected with PC2 mRNA was assayed using small peptide fluorogenic substrates. Boc.Gln.Arg.Arg aminomethyl coumarin was hydrolysed in a Ca2+‐dependent manner, but substrate analogues bearing a single basic aminoacid were not. The substrate specificity, inhibitor profile, and pH optimum of 5.5 were compatible with an involvement of PC2 in prohormone processing in mammalian cells.
Heterologous in vitro transcription from two archaebacterial promoters
Authors: Hüdepohl Uwe , Gropp Felix , Horne Mary , Zillig Wolfram
Published: July 2002

A cell‐free extract of Sulfolobus shibatae is able to specifically initiate transcription in vitro at the promoter of the plasmid‐encoded gene for the major gas vesicle protein of Halobacterium halobium and at the promoter for the transcript T4 of the temperate H. halobium phage ΦH. The corresponding promoter from the virulent phage mutant ΦHL1 yields enhanced transcription in the heterologous system, in agreement with strongly increased in vivo expression.
Functional relationships between cyclodextrin glucanotransferase from an alkalophilic Bacillus and α‐amylases Site‐directed mutagenesis of the conserved two Asp and one Glu residues
Authors: Nakamura Akira , Haga Keiko , Ogawa Shigeyuki , Kuwano Kayoko , Kimura Kenji , Yamane Kunio
Published: July 2002

Comparison of the amino acid sequences of cyclodextrin glucanotransferases (CGTases) with those of α‐amylases revealed that two Asp and one Glu residues, which are considered to be the catalytic residues in α‐amylases, were also conserved in CGTases. To analyze the function of the three conserved amino acid residues in CGTases, site‐directed mutagenesis was carried out. The three mutant CGTases, in which Asp229, Glu257 and Asp328 were individually replaced by Asn or Gln, completely lost both their starch‐degrading and β‐cyclodextrin‐forming activities, whereas another mutant CGTase, in which Glu264 replaced by Gln, retained these activities. The three inactive enzymes retained the ability to be bound to starch. These results suggest that Asp229, Glu257 and Asp328 play an important role in the enzymatic reaction catalyzed by CGTase and that a similar catalytic mechanism is present in both CGTases and α‐amylases.
Crossing three membranes Channel formation by aerolysin
Authors: Buckley J.Thomas
Published: July 2002

Aerolysin is a channel‐forming toxin responsible for the pathogenicity of Aeromonas hydrophila. It crosses the inner and outer membranes of the bacteria in separate steps and is released as a 52‐kDa inactive protoxin which is activated by proteolytic removal of approximately 40 amino acids from the C terminus. The toxin binds to the erythrocyte transmembrane protein glycophorin and oligomerizes before inserting into the membrane, producing a voltage gated, anion selective channel about 1 nm in diameter. Remarkably, proaerolysin appears to be dimeric, whereas the oligomeris a heptamer. Using chemical modification and site‐directed mutagenesis, we have identified some of the regions of the molecule which appear to be involved in secretion and in channel formation.
Platelet‐derived endothelial cell growth factor Pharmacokinetics, organ distribution and degradation after intravenous administration in rats
Authors: Waltenberger Johannes , Usuki Kensuke , Fellström Bengt , Funa Keiko , Heldin Carl-Henrik
Published: July 2002

Platelet‐derived endothelial cell growth factor (PD‐ECGF) stimulates chemotaxis of endothelial cells in vitro and has angiogenic activity in vivo. Recently PD‐ECGF was shown to have thymidine phosphorylase activity. In order to study possible therapeutic applications of PD‐ECGF we used a rat model to determine its pharmacokinetics and tissue distribution after intravenous injection. [125I]PD‐ECGF disappeared from the plasma in a biphasic manner, with estimated distribution and elimination half‐lives of 17 min and 7 h, respectively. PD‐ECGF was metabolized in the liver, excreted via the bile, and not accumulated in any organ system. The stability and long half‐life in the circulation, together with the specificity for endothelial cells, suggest that PD‐ECGF may be useful as a therapeutic agent to stimulate re‐endothelialization in vivo, or, in view of its thymidine phosphorylase activity, in chemotherapy, by decreasing the pool of available thymidine.
Molecular cloning of the human α2(IX) collagen cDNA and assignment of the human COL9A2 gene to chromosome 1
Authors: Perälä Merja , Hänninen Marketta , Hästbacka Johanna , Elima Kati , Vuorio Eero
Published: July 2002

Type IX collagen, a heterotrimer of αl(IX), α2(IX) and α3(IX) chains, is a cartilage‐specific fibril‐associated collagen. In the process of characterizing genomic clones for the mouse α2(IX) collagen gene four pairs of oligonucleotide primers designed for amplification of murine exon sequences were also utilized to construct cDNA clones for human α2(IX) collagen spanning > 90% of the coding region. The amino acid and nucleotide sequence identities between human and chick are 78% and 71%, respectively. Localization of the COL9A2 gene to human chromosome 1 was subsequently performed using a panel of DNAs from human/rodent somatic cell hybrids.
Requirement of phosphatidylglycerol for flagellation of Escherichia coli
Authors: Tomura Arihiro , Ishikawa Toyoko , Sagara Yasuhiro , Miki Takeyoshi , Sekimizu Kazuhisa
Published: July 2002

We report that phosphatidylglycerol is required for flagellation of Escherichia coli. Cells carrying the pgsA3 mutation did not form swarm rings in semisolid agar. P1 transduction experiments revealed that the potential for phosphatidylglycerol synthesis and for the formation of swarm rings was co‐transducible. The pgsA3 mutant transformed with the wild type pgsA
+ gene cloned into the R‐plasmid vector had the potential for both phosphatidylglycerol synthesis and cell motility. Electronmicroscopic and SDS‐PAGE analyses showed that the pgsA3 mutation causes the lack of flagellation.
The 100 kDa F‐actin capping protein of Dictyostelium amoebae is a villin prototype (‘protovillin’)
Authors: Hofmann A. , Noegel A.A. , Bomblies L. , Lottspeich F. , Schleicher M.
Published: July 2002

The 100 kDa actin‐binding protein from Dictyostelium amoebae is an F‐actin capping protein that displays neither severing nor crosslinking nor nucleating activities [Hofmann et al. (1992) Cell Motil. Cytoskel. 23,133‐144]. Cloning and sequencing of the gene revealed that the protein is highly homologous to vertebrate villin, a unique component of brush border microvilli and contains six domains fused to a villin‐like headpiece domain via a threonine/proline rich neck region. The functional differences and similarities between the 100 kDa protein and villin are reflected in the amino acid sequences. We draw from the data the following conclusions, (i) The presence of a six domain protein in Dictyostelium suggests that in contrast to the current view gene duplications must have happened before Dictyostelium branched off during evolution, (ii) The villin‐like molecule in Dictyostelium appears to be a premature villin (‘protovillin’) which is able to cap actin filaments but still lacks the other villin‐type actin‐binding activities. This renders capping of actin filaments as the evolutionarily oldest function of an F‐actin binding protein.
The nuclear pore complex
Authors: Hurt Eduard C.
Published: July 2002

Over the past years, significant progress has been made both in the analysis of the structural and molecular organization of the nuclear pore complex (NPC) and the mechanism of nuclear transport. In this minireview, I will focus on some of the recent developments in this field. Structural studies employing high resolution EM have revealed a detailed view of the three‐dimensional organization of the NPC. In addition, an isolation procedure which yields highly enriched NPCs from yeast has given insight into the molecular complexity of the NPC organization. By biochemical, immunological and genetic approaches, a series of novel pore proteins were identified. Exploiting yeast as a genetic system, several mutants defective in nuclear import of proteins and export of RNA were selected. By in vitro nuclear transport assays, soluble cytoplasmic factors including NLS (nuclear localization sequence) binding proteins and heat shock proteins required for nuclear accumulation were found. The aim of the future research must be to put these various components of the NPC and nuclear transport machinery in a topological and functional context.