bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid method for construction of yeast artificial chromosome human DNA libraries involving the trapping of cells in agarose films
Authors: Kai Tatsuo , Song Jac-chan , Tashiro Hiroyuki , Saka Fujiko , Ozawa Kazuo , Miwa Tokiko , Kitabayashi Issay , Soeda Ei-ichi , Yokoyama Kazushige
Published: July 2002

A simple method for molecular cloning of fragments of more than one hundred kilobase pairs of exogenous DNA, by the encapsulation of cell in agarose beads, was reported previously for the construction of a human genomic DNA library in a yeast artificial chromosome (YAC) vector in situ YAC construction [1]. The efficiency of this procedure is impaired by the step in which agarose beads that contain human DNA fragments are melted before transformation. The incomplete solubility of the ligated human DNA fragment. YAC vector often results in lower than desirable frequencies of transformation. To overcome this problem we have developed a new improved method that involves use of an agarose film. The technical manipulations involved in the construction of clones of very large segments of human DNA are discussed.
Comparison of the γ‐crystallins isolated from eye lenses of shark and carp Unique secondary and tertiary structure of shark γ‐crystallin
Authors: Chiou S.-H. , Chen S.-W. , Itoh T. , Kaji H. , Samejima T.
Published: July 2002

γ‐Crystallin isolated from the shark of cartilaginous fishes was compared with the cognate γ‐crystallin from the carp of bony fishes. Distinct differences in amino acid compositions, primary, secondary and tertiary structures were found. The most salient features of shark γ‐crystallin lie in the fact that this crystallin possessed a significant α‐helical structure in the peptide backbone as revealed by circular dichroism study, in contrast to those orthologous γ‐crystallins from other vertebrate species including bony fishes which all show a predominant β‐sheet secondary structure. The tertiary structure as reflected in the intrinsic microenvironments of various aromatic amino acids in the native crystallins also shows unambiguous differences between these two classes of γ‐crystallins. N‐Terminal sequence analysis corroborates the structural differences between shark and carp γ‐crystallins. γ‐Crystallin from the more primitive shark seems to be more in line with the main evolutionary phylogeny leading to the modern mammalian γ‐crystallin.
Na,K‐ATPase labelled with 5‐iodoacetamidofluorescein: E2‐E1 conformational transition induced by different nucleotides
Authors: Lopina O.D. , Pindel E.V. , Boldyrev A.A.
Published: July 2002

A conformational transition between E2 and E1 forms of Na, K‐ATPase induced by different nucleotides has been studied under steady state conditions using the enzyme labelled with 5‐iodoacetamidofluorescein. In the presence of K+ the plot of fluorescence as a function of [ATP], [ADP] or [CTP] (in a range of 5 μM‐12 mM) is a biphasic one. A similar dependence for AMP, ITP, GTP and UTP demonstrates a hyperbolic behaviour. The data suggest that the shift in the equilibrium between E2 and E1 forms of Na,K‐ATPase towards the E1 conformation is induced by ATP binding both with high and low affinity sites. Two structural features of ATP are apparently important for its interaction with more than one type of ATP binding sites or for providing for E2‐E1 transition induced by this interaction: (i) β‐phosphate group in the terminal part of the molecule, (ii) unprotonated N1 and/or NH2‐group in the 6th position of the purine base.
Regulation of ATP hydrolysis in liver mitochondria from ground squirrel
Authors: Bronnikov G.E. , Vinogradova S.O. , Chernyak B.V.
Published: July 2002

The uncoupler‐induced inactivation of H+‐ATPase in liver mitochondria from ground squirrel has been studied. The dependence of this process on ΔgmH+, pH and ATP indicates that it is caused by the protein inhibitor. This conclusion is also supported by the protective effect of Zn2+ and Cu2+. The inactivation can be induced by Ca2+ at low concentrations in the presence of phosphate. It is shown that the protein inhibitor inactivates ATPase almost completely under optimal conditions while its effect in mice or rat liver mitochondria does not exceed 30%. The potential efficiency of the inhibitor's action does not depend on either the season or the state of animals (hibernating or active). At the same time, the sensitivity of this system to Ca2+ is significantly lower in active (summer) animals.
Nature of primary product(s) of D‐glucose 6‐phosphate dehydrogenase reaction 13C and 31P NMR study
Authors: Jarori Gotam K. , Maitra Pabitra K.
Published: July 2002

Glucose 6‐phosphate dehydrogenase catalyzes the oxidation of glucose 6‐phosphate, resulting in the formation of 6‐phosphogluconolactone, As this compound is unstable, it has not been characterized directly. NMR provides a way to directly monitor all components of a reaction and study their structure. Here we report some results on the glucose 6‐phosphate dehydrogenase reaction using 31P and 13C‐NMR. Our results indicate that two different lactones, namely γ (1=4 and δ (1=5) 6‐phosphogluconolactones, are formed as products in this reaction. This is in contrast to an earlier suggestion that glucose 6‐phosphate dehydrogenase produces only the δ‐lactone. On the basis of these results, a new mechanisms for dehydrogenation of the sugar phosphate is proposed.
Interaction of cytochrome P‐450scc with cytochrome bs
Authors: Usanov Sergei A. , Chashchin Vadim L.
Published: July 2002

Spectrophotometric, affinity chromatography and cross‐linking experiments provided evidence that cytochroms P‐450scc from bovine adrenocortical mitochondria forms a light complex with cytochrome, bs
from rabbit liver microsomes. In the reconstituted system cholesterol side chain activity of cytochrome P‐450scc was enhaced by the addition of cytochrome bs
.
Purification of SecE and reconstitution of SecE‐dependent protein translocation activity
Authors: Tokuda Hajime , Akimaru Jiro , Matsuyama Shin-ichi , Nishiyama Ken-ichi , Mizushima Shoji
Published: July 2002

SecE was solubilized from SecE‐overproducing E. coli cells and purified through ion exchange and size exclusion chromatographies. When the solubilized membrane containing overproduced amounts of SecY and SecE was fractionated by means of size exclusion chromatography, the two proteins were eluted in different fractions with slight overlapping. Proteoliposomes active in protein translocation were reconstituted from these fractions only when both SecE and SecY were present. When reconstituted was carried out with the purified SecE and fractions containing SecY but only a small amount of SecE, the resultant proteoliposomes exhibited appreciable translocation activity, indicating that SecE is essential for protein translocation. The translocation activity of proteoliposomes was proportional to the amount of purified SecE used for reconstitution. SecE dependent protein translocation absolutely required ATP and SecA.
Anaerobically induced expression of the nitrite reductase cytochrome c‐551 operon from Pseudomonas aeruginosa
Authors: Arai Hiroyuki , Igarashi Yasuo , Kodama Tohru
Published: July 2002

The nitrite reductase gene (denA) and the cytochrome c‐551 gene (denB) are located only 50 bp apart from each other in the Pseudomonas aeruginosa chromosome. We report evidence that these two genes are co‐transcribed as an operon only under anaerobic (denitrifying) conditions. The nucleotide sequence of the promoter (regulatory) region of the operon is highly AT‐rich and contains a sequence closely resembling the consensus FNR binding site in E. coli.
Tyrosine phosphorylation of human urokinase‐ type plasminogen activator
Authors: Barlati Sergio , Paracini Francesco , Belloti Daniela , DePetro Giuseppina
Published: July 2002

Immunoblatting analysis of purified human urokinase plasminogen activator (u‐PA), gives a positive signal when reacted with anti‐phosphotyrosine monoclonal antibodies (MoAb anti P‐Tyr); competition with α‐phospha‐DL‐tyrosine (P‐Tyr) but not α‐phosphia‐DL‐threonine or serine (P‐Treo, P‐Ser) completely suppresses this signal. Either the 55 kDa u‐PA form and the lower M
r form (33 kDa) derived from the 55 kDa u‐PA are Tyrphosphorylated also the u‐PA secreted in the culture media of human fibrosarcoma cells (HT‐1080) is phosphorylated in tyrosine as well as u‐PA present in tissue extracts of tumors induced in nude mice by HT‐1080 cells. These data show that urine purified human u‐PA and u‐PA produced by humans fibrosarcoma cells, in vitro and in vivo, are phosphorylated in tyrosine; furthermore our data show that u‐PA is the major Tyr‐phosphorylated protein present in these human tumor cells.
A new class of potent thrombin inhibitors that incorporates a scissile pseudopeptide bond
Authors: DiMaio John , Ni Feng , Gibbs Bernard , Konishi Yasuo
Published: July 2002

A synthetic hirudin peptide analog corresponding to N′‐acetyl [D‐Phe45, ArgΨ(COCH2)47, Gly48]desulfo hirudin55–65 (P79) was synthesized. Comparative kinetic studies showed that while recombinant hirudin (HV2) is a slow‐tight binding inhibitor, P79 behaves as a classical competitive inhibitor of human α‐thrombin (K
i=3.7±0.3 × 10−10 M) and bovine α‐thrombin (1.8±0.7 × 10−9 M). P79 showed saturable inhibition of plasma APTT. The P1 subsite of P79 is isosteric with the glycine residue of the natural thrombin substrate fibrinogen, but is proteolytically stable due to the incorporation of a ketomethylene pseudopeptide bond. The model active site‐directed tripeptide [D‐Phe‐Pro‐ArgΨ(COCH2)CH3COOCH3, P79L] corresponding to the amino terminal of P79 also binds competitively to the active site of α‐thrombin and inhibited the proteolysis of a tripeptidyl substrate with a K
1=17.9±2.1 μM (human) and 10.3±3.6 μM (bovine) α‐thrombin. NMR experiments indicated that P79L and the corresponding amino terminal residues of P79 occcupy a mutually exclusive binding site on bovine α‐thrombin while the carboxyl terminal tail of the latter adopts a similar bound conformation as the fragment hirudin??? which is known to interact with the ‘anion’ exosite. Taken together these results provide conclusive evidence that the high antithrombin activity of N???‐acetyl[D‐Phe45, ArgΨ(COCH2)47, Gly48]desulfo hirudin45–65 stems from the concurrent interaction with the catalytic site and the putative ‘anion’ exosite through its respective NH2‐ and COOH‐terminal recognition sites.
Extracellular ATP itself elicits superoxide generation in guinea pig peritoneal macrophages
Authors: Nakanishi Masayuki , Takihara Hironori , Minoru Yoshida , Yagawa Katsuro
Published: July 2002

Extracellular ATP itself elicited the generation of superoxide (O−
2) in guinea pig peritoneal macrophages associated with an increase in cytosolic calcium ([Ca2+]). The ATP‐Induced O−
2 generation was completely inhibited by pretreatment with pertussis toxin (PT) accompanied by the suppression of (Ca2+), mobilization. Pre‐exposure to a small amount of phorbol myristate acetate (PMA) primed the ATP‐induced generation of O−
2 without it change of (Ca2+). The results suggest that ATP‐induced O−
2 generation is mediated by (Ca2+), mobilization and by PT‐sensitive G protein.
19F NMR evidence for interactions between the c‐AMP binding sites on the c‐AMP receptor protein from E. coli
Authors: Hinds Mark G. , King Rodney W. , Feeney James
Published: July 2002

The 19F NMR spectra of 3‐fluorotyrosine containing c‐AMP receptor protein (CRP) from E. coli have been recorded in the presence of increasing amounts of c‐AMP. One of the signals (from Tyr B) shifts upfield by 0.6 ppm in the presence of excess c‐AMP and shows both slow and fast exchange behaviour during the titration. This is evidence for interactions between the two c‐AMP binding sites on the CRP dimer leading to different dissociation rate constants (≤ 75 s−1; ≥ 350 s−1) for complexes containing one and two c‐AMP molecules.
Cloning of the protein D2 gene of Pseudomonas aeruginosa and its functional expression in the imipenem‐resistant host
Authors: Yoneyama Hiroshi , Nakac Taiji
Published: July 2002

Protein D2 forms the water‐filled pore across the outer membrane of Pseudomonas aeruginosa and allows the penetration of imipenem. We cloned the protein D2 gene by the antibody screening technique. When the imipenem‐resistant mutant lacking protein D2 harbored the plasmid with the cloned D2 gene, the mutant overproduced protein D2 in the outer membrane. These transformants exhibited fully‐restored imipenem susceptibility. The results prove unequivocally that protein D2 forms the imipenem‐permeable pore in the P. aeruginosa outer membrane.
Energetic cooperation via ion‐permeable junctions in mixed animal cell cultures
Authors: Aslanidi K.B. , Boitsova L.Ju. , Chailakhyan L.M. , Kublik L.N. , Marachova I.I. , Potapova T.V. , Vinogradova T.A.
Published: July 2002

Low ouabain concentration (1 × 10−4 M) is shown to decrease intracellular K+ (K+
in) and to increase intracellular Na+ (Na+
in) in human fibroblast cell cultures. The same ouabain concentration was without effect upon K+
in ad Na+
in in rodent cultures such as BHK‐21, mouse fibroblasts and rat glyoma C6 cells. K+
in and Na+
in in the mixed cultures of human and BHK‐21 fibroblasts or human and mouse fibroblasts were found to be resistant to 1 × 10−4 M ouabain whereas that of the mixtures of human and rat glyoma C6 cells proved to be ouabain‐sensitive. The gap‐junction‐mediated dye transfer was revealed between human and BHK‐21 cells. Such an effect was very small in the human‐C6 cell mixed culture. It is concluded that cells with active ion pumps can support the maintenance of K+ and Na+ gradients in cells with inactive pumps, provided that effective ion transport via gap junctions takes place.
F0F1‐ATPase from Vibrio alginolyticus Subunit composition and proton pumping activity
Authors: Dmitriev O.Yu. , Krasnoselskaya I.A. , Papa S. , Skulachev V.P.
Published: July 2002

An F0F1‐ATPase was isolated from the membranes of the marine bacterium Vibrio alginolyticus. Homology between the subunits of the F0‐complexes from E. coli and V. alginolyticus was found using antibodies against subunits a, b and c of the E. coli F0F1‐ATPase. The F0F1‐complex from V. alginolyticus was reconstituted into proteoliposomes, which were competent in ATP‐dependent proton uptake. This process was inhibited by triphenyltin, DCCD, and venturicidin. Na+ did not affect proton translocation.
Interaction of HIV‐1 with susceptible lymphoblastoid cells 1H NMR studies
Authors: Luciani A.M. , Rosi A. , Maggiorella M.T. , Federico M. , Sulli N. , Verani P. , Rossi G.B. , Viti V. , Guidoni L.
Published: July 2002

Different strains of HIV susceptible lymphoblastoid cells have been infected by HIV‐1 and examined by means of 1H NMR spectroscopy at different times after infection, taking advantage of the presence of high resolution lipid signals from the plasma membrane of tumor cells. A transient decrease in intensity of fatty acid signals, originated by changes in membrane structure, has been observed early after viral infection. Marked alterations in membrane‐dependent steps of phospholipid synthesis can also be inferred by the observed transient depression in peaks from choline‐based metabolites. Spectral modifications deriving from charges in lipid metabolism are also produced both in infected cells a few days after infection and in permanently infected cells. 1H NMR can, therefore, monitor structural and metabolic effects induced by HIV infection.
Radiation inactivation of estrogen receptor in intact human breast cancer cells (MCF‐7) Lack of energy transfer to the estradiol binding domain
Authors: Toulas Christine , Beauregard Guy , Delassus Felicité , Thauvette Louise , Potier Michel , Bayard Francis , Faye Jean-Charles
Published: July 2002

Whole MCF‐7 human breast‐cancer cells were irradiated at −78°C in a calibrated Gammacel 6OCo irradiator. Freezing or storing conditions induce neither an alteration of the viability of cells nor a change in estradiol binding activity. Hexosaminidase was used as internal marker, and we measured the radiation inactivation size (RIS) of the estrogen receptor in whole cells. After various cell treatments, the estradiol binding unit always presents a molecular mass of 25 kDa. This value, which corresponds to the size of the defined hormone binding domain of the estrogen receptor, suggests that the energy delivered to the protein by the radiation is efficient to inactivate estradiol binding only when the hit occurs directly in the smaller hormone binding domein.
Autoprocessing of Drosophila copia gag precursor to generate a unique laminate structure in Escherichia coli
Authors: Yoshioka Katsuji , Kanda Hiroma , Kondo Shunzo , Togashi Shin , Miyake Tadashi , Shiba Tadayoshi
Published: July 2002

Drosophila copia protease is likely to be encoded in the gag gene. We have expressed copia gag polyprotein precursor in E. coli. The gag precursor was correctly processed to generate a unique laminate structure in E. coli. The processing was almost completely blocked by a mutation at the putative active site of copia protease, and resulted in accumulation of the precursor. Furthermore, the laminate structure was not found in E. coli expressing the mutant precursor. These results indicate that the protease is involved in cleaving the gag precursor itself. Also, the assembly of copia gag protein should correlate to the autoprocessing of copia gag polyprotein precursor.
Purification and characterization of N‐acyl‐D‐glutamate deacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A‐6
Authors: Sakai Kenji , Imamura Kazuyuki , Sonoda Yuji , Kido Hiroyuki , Moriguchi Mitsuaki
Published: July 2002

The purification and properties of N‐acyl‐D‐glutamate deacylase from the cell extracts of Alcaligenes xylosoxydans subsp. xylosoxydans A‐6 were studied. The two active fractions (peaks I and II) were obtained by a Mono Q column chromatography. The predominant enzyme (peak I) has been purified, 1960‐fold to homogeneity and characterized. The enzyme was a monomer with a molecular weight of 59 000. The optimum pH and the isoelectric point were 8.0 and 5.5, respectively. The enzyme catalyzed the hydrolysis of N‐acyl derivatives of D‐glutamate. The K
ms for N‐acetyl, N‐butyryl and N‐propionyl derivatives of D‐glutamate were 0.129, 0.066 and 0.01 mM, respectively.
Laminin A‐related domains in crb protein of Drosophila and their possible role in epithelial polarization
Authors: Patthy László
Published: July 2002

It is shown that four domains of the integral membrane protein crb of Drosophila are homologous with five C‐terminal domains of the laminin A chain and merosin. Since the latter domains of laminin A have been implicated in determining epithelial polarity, the homology may suggest a similar function for these domains or crb. Such a function would be consistent with the known importance of crb for organization of epithelia in Drosophila.
Evidence for a ribosome‐associated thiol protease cleaving wheat germ methionyl‐tRNA synthetase
Authors: de Vencay Jacques Archambault , Cenatiempo Yves , Julien Raymond
Published: July 2002

A wheat germ protease is responsible for M
r 105000 methionyl‐tRNA synthetase hydrolysis, generating two fragments of M
r 82000 (harbouring the catalytic domain) and 20000, respectively. Specificity of the protease was sought for using different kinds of protein substrates. It turned out that charged peptides were preferentially cleaved and that no proteolysis occurred when proteins were replaced by small synthetic substrates, harbouring target sites similar to those cleaved in proteins. The protease could be a ribosomal protein, since it remained associated to ribosomal structure, even after treatment by deoxycholate, Triton X‐100, 800 mM KCl and puromycin. Nevertheless, it was still active after ribonuclease treatment of the ribosomes. An identical protease activity was found in rat liver, but not in E. coli.
6‐Hydroxymellein synthetase as a multifunctional enzyme complex in elicitor‐treated carrot root extract
Authors: Kurosaki Fumiya , Itoh Masataka , Yamada Mitsuhiro , Nishi Arasuke
Published: July 2002

Synthetic activity of a polyketide compound 6‐hydroxymellein was induced in elicitor‐treated carrot root tissues. The activity was significantly inhibited by an antiserum raised against the acyl carrier protein (ACP) of fatty acid synthetase, suggesting that the enzyme(s) for 6‐hydroxymellein synthesis require(s) a functional unit similar to ACP. However, the synthetic activity was not stimulated by the addition of ACP purified from Escherichia coli and was not lost even after fractionation by gel‐filtration chromatography. The active fraction obtained by gel‐filtration (136 kDa) was subjected to immunoblot analysis, and a 128 kDa polypeptide in the fraction was found to cross‐react with anti‐ACP serum. These observations suggest that the biosynthesis of 6‐hydroxymellein in carrot cells is catalyzed by an enzyme consisting of a single peptide chain.
Electron microscopy of the complexes of ribulose‐1,5‐bisphosphate carboxylase (Rubisco) and Rubisco subunit‐binding protein from pea leaves
Authors: Tsuprun V.L. , Boekema E.J. , Samsonidze T.G. , Pushkin A.V.
Published: July 2002

The structure of ribulose‐1,5‐bisphosphate carboxylase (Rubisco) subunit‐binding protein and its interaction with pea leaf chloroplast Rubisco were studied by electron microscopy and image analysis. Electron‐microscopic evidence for the association of Rubisco subunit‐binding protein, consisting of 14 subunits arranged with 72 point group symmetry, and oligomeric (L8S8) Rubisco was obtained.
cDNA cloning of a human androgen‐induced mRNA exhibiting an early and protein synthesis‐independent induction
Authors: Colletta Anthony A. , Kealey Terence
Published: July 2002

The detailed mechanism of action of androgens remains unknown. We have used an androgen‐dependent human prostate cancer cell line and a subtractive cDNA hybridisation strategy to enrich for androgen‐dependent sequences. This yielded a cDNA clone which exhibits the characteristics of a primary trans‐activated target for androgens. This androgen‐regulated gene encodes a polyadenylated 4.5 kb mRNA which is induced 30–50‐fold within 3 h of treatment with 10 nM dihydrotestosterone. The induction does not require continued protein synthesis as it is maintained in the presence of protein synthesis inhibitors.
The DNA sequence specificity of cyanomorpholinoadriamycin
Authors: Cullinane Carleen , Phillips Don R.
Published: July 2002

Cyanomorpholinoadriamycin (1 μM) was reacted in a transcription buffer with DNA of an initiated transcription complex. Subsequent elongation of the initiated complex revealed permanent transcriptional blockages at 16 sites after only 5 min of drug‐DNA reaction time. The most dominant sites were immediately prior to 5′‐CC (six) and 5′‐GG (six) sequences of the non‐template strand, consistent with the presence of intrastrand crosslinking between adjacent guanine residues. Minor levels of blockage were at 5′‐GC and 5′‐CG sequences and may reflect low levels of interstrand crosslinking.
Analysis of the polyadenylation consensus sequence context in the genes of nuclear encoded mitochondrial proteins
Authors: Juretić Nenad , Theus Michael
Published: July 2002

A compilation of the pre‐mRNA ends of the genes of nuclear encoded mitochondrial proteins resulted in a consensus sequence of the type (T/A)NTTNNNNNTTTN. Nucleotide positions +8, +13, +14, +16 and +17 downstream of the AATAAA sequence show also a predominance of nucleotide T. This consensus sequence suggests the importance of the immediate surroundings of the cannonical polyadenylation signal sequence AATAAA on the efficiency ofthe cleavage and polyadenylation of this specific group of pre‐mRNAs.
Differential expression of laminin chains in hepatic lipocytes
Authors: Loreal Olivier , Levavasseur Françoise , Rescan Pierre-Yves , Yamada Yoshihiko , Guillouzo André , Clement Bruno
Published: July 2002

The lipocyte is an important source of laminin in the normal liver. We have investigated the expression of the 3 chains of laminin in isolated rat lipocytes. Both B1 and B2 chains, but not A, were found in medium from 5‐day‐old lipocyte primary cultures by immunoblotting and immunoprecipitation of 35S‐labeled proteins after reducing SDS‐polyacrylamide gel electrophoresis. An additional polypeptide of M
r=380 000 was identified by immunoprecipitation. Under non‐reducing conditions only one M
r=900 000 band was revealed. High levels of B1 and B2 mRNAs were also demonstrated in 5‐day‐old cultured lipocytes while at the time of seeding, only B2 chain mRNAs were clearly detectable. A chain mRNA was constantly absent. These results suggest that lipocytes produce a variant form of laminin in primary culture and that the M
r=380 000 polypeptide could be unrelated to the A chain of laminin.
Hydrolysis of branched cyclodextrins by a cyclodextrin‐hydrolyzing enzyme from Bacillus sphaericus E‐244
Authors: Oguma Tetsuya , Kikuchi Mamoru , Mizusawa Kiyoshi
Published: July 2002

The action of a cyclodextrin‐hydrolyzing enzyme from Bacillus sphaericus E‐244 on branched α‐ and β‐cyclodextrins was investigated. Glucosyl‐α‐cyclodextrin (6‐O‐α‐D‐glucosylcyclomaltohexaose) and maltosyl‐α‐cyclodextrin (6‐O‐α‐D‐maltosylcyclomaltohexaose) were hydrolyzed to 63‐O‐α‐D‐glucosylmaltohexaose and 63‐O‐α‐D‐maltosylmaltohexaose, respectively. Glucosyl‐β‐cyclodextrin (6‐O‐α‐D‐glucosylcyclomaltoheptaose) and maltosyl‐β‐cyclodextrin (6‐O‐α‐D‐Maltosylcyclomaltoheptaose) were also transformed mainly to 64‐O‐α‐D‐glucosylmaltoheptaose and 64‐O‐α‐D‐maltosylmaltoheptaose, respectively. These results suggest that the cyclodextrin‐hydrolyzing enzyme cleaves branched α‐ and β‐cyclodextrins at an α‐1,4 linkage which is located furthest from the branching point on the cyclodextrin ring.
Comparative study of subunits of phenylalanyl‐tRNA synthetase from Escherichia coli and Thermus thermophilus
Authors: Bobkova Ekaterina V. , Mashanov-Golikov Anton V. , Wolfson Aleksey , Ankilova Valentina N. , Lavrik Olga I.
Published: July 2002

FPLC separation of α‐ and β‐subunits of phenylalanyl‐tRNA synthetases from E. coli MRE‐600 and Thermus thermophilus HB8 has been carried out in the presence of urea. Native α‐subunits of both enzymes were primarily α2‐dimers and tended to aggregate. Most E. coli enzyme β‐subunits were monomeric and only a small fraction was represented by β2‐dimers. All thermophilic β‐subunits were β‐dimers. It was shown that monomers and all forms of homologous subunits had no catalytic activity in tRNAPhe aminoacylation. For the enzymes and their subunits, titration curves were obtained and isoelectric points were determined. The comparison of the relative surface charges indicated similarity of the surfaces of entire enzymes and the corresponding β‐subunits. α‐Subunits displayed a distinctly different pH dependence of the surface charge. A spatial model of the oligomeric structure and a putative mechanism for its formation are discussed.
Induction of neurite outgrowth of PC12 cells by an inhibitor of vacuolar H+‐ATPase, bafilomycin A1
Authors: Tamura Hiro-omi , Ohkuma Shoji
Published: July 2002

Bafilomycin A1, a selective inhibitor of vacuolar H+‐ATPase, induced neurite outgrowth of PC12 cells dose‐ and time‐dependently: more than 50% of the cells extended neurite‐like spikes after 24 h treatment with 100 nM bafilomycin A1. Its dose‐response ran roughly parallel to that of a bafilomycin A1‐induced lysosomal pH increase. It was inhibited by LiCl, an inhibitor of the phosphorylation of microtubule‐associated proteins and, like nerve growth factor (NGF)‐induced neurite outgrowth, it was also inhibited by cycloheximide and actinomycin D. But, unlike the NGF‐effect, it was not associated with rapid induction of c‐fos.
Specific binding of nucleotides and NAD+ to Clostridium difficile toxin A
Authors: Lobban M.D. , Borriello S.P.
Published: July 2002

Binding of nucleotides, a tetrapolyphosphate, and NAD+ to purified toxin A of Clostridium difficile was determined by monitoring changes in intrinsic fluorescence following excitation at 280 nm, and recording emissions at 340 nm. Binding was specific for concentration over the range 5 to 100 μM for ATP, GTP, and their respective non‐hydrolysable analogues AMP‐PNP and Gpp(NH)p, tetrapolyphosphate and NAD+.
Cloning and characterisation of a gene encoding the 11.5 kDa zinc‐binding protein (parathymosin‐α)
Authors: Trompeter Hans-Ingo , Söling Hans-Dieter
Published: July 2002

A cDNA clone encoding the 11.5 kDa zinc‐binding protein (ZnBP) was used to screen a genomic and a subgenomic rat liver library to isolate the corresponding genomic DNA. Positive clones were restriction‐mapped and sequenced to give the primary structure of the ZnBP gene. A putative promotor region was detected.
Transcription of the gene for the merozoite surface antigen MSA2 of the human malaria parasite Plasmodium falciparum during the asexual cycle
Authors: Barron Carlos
Published: July 2002

The transcription of the Plasmodium falciparum gene for the MSA2 antigen has been studied throughout the parasite's asexual growth cycle. For this purpose poly(A)+ RNA from different times of the cycle was tested for the presence of the mRNA encoding MSA2 by in vitro translation and subsequent analysis of the translation products by immunoprecipitation with an antibody against MSA2. The results revealed that this mRNA is present in trophozoites, reaches the highest concentration during the transition from the trophozoite into the schizont stage, and persists until the cycle end. Minute amounts of this mRNA were also detected in rings. In addition, the data confirmed that the primary translation product is not proteolytically processed at any time of the cycle.
Decreased expression of FcγRIII mRNA in leukemic granulocytes
Authors: Monteiro Joanita , Advani Suresh , Gothoskar Balwant , Zingde Surekha
Published: July 2002

Morphologically mature granulocytes from patients with chronic myeloid leukemia show significant impairment in their ability to internalize aggregated IgG, a ligand that is rapidly phagocytosed by normal human granulocytes. With a view to understand the molecular basis of this defect, normal and leukemic granulocytes were examined for the steady‐state levels of mRNA for FcγRIII, a membrane‐associated receptor that initially binds and traps the IgG‐opsonized antigens. Northern blot analyses revealed that the level of the specific mRNA in CML granulocytes was between 0.08 and 0.69 times that seen in the normal granulocytes. This could be one of the contributory factors for the observed endocytic defect in the leukemic granulocytes.
Effect of TGFβ on liver genes expression Antagonistic effect of TGFβ on IL‐6‐stimulated genes in Hep 3B cells
Authors: Morrone G. , Poli V. , Hassan J.H. , Sorrentino V.
Published: July 2002

The effect of transforming growth factor beta (TGFβ) on the expression of a group of liver genes has been investigated in the hepatoma cell line Hep 3B. TGFβ induces a decrease of the basal level of apolipoprotein A‐II (ApoA‐II), retinol binding protein (RBP) α‐fetoprotein (αFp). Furthermore, TGFβ efficiently antagonizes the IL‐6‐induction of hemopexin (Hpx) and haptoglobin (Hp) and α1‐acid glycoprotein (AGP). These effects of TGFβ are apparently mediated by post‐transcriptional mechanism(s). These findings, together with previously reported data on the inhibitory effect of TGFβ on acute phase genes (e.g. ApoA‐I and albumin), suggest a role for TGFβ in the regulation of expression of liver genes.
Development of insulin sensitivity in rat skeletal muscle Studies of glucose transporter and insulin receptor mRNA levels
Authors: Wallace Shona , Campbell Gillian , Knott Rachel , Gould Gwyn W. , Hesketh John
Published: July 2002

Expression of GLUT‐4 and insulin receptor mRNAs was investigated in rat skeletal muscle by Northern hybridization. GLUT‐4 mRNA was barely detectable in foetal muscle, was expressed at low levels by 1–8 days and at 2–3‐fold higher levels during and after weaning (18–40 days). In contrast there was little change in insulin receptor mRNA levels prior to weaning and a reduction in mRNA abundance between 18 and 40 days. Weaning rats on to a diet rich in fat prevented the increase in GLUT‐4 abundance seen between 15 and 29 days in animals weaned on a high‐carbohydrate diet.
Stability and detection of recombinant pre‐pro‐concanavalin A after cytoplasmic expression in Escherichia coli
Authors: Min Wang , Jones D.Hugh
Published: July 2002

The cDNA for pre‐pro‐concanavalin A (pre‐pro‐Con A) from Canavalia ensiformis was used to construct two cytoplasmic expression vectors: pKconA (no transcription terminator) and pTKconA (containing the cry transcription terminator). The latter produced 2‐ to 3‐fold greater amounts of pre‐pro‐Con A. This product containing the plant signal can be detected by Western blotting only after electrophoretic transfer in the presence of SDS, indicating reduced solubility. The signal is not removed and pre‐pro‐Con A is clearly stable after expression in E. coli JM109. The protein is cleaved and ligated as in the plant, in contrast to a recent report.
Annexin II inhibits calcium‐dependent phospholipase A1 and lysophospholipase but not triacyl glycerol lipase activities of rat liver hepatic lipase
Authors: Bohn Ernst , Gerke Volker , Kresse Hans , Löffler Bernd-Michael , Kunze Hans
Published: July 2002

A member of the annexin family (the heterotetrameric annexin II2pl 12 complex purified from porcine intestinal epithelium) was tested for its ability to affect different calcium‐dependent intrinsic lipolytic activities of rat liver hepatic lipase (HL). Whereas annexin II in the presence of calcium failed to interfere with HL triacyl glycerol lipase (EC 3.1.1.3) activity, it inhibited HL phospholipase A1 (EC 3.1.1.32) and lysophospholipase (EC 3.1.1.5) activities. Inhibition could be overcome by increasing the substrate concentration. Under phospholipase A1 assay conditions, annexin II did not bind to the purified HL enzyme. These results therefore suggest that only inhibitor/substrate interactions lead to inhibition of HL phospholipase A1 and lysophospholipase activities, an obviously general mechanism of phospholipase inhibition by annexins. Possible implications of HL inhibition in vivo by annexins are discussed.
Transcription mapping of the Ori L region reveals novel precursors of mature RNA species and antisense RNAs in rat mitochondrial genome
Authors: Sbisà E. , Tullo A. , Nardelli M. , Tanzariello F. , Saccone C.
Published: July 2002

We have identified new transcripts in the region surrounding the L‐strand replication origin (Ori L) of rat liver mitochondrial DNA. In particular, we have detected previously unidentified intermediates of RNA processing on both the heavy and the light strands, such as precursors of the ND2 mRNA plus the Trp‐tRNA and precursors of the tRNAs clustered in the Ori L region. This indicates that the mechanism of RNA processing in mitochondria proceeds step‐wise producing a variety of precursors of the mature forms. The other striking finding is the detection of antisense RNA species in the region of L‐strand replication. Since a variety of antisense transcripts were also found in the D‐loop region of rat mitochondrial DNA, we suggest that they might play a regulatory role in the replication and expression of the mitochondrial genome.
Bestatin inhibits covalent coupling of [3H]LTA4 to human leukocyte LTA4hydrolase
Authors: Evans Jilly F. , Kargman Stacia
Published: July 2002

The covalent coupling of [3H]LTA4 to human leukocyte LTA4 hydrolase is inhibited in a concentration‐dependent fashion by pre‐incubation with bestatin. This inhibition correlated with the concentration‐dependent inhibition by bestatin of LTB4 and LTB5 synthesis by LTA4 hydrolase. Epibestatin, a diastereomer of bestatin, neither inhibited LTB4 or LTB5 production by LTA4 hydrolase nor prevented the covalent coupling of [3H]LTA4 to the enzyme. In contrast, captopril inhibited both LTB4 synthesis by LTA4 hydrolase and covalent coupling of [3H]LTA4 to the enzyme.
The major form of protein tyrosine kinase in the dog prostate is expressed by a 50 kDa polypeptide
Authors: Nguyen Linh T. , Durocher Yves , Chapdelaine Alcide , Chevalier Simone
Published: July 2002

We have already reported that the protein tyrosine kinase (PTK) activity in the dog prostate is distributed in cytosolic (75%) and particulate (Triton X‐100‐solubilized) fractions and that upon gel filtration, both PTKs migrate as entities of M
r 44 000 [(1991) Biochem. Cell. Biol. 69, 146–153]. Herein we demonstrate by immunoprecipitation with anti‐phosphotyrosine antibodies that the soluble PTK has the ability to undergo self‐phosphorylation. In addition, the polypeptide responsible for that enzymatic activity has been identified by 2 approaches; (1) a two‐dimensional electrophoresis, in which the first dimension performed in non‐denaturing conditions allowed the localization of the native enzyme, while the second dimension (SDS‐PAGE) permitted the analysis of alkali‐resistant phosphoproteins corresponding to the activity; (2) protein renaturation after SDS‐PAGE followed by in situ phosphorylation (with [γ‐32P]ATP) of polyGT electrophoresed together with the enzyme preparation; the exclusive presence of the radiolabeled phosphotyrosine in the renatured protein confirmed its enzymatic nature. Using these methods, the major form of PTK in the dog prostate was shown to be expressed by a 50 kDa polypeptide which possesses autophosphorylation sites and which is present in the cytosol as an active monomer.
Increase in protein kinase C activity is associated with human fibroblast growth inhibition
Authors: Rogalsky Vitaly , Todorov German , Den Tiberius , Ohnuma Takao
Published: July 2002

Protein kinase C(PKC) activity and DNA synthesis were measured in human fetal bone marrow fibroblasts following treatment with tumor necrosis factor alpha (TNFα)(500 U/ml) or conditioned media containing natural cell proliferation inhibitor (CM‐NCPI). Treatment with TNFα led to growth stimulation (120 ± 7% of control in 24 h, 141 ± 6% in 72 h). At the same time particulate PKC activity diminished, reaching 55 ± 8% of control in 24 h and remaining at this level at 72 h. CM‐NCPI treatment of the cells resulted in a decrease in DNA synthesis (by 39 ± 6% in 2 h, by 58 ± 5% in 24 h, and by 78 ± 8% in 72 h). This was accompanied by a significant rise in particulate PKC activity which increased over 3‐fold in 2 h, over 5‐fold in 24 h, and up to 11‐fold in 72 h. This 11‐fold elevation was maintained after 2 week exposure of the fibroblasts to CM‐NCPI. The PKC inhibitor neomycin abolished CM‐NCPI induced growth inhibition, whereas PKC activator 12‐O‐tetradecanoylphorbol 13‐acetate intensified it. These results suggest that CM‐NCPI acts as PKC activator and that negative growth regulation by extracellular agents may involve stimulation of PKC activity.
Formation and quantification of protein complexes between peroxisomal alcohol oxidase and GroEL
Authors: Evers Melchior E. , Langer Thomas , Harder Wim , Hartl Franz-Ulrich , Veenhuis Marten
Published: July 2002

We have studied the use of yeast peroxisomal alcohol oxidase (AO) as a model protein for in vitro binding by GroEL. Dilution of denatured AO in neutral buffer leads to aggregation of the protein, which is prevented by the addition or GroEL. Formation of complexes between GroEL and denatured AO was demonstrated by a gel‐shift assay using non‐denaturing polyacrylamide gel electrophoresis, and quantified by laser‐densitometry of the gels. In the presence of MgAMP‐PNP or MgADP the affinity of GroEL for AO was enhanced. Under these conditions up to 70% of the purified GroEL formed a complex with this protein. Release was stimulated at room temperature by MgATP, and was further enhanced by addition or GroFS.
A relationship between the starting secondary structure of recombinant porcine growth hormone solubilised from inclusion bodies and the yield of native (monomeric) protein after in vitro refolding
Authors: Puri N.K. , Cardamone M.
Published: July 2002

Recombinant porcine growth hormone (rPGH) was solubilised from inclusion bodies (IB's) using either 6 M guanidinium hydrochloride (GnHCl), 7.5 M urca or by a novel method using a cationic surfactant, cetyltrimethylammonium chloride (CTAC). Circular dichroism (CD) analysis of the secondary (2°) structure of the urea‐ and GnHCl‐solubilised rPGH showed the absence of α‐helical content with the majority of the molecule existing in a ‘random coil’ structure. In contrast, the CTAC‐solubilised rPGH displayed significant starting 2° structure (10–15% α helix; 30–40% β structure). The three rPGH preparations were refolded in vitro against weak urea, GnHCl or aqueous buffers, resulting in an average refolding efficiency of 50% native (monomeric) rPGH for CTAC solubilised IB's and only 20% for urea or GnHCl solubilised IB's. We conclude that the method of solubilisation of IB's and the resultant difference in the starting 2° structure of rPGH, particularly α‐helical content, is a major in vitro factor that apparently predetermines the aggregation/refolding behaviour rPGH irrespective of refolding environment.
Cloning and sequence analysis of a cDNA clone from Arabidopsis thaliana homologous to a proteasome α subunit from Drosophila
Authors: Genschik Pascal , Philipps Gabriel , Gigot Claude , Fleck Jacqueline
Published: July 2002

A cDNA clone isolated from an Arabidopsis thaliana cell suspension culture library showed considerable similarities to the proteasome 28 kDa α subunit of Drosophila [(1990) Gene 90, 235–241]. The 250 amino acid‐long protein encoded by Arabidopsis TAS‐g64 clone has important homologies in its primary structure and in the predicted secondary structure with the PROS‐28.1 clone from Drosophila. The only divergence observed between the two sequences is for the 20 C‐terminal amino acids. This subunit might share important functions in both kingdoms, as revealed by the important conservation between plants and animals. In plant cells it is encoded by a single‐copy gene and probably regulated by stress and/or division.
Studies on antisense inhibition of translation in vitro Anomalies and re‐evaluation
Authors: Ricker Robert D. , Kaji Akira
Published: July 2002

Experiments were carried out to better characterize antisense control of translation, Results in an E. coli system confirmed specific inhibition of poly(U) translation. At low concentrations, certain homopolymers (including poly(rA)) stimulated translation, Oligo(dA) was inhibitory at 1158. Translation of globin mRNA in reticulocyte lysates indicated that ssDNA 15‐mers targeted at β‐globin mRNA inhibited both α‐ and β‐globin production. Sequences targeted immediately downstream of the AUG were the least effective in inhibition. These and other anomalies are discussed here in relation to those of others, emphasizing caution in performing antisense experiments.
Alcohol dehydrogenase (ADH) Influence of homo‐ and heterologous ADH administration on albino rats craving for alcohol and on ADH isozyme activity in the liver
Authors: Ashmarin I.P. , Danilova R.A. , Pshezhetsky A.V. , Fedorova I.M. , Obukhova M.F. , Sagimbaeva Sh.K.
Published: July 2002

The effects of homo‐ and heterologous alcohol dehydrogenase (ADH) administration into albino rats were investigated. It was found that homologous ADH increases and heterologous ADH decreases the craving for ethanol. The latter effect was accompanied by the appearance of anti‐ADH‐3 antibodies and by a decrease in ADH‐3 activity in the liver. Craving for alcohol decreased after both active and passive immunization against ADH.
The lysine‐rich H1 histones from the slime moulds, Physarum polycephalum and Dictyostelium discoideum lack phosphorylation sites recognised by cyclic AMP‐dependent protein kinase in vitro
Authors: Heads Richard J. , Carpenter Brian G. , Rickenberg Howard V. , Chambers Timothy C.
Published: July 2002

Calcium chloride‐extracted histones were prepared from nuclei of the slime moulds, Physarum polycephalum and Dictyostellum discoideum, and phosphorylation by purified preparations of cyclic AMP‐dependent protein kinase (cAMP‐d PK) and growth‐associated H1 histone kinase (HKG) examined and compared. Among the major histone fractions and other proteins in the two preparations, the H1 histones from both organisms were found to be effective and exclusive substrates for HKG, cAMP‐d PK, which phosphorylates mammalian H1 histone and certain, in particular H2B, of the mammalian core histones, phosphorylated several of the core histones from both slime moulds but did not phosphorylate H1 histone from either. The slime mould H1s remained ineffective substrates for cAMP‐d PK even after extensive alkaline phsophatase treatment of the histone preparations. Additional studies demonstrated that the lack of slime mould H1 phosphorylation by cAMP‐d PK was not due to competition of the H1 molecules with the core histones for the kinase. Our studies suggest that H1 histones from these organisms, whilst clearly containing sites for phosphorylation by HKG, apparently lack phosphorylation sites recognised by cAMP‐d PK. Thus, the mediation of specific nuclear functions by cAMP‐dependent phosphorylation of H1 in higher organisms may not occur or be required in these lower eukaryotes.
A rapid‐kinetic study of the class C β‐lactamase of Enterobacter cloacae 908R
Authors: Monnaie Didier , Virden Richard , Frère Jean-Marie
Published: July 2002

The individual rate constants for acylation and deacylation (k
2 and k
3, respectively) of the class C β‐lactamase of Enterobacter cloacae 908R by ampicillin and carbenicillin have been determined. For several other β‐lactams, the value of k
2 was too high to be determined and the k
2/k
3 ratio could be larger than 10,000. Branched pathways were also shown to occur with several penicillins and cephalosporins.
Depicting topology and handedness in jellyroll structures
Authors: Stirk H.J. , Woolfson D.N , Hutchinson E.G. , Thornton J.M.
Published: July 2002

The jellyroll structure is a special case of the Greek key topology and, to date, has only been observed in complete form in one of its four possible arrangements. Like other elements of super‐secondary structure involving the β‐strand (eg. the βαβ unit) the known structure forms a right‐handed superhelix. The possibility of losing such tertiary information and other problems associated with representing these structures by two‐dimensional topology diagrams are discussed. A series of rules are presented which allow this three‐dimensional information to be represented in two‐dimensional topology diagrams from which the handedness of a jellyroll structure can be determined.
The formate complex of the cytochrome bo quinol oxidase of Escherichia coli exhibits a ‘g = 12’ EPR feature analogous to that of ‘slow’ cytochrome oxidase
Authors: Calhoun Melissa W. , Gennis Robert B. , Salerno John C.
Published: July 2002

The cytochrome bo quinol oxidase of Escherichia coli is homologous in sequence and in structure to cytochrome aa
3 type cytochrome oxidase in subunit 1, which contains the catalytic core. The cytochrome bo enzyme forms a formate complex which exhibits ‘g = 12’ and ‘g = 2.9’ EPR signals at X band; similar signals have previously been observed only in association with the ‘slow’ and formate‐ligated states of cytochrome oxidase. These signals arise from transitions within integral spin multiplets identified with the homologous heme‐copper binuclear catalytic centers in both enzymes.
No evidence for calpain I involvement in fodrin rearrangements linked to regulated secretion
Authors: Perrin Dominique , Söling Hans-Dieter
Published: July 2002

Stimulation of secretion in chromaffin and parotid acinar cells is associated with dramatic rearrangements of the subplasmalemmal cytoskeleton, notably of fodrin and F‐actin. It has been proposed that a proteolytic cleavage of fodrin resulting from an activation of the neutral calcium activated protease (calpain) could be responsible for these changes. Using an affinity‐purified anti‐α‐fodrin antibody, several cleavage products of fodrin could clearly be detected following incubation of total cell homogenates from chromaffin and parotid acinar cells with purified calpain I. On the other hand, maximum stimulation of secretion of chromaffin cells by nicotine, and of parotid acinar cells by carbachol plus isoproterenol, was not associated with an increased appearance of cleavage products of fodrin. This result is not compatible with the ‘proteolytic cleavage’ hypothesis.