bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of human prorenin gene expression by antisense oligonucleotides in transfected CHO cells
Authors: CUMIN Frederic , ASSELBERGS Fred , LARTIGOT Michèle , FELDER Eduard
Published: March 2005

Four phosphorothioate oligonucleotides whose sequences are complementary to the 5′ untranslated region, the initiation codon or the coding region of human prorenin mRNA, were studied for their capacity to inhibit gene expression in stably transfected Chinese hamster ovary (CHO) cells constitutively producing human prorenin. In contrast to oligomers complementary to the initiation codon and the coding region, antisense oligomers directed towards the 5′ untranslated region have no inhibitory effects. The intracellular delivery of a biotinylated phosphorothioate oligonucleotide (biotin‐CATCCATGCTTCCCTC) was monitored in immunofluorescence studies. In the absence of a cationic liposome preparation, LipofectinTM, the oligomer failed to penetrate the cells. In the presence of LipofectinTM, the 35S‐labelled oligomer entered the cells and was distributed in proportions of 54% to the nuclei and 35% to the cytosol.

The effects of regular oligonucleotides and of 3′‐end‐modified phosphodiester oligonucleotides on prorenin production were tested. Terminal modification by biotinylation at the 5′‐end and/or 3′‐dodecyl esterification stabilized oligonucleotides towards exonucleases, but did not translate into a significant inhibition of prorenin production and did not improve the intracellular delivery and or stability of the oligomers.

We have shown that it is possible to inhibit prorenin production intracellularly using specific antisense oligonucleotides. Stability and delivery are crucial factors in the design of potent and specific compounds directed at prorenin mRNA.
The purification of a cd1‐type nitrite reductase from, and the absence of a copper‐type nitrite reductase from, the aerobic denitrifier Thiosphaera pantotropha; the role of pseudoazurin as an electron donor
Authors: MOIR James W. B. , BARATTA Daniela , RICHARDSON David J. , FERGUSON Stuart J.
Published: March 2005

Thiosphaera pantotropha has been reported to contain a copper‐type nitrite reductase on the basis that the copper chelator diethyldithiocarbamate inhibited the overall process of denitrification. It is now shown that nitrous oxide reduction is 100% inhibited by 10 mM diethyldithiocarbamate or 100 μM azide. We also found that both these inhibitors partially inhibited nitrite reduction in this organism.

We purified the nitrite reductase of T. pantotropha and found that it was of the cytochrome cd1 type, contrary to the published report of it being a copper‐type nitrite reductase. This is of importance since T. pantotropha is capable of acrobic nitrite reduction. The only detectable nitrite reduction in anaerobically or aerobically grown cells is the cd1 type.

We also purified a small copper‐containing protein, pseudoazurin. Pseudoazurin was found to be capable of donating electrons to the cd1‐type nitrite reductase in vitro, and its copper centre was chelated by diethyldithiocarbamate. Since nitrite reduction is partially inhibited by diethyldithiocar‐bamate, it is thought that pseudoazurin is an electron donor to nitrite reductase in vivo.
Sequence‐specific DNA‐triplex formation at imperfect homopurine‐homopyrimidine sequences within a DNA plasmid
Authors: XODO Luigi E. , ALUNNI‐FABBRONI Marianna , MANZINI Giorgio , QUADRIFOGLIO Franco
Published: March 2005

The ability of pyrimidine‐rich and purine‐rich oligodeoxynucleotides to form stable triple‐helical structures with imperfect R · Y target sites, containing C interruptions in the purine strand (CG inversions) and located within Bluescript KS+, a plasmid of 2959 bp, has been investigated by electrophoresis, ultraviolet absorbance and cleavage‐protection experiments. First, we synthesized double‐stranded oligonucleotides corresponding to the plasmid sites and studied their interaction with oligopyrimidines which oppose either G or T to CG inversions. The resulting imperfect DNA triplexes were detected by gel‐mobility shift. Their melting profiles were found to be biphasic, and the triplex‐to‐duplex plus single‐strand transition was affected by hysteresis. The 21‐nucleotide triplex containing three GC · G mismatched triads had tm= 45°C, while the same triplex, but with three GC · T triads, had tm= 31°C. Moreover, replacing C with 5‐methylcytosine in the third strand resulted in a significant stabilization of the defective triplex, tm= 49°C. The potential of the 21‐nucleotide oligopyrimidines to recognize and bind in a sequence‐specific manner to imperfect R · Y sequences in Bluescript KS+ has been investigated by means of a restriction‐endonuclease‐protection assay, taking advantage of the fact that one R · Y sequence of Bluescript KS+ was partially overlapped with a HaeII site. Effective endonuclease inhibition was observed with oligopyrimidines opposing G‐to‐GC inversions, at 10–50 μM. By contrast, the oligopyrimidine opposing T‐to‐CG inversions did not exhibit any interference with endonuclease activity in our standard conditions. Finally, we have tested the ability of purine‐rich strands to bind the R · Y sites of Bluescript KS+. A very weak cleavage protection was observed by using an oligomer (130 μM) with a polarity antiparallel to the purine strand of the target site. The resulting Y · R · R triplex was stabilized by CG · G (GC · G) and TA · A base triplets. This triplex denatures with a low cooperative melting profile suggesting the absence of strong interactions between the third strand and the target site.
Production of recombinant notechis 11′2L, an enzymatically active mutant of a phospholipase A2 from Notechis scutatus scutatus venom, as directly generated by cleavage of a fusion protein produced in Escherichia coli
Authors: HODGSON Darren , GASPARINI Sylvaine , DREVET Pascal , DUCANCEL Frédéric , BOUET Françoise , BOULAIN Jean‐Claude , HARRIS John B. , MENEZ André
Published: March 2005

We have constructed an expression vector to produce, in Escherichia coli, a fusion protein containing successively two IgG binding domains from staphyloccocal protein A, a nine‐amino‐acid linker peptide terminating in a methionine residue and the phospholipase A2 notechis 11′2L, an isoform of notexin of Notechis scutatus scutatus, venom. Notechis 11′2L is a mutant of the naturally occuring notechis 11′2 [Bouchier, C., Boyot, P., Tesson, F., Trémeau, O., Bouet, F., Hodgson, D., Boulain, J. C. & Ménez, A. (1991) Eur. J. Biochem. 202, 493–500] in which Met8 has been replaced by Leu. The fusion protein was recovered in the periplasmic extract with a yield of 0.25 mg/I culture. It was hydrolyzed with cyanogen bromide, yielding a protein having the molecular mass, amino acid composition and N‐terminal sequence of notechis 11′2L. Notechis 11′2L and the wild notechis 11′2 displayed identical circular dichroic spectra and shared similar enzymatic, myotoxic and antigenic properties, suggesting that the recombinant notechis 11′2L was directly generated in a correctly folded form.
Dissecting the contributions of a specific side‐chain interaction to folding and catalysis of Bacillus stearothermophilus lactate dehydrogenase
Authors: NICHOLLS David J. , WOOD I. Stuart , NOBBS Timothy J. , CLARKE Anthony R. , HOLBROOK J. John , ATKINSON Tony , SCAWEN Michael D.
Published: March 2005

X‐ray crystallography predicts hydrogen‐bonding interactions between the side chains of Thr198 and two other amino acid residues, Glu194 (adjacent to the catalytic His195) and Ser318 (on the α‐H helix which rearranges on substrate binding). In order to investigate the contribution of this conserved amino acid residue, Thr198, two mutants of Bacillus stearothermophilus lactate dehydrogenase were created (Val198 and Ile198). The steady‐state kinetic parameters for both mutant enzymes were very similar with increased substrate Km and reduced kcat when compared with the wild‐type enzyme. The mutation Val198 allowed non‐productive binding of pyruvate to the unprotonated form of His195. Steady‐state kinetic parameters determined for the Val198 mutant enzyme in high solvent viscosity suggested both an altered rate‐limiting step in catalysis and implicated Thr198 in allosteric activation by the effector fructose 1,6‐bisphosphate (Fru1,6P2). A shift in the Fru1,6P2 activation constant for the Val198 mutant enzyme suggested that Thr198 stabilises the catalytically competent (Fru1,6P2‐activated) form of the enzyme by 6.6kJ/mol. However, Thr198 was not important for maintaining the thermal stability of the Fru1,6P2‐activated form. Equilibrium unfolding in guanidinium chloride indicated that Thr198 contributes 17.2 kJ/mol subunits towards the tertiary structural stability. The results emphasise the importance of the side chain‐hydroxyl group of Thr198 which is required for (a) productive substrate binding, (b) allosteric activation and (c) protein conformational stability. The characteristics of the B. stearothermophilus lactate dehydrogenase mutations reported here were significantly different from those of the same mutations made in the corresponding position of the analogous enzyme Thermus flavus malate dehydrogenase [Nishiyama, M., Shimada, K., Horinouchi, S., & Beppu, T. (1991) J. Biol. Chem. 266, 14294–14299].
Purification and characterization of a nitrous oxide reductase from Thiosphaera pantotropha
Authors: BERKS Ben C. , BARATTA Daniela , RICHARDSON David J. , FERGUSON Stuart J.
Published: March 2005

The aerobic denitrifier Thiosphaera pantotropha is able to reduce simultaneously nitrous oxide and oxygen even after anaerobic growth [Bell, L. C. & Ferguson, S. J. (1991) Biochem J. 273, 423–427]. A nitrous oxide reductase was purified from anaerobically grown T. pantotropha cells. It is argued, on the basis of inhibitor sensitivities and from immunological evidence, that the same nitrous oxide reductase is involved in nitrous oxide reduction in aerobically grown cells. The purified nitrous oxide reductase was shown to have molecular properties very similar to nitrous oxide reductases previously isolated from anaerobically denitrifying bacteria. The visible absorption spectra of the T. pantotropha enzyme resemble those of the oxygen‐affected form of nitrous oxide reductases from other organisms. It is thus concluded that the T. pantotropha nitrous oxide reductase is not peculiarly resistant to the structural changes caused by oxygen. The activity of the purified T. pantotropha nitrous oxide reductase was reconstituted in vitro using horse heart cytochrome c, T. pantotropha cytochrome c551 and T. pantotropha pseudoazurin as electron donors. It is suggested on this basis that either of the T. pantotropha electron‐carrier proteins are possible physiological electron donors to T. pantotropha nitrous oxide reductase. Oxygen was shown not to inhibit the in‐vitro reduction of nitrous oxide with horse heart ferrocytochrome c as electron donor to the reductase.
Cloning and expression of Saccharomyces cerevisiae copper‐metallothionein gene in Escherichia coli and characterization of the recombinant protein
Authors: SAYERS Zehra , BROUILLON Patricia , VORGIAS Constantin E. , NOLTING Hans F. , HERMES Christoph , KOCH Michel H. J.
Published: March 2005

The gene sequences for intact and truncated forms of copper‐binding metallothionein from Saccharomyces cerevisiae were cloned and overexpressed in Escherichia coli BL21(DE3)pLysE cells. In contrast to several other genes, the intact and truncated metallothionein genes are amplified in the polymerase chain reaction when Mg2+ is replaced by Co2+. The recombinant truncated protein binds copper in vivo and in vitro. A ratio of 8 Cu/12 cysteines was determined from atomic absorption, X‐ray fluorescence and amino acid analysis. Extended X‐ray absorption spectroscopy indicates that all Cu is in Cu(I) form and coordinated to three S atoms.
Expression and purification of a trefoil peptide motif in a β‐galactosidase fusion protein and its use to search for trefoil‐binding sites
Authors: CHINERY Rebecca , POULSOM Richard , ELIA George , HANBY Andrew M. , WRIGHT Nicholas A.
Published: March 2005

The cysteine‐rich trefoil motif of rat intestinal trefoil factor (rITF) was cloned and expressed in Escherichia coli. A 270‐bp cDNA fragment including the signal sequence and the trefoil motif was cloned into the expression vector pAX5+ to direct the expression of a β‐galactosidase collagenhinged fusion protein in E. coli. Cultures harbouring the recombinant plasmid produced a soluble novel protein with a molecular mass of 134.5 kDa, as predicted for the trefoil‐motif‐containing fusion protein. Purification of the rITF moiety was achieved by p‐aminophenyl‐thio‐β‐d‐galactoside(APTG)‐affinity chromatography, collagenase digestion of the hybrid molecule, and removal of the β‐galactosidase‐hinge molecule by a further APTG‐affinity step. It was demonstrated that intrachain disulphide‐bond formation in rITF occurred during the procedure, so no refolding steps were required. Analysis by immunoblotting revealed that the fusion protein and the cleaved trefoil‐motif‐containing protein were recognised by an antibody raised against the chemically synthesised peptide.

The trefoil motif present in the fusion protein was used to localise putative trefoil‐binding sites in sections of frozen rat tissue. Binding was demonstrated using the β‐galactosidase portion of the fusion protein as a reporter moiety, either directly with 5‐bromo‐4‐chloro‐3‐indolyl‐β‐d‐galactoside, or indirectly using a monoclonal antibody to β‐galactosidase and indirect immunohistochemistry. Binding sites were localised to the foveolar and surface epithelium of rat stomach, the collecting ducts of the kidney and within colonic crypts. The presence of a trefoil motif was necessary for binding. The use of β‐galactosidase fusion proteins for histochemical localisation of peptide‐binding sites should prove more generally useful.
Acceleration of bovine neurofilament L assembly by deprivation of acidic tail domain
Authors: NAKAMURA Yu , TAKEDA Masatoshi , AIMOTO Saburo , HARIGUCHI Shiro , KITAJIMA Shogo , NISHIMURA Tsuyoshi
Published: March 2005

Functions of the tail region of neurofilament L have, to date, not been clearly elucidated. Bovine neurofilament L was cleaved into tail‐less neurofilament L (50 kDa) and a tail fragment (19 kDa), by thrombin. Tail‐less neurofilament L was deficit of the highly acidic domain of the tail region (∼77% of the entire tail region). Assembly of tail‐less neurofilament L was observed to be accelerated by both fluorometric and centrifugal measurements, compared with intact neurofilament L. The critical concentration of tail‐less neurofilament L, which constitutes the constant unassembled pool, was approximately 0.25‐times lower than that of neurofilament L. Under physiological conditions, tail‐less neurofilament L formed a ribbon‐like structure, wheres tail‐less neurofilament L could from 10‐nm filaments in an extremely low ionic‐strength buffer in the presence of 1 mM MgCl2. An affinity‐purified antibody directed against the tail fragment also accelerated neurofilament L assembly. The tail fragment neither coassembled with neurofilament L nor affect neurofilament L assembly. The acidic domain of the tail region may regulate neurofilament assembly and may be involved in 10‐nm filament formation under physiological conditions.
Characterization of a linear epitope within the human pancreatic 64‐kDa glutamic acid decarboxylase and its autoimmune recognition by sera from insulin‐dependent diabetes mellitus patients
Authors: MAUCH Ludwig , ABNEY Charles C. , BERG Heike , SCHERBAUM Werner A. , LIEDVOGEL Bodo , NORTHEMANN Wolfgang
Published: March 2005

A 2.0‐kb cDNA coding for the full‐length 64‐kDa human glutamic acid decarboxylase (GAD64) was isolated from a pancreatic carcinoma cDNA library by oligonucleotide screening, polymerase‐chain‐reaction amplification and subsequently characterized by sequence analysis. Five overlapping fragments of GAD64 cDNA were constructed into the vector pH6EX3, allowing the highly efficient expression of corresponding fusion proteins with a histidine hexapeptide as an affinity ligand at their N‐termini in Escherichia coli. The recombinant GAD64 fragments were analysed by Western blotting using sera from patients with early onset of insulin‐dependent diabetes mellitus (IDDM). We found that at least 20% of the patients with an onset of IDDM have developed autoantibodies which can specifically recognize a linear antigenic epitope within the GAD64. With a selected IDDM serum, an antigenic epitope was localized in a region of 31 amino acids located at the C‐terminus of GAD64, using epitope mapping techniques, and it was characterized. The possibility of using recombinant GAD64 for the development of an immunoassay for a predictive diagnosis of IDDM is discussed.
Molecular cloning of a 12‐lipoxygenase cDNA from rat brain
Authors: WATANABE Takashi , MEDINA Juan F. , HAEGGSTRÖM Jesper Z. , RÅDMARK Olof , SAMUELSSON Bengt
Published: March 2005

A cDNA encoding an arachidonate 12‐lipoxygenase from rat brain was obtained by polymerase chain reaction cloning. Primers specific for porcine leukocyte 12‐lipoxygenase cDNA were used to isolate the initial polymerase‐chain‐reaction product (395 bp). The final sequence of the rat 12‐lipoxygenase cDNA coding region (1989 bp) was verified by analysis of several separate polymerase‐chain‐reaction products. The open reading frame corresponded to a protein of 662 amino acid residues, with a calculated molecular mass of 75 305 Da. Also the rat 12‐lipoxygenase contained the six conserved histidines, characteristic for all cloned lipoxygenases. It displayed the highest degree of identity to porcine leukocyte 12‐lipoxygenase (71%) and to human 15‐lipoxygenase (75%), with less resemblance to human platelet 12‐lipoxygenase (59%) or rat leukocyte 5‐lipoxygenase (41%).

The recombinant enzyme was expressed in Escherichia coli and incubated with arachidonic acid. Primarily 12‐lipoxygenase (but also some 15‐lipoxygenase) enzyme activity was obtained. A part of the brain 12‐lipoxygenase cDNA was used as probe in Northern blots. A 2.7‐kb mRNA was more abundant in RNA from rat leukocytes, lung, and aorta, than in RNA from rat brain. Sequencing of parts of the corresponding cDNAs (from leukocytes and lung), and comparison to the brain 12‐lipoxygenase sequence, indicated that these mRNAs from the different rat tissues were identical.
Characterization, purification and cDNA cloning of a rat perchloric‐acid‐soluble 23‐kDa protein present only in liver and kidney
Authors: LEVY‐FAVATIER Florence , CUISSET Laurence , NEDELEC Brigitte , TICHONICKY Lydie , KRUH Jacques , DELPECH Marc
Published: March 2005

A novel protein was extracted with 5% perchloric acid from rat liver and kidney. It is absent from other rat organs. Its apparent molecular mass is 23 kDa as determined by HPLC gel filtration. A single band, corresponding to 10 kDa, was observed after SDS/PAGE, suggesting that the protein consists of two subunits with similar molecular masses.

This protein can neither be phosphorylated by ATP, nor acetylated. The sequence of the cDNA encoding this protein was determined. Southern‐blot analysis showed that the corresponding gene spanned at least 10 kb and contained at least five introns. Zoo‐blot analysis at medium stringency strongly suggests that the gene has been conserved during evolution. Tne amino‐acid sequence of this protein with a highly conserved region is similar to that of a heat‐shock protein.
Horse plasma gelsolin labelled with fluorescein isothiocyanate responds to calcium and actin
Authors: KOEPF Edward K. , BURTNICK Leslie D.
Published: March 2005

Reaction between horse plasma gelsolin and fluorescein‐5‐isothiocyanate (FITC) resulted in incorporation of 4.8±0.6 fluorescein groups/gelsolin molecule. The sites of modification were not clustered in any one portion of the gelsolin polypeptide chain; all major peptides produced by proteolytic digestion with α‐chymotrypsin exhibited a fluorescence characteristic of fluorescein. FITC‐gelsolin has a peptide‐backbone circular dichroism spectrum at 20°C that is indistinguishable from that of native gelsolin, but FITC‐gelsolin is considerably more resistant than native gelsolin to thermally induced precipitation. FITC‐gelsolin is fully able to carry out severing of F‐actin filaments, the prime function of gelsolin in plasma. An opening up of the structure of gelsolin on binding Ca2+ is evident from an increased susceptibility of FITC‐gelsolin to quenching by I−. Ca2+ dependence of the interaction between gelsolin and actin is evident in titrations both of intensity and polarization of the fluorescence of FITC‐gelsolin solutions. A Ca2+‐sensitive interaction between gelsolin and tropomyosin also is observed.
Mitochondrial cardiolipin in diverse eukaryotes
Authors: SCHLAME Michael , BRODY Stuart , HOSTETLER Karl Y.
Published: March 2005

Cardiolipin, a unique dimeric phospholipid of bacteria and mitochondria, can be synthesized by two alternative pathways discovered in rat and Escherichia coli, respectively. In mitochondrial preparations from fungi (Saccharomyces cerevisiae, Neurospora crassa), higher plants (Phaseolus aureus), molluscs (Mytilus edulis) and mammals (rat liver, bovine adrenal gland), cardiolipin was synthesized from CDP‐diacylglycerol and phosphatidylglycerol, suggesting a common eukaryotic mechanism of cardiolipin formation which is in contrast to the prokaryotic biosynthesis from two molecules of phosphatidylglycerol. All mitochondrial cardiolipin synthases were inhibited by lyso‐phosphatidylglycerol, were insensitive to N‐ethylmaleimide and required divalent cations, although they had different cation specificities.

The molecular species of cardiolipin from rat liver, bovine heart, S. cerevisiae and N. crassa were analysed by high‐performance liquid chromatography of the derivative 1,3‐bis[3′‐sn‐phosphati‐dyl]‐2‐benzoyl‐sn‐glycerol dimethyl ester. Cardiolipins from these organisms contained mainly mo‐nounsaturated or diunsaturated chains with 16 or 18 carbon atoms, resulting in a relatively homogeneous distribution of double bonds and carbon numbers among the four acyl positions. About half of the molecular species were symmetrical, i.e. they combined two identical diacylglycerol moieties. In N. crassa, the same species pattern was found at growth temperatures of 25°C and 37°C. Tentative molecular models were created for the most abundant molecular species and subjected to energy minimization. Geometric data, derived from these models, suggested similarities in the gross structure of the major cardiolipin species from different sources.
Purification, characterisation and crystallisation of selenomethionyl recombinant human interleukin‐5 from Escherichia coli
Authors: GRABER Pierre , BERNARD Alain R. , HASSELL Anne M. , MILBURN Michael V. , JORDAN Steven R. , PROUDFOOT Amanda E. I. , FATTAH Dilniya , WELLS Timothy N. C.
Published: March 2005

Interleukin‐5 (IL‐5) plays a key role in the proliferation and differentiation of eosinophils. To aid the solution of the crystallographic three‐dimensional structure, we have expressed large quantitites of recombinant human IL‐5 (hIL‐5) in a methionine auxotroph strain of Escherichia coli (DL41) grown on an enriched seleno‐dl‐methionine‐containing medium. Cell densities of A650= 10 have been achieved. The selenomethionyl‐labelled hIL‐5 (Se‐hIL‐5) has been purified and found to contain 3.6 selenium atoms/dimer, and 0.4 methionine residues/dimer. In a B‐cell growth factor assay, the Se‐hIL‐5 is significantly more active than the non‐labelled hIL‐5. Electrospray mass spectrometry shows two major peaks, with relative molecular masses of 26 326 ± 6 and 26 280 ± 8 corresponding to the 4Se and 3Se/1S forms of hIL‐5. Unlike the methionine‐containing hIL‐5, the N‐terminal selenomethionine is neither oxidised nor carbamoylated and can only be resolved into two species in isoelectric focusing gel electrophoresis. Se‐hIL‐5 crystallises in the same space group and unit cell as hIL‐5. Difference Fourier calculations identify two of the selenomethionines corresponding to Met107 in the dimer. However, the N‐terminal is disordered in the crystal, and the N‐terminal selenomethionines are not resolved in the difference Fourier.
CCACC‐binding or simian‐virus‐40‐protein‐1‐binding proteins cooperate with human GATA‐1 to direct erythroid‐specific transcription and to mediate 5′ hypersensitive site 2 sensitivity of a TATA‐less promoter
Authors: ELEOUET Jean‐François , ROMÉO Paul‐Henri
Published: March 2005

Previous studies have shown that a ‐ 112 to + 78 DNA fragment from the erythroid promoter of the human porphobilinogen deaminase (PBGD) gene has erythroid‐specific activity. This PBGD‐(‐112 to + 78) promoter contains a CCACC binding site (position ‐ 100), a GATA binding site (position ‐ 70) and an initiator element around the cap site. Using a cotransfection assay, we find that the human factor GATA‐1 trans‐activates the PBGD‐(‐ 112 to + 78) promoter in non‐erythroid cells. We show that, if trans‐activation is abolished by mutations that destroy either the ‐ 100 CCACC binding or the ‐ 70 GATA binding sites, replacement of the ‐ 100 CCACC binding site by a simian‐virus‐40‐protein‐1 (Sp1) binding site maintains both the erythroid‐specific activity of this promoter and the human GATA‐1 trans‐activation. Thus, human GATA‐1 acts on the PBGD promoter in association with Sp1 or CCACC binding proteins. This PBGD‐(‐ 112 to + 78) promoter is activated 20‐fold by a cis‐linked 5′ hypersensitive site 2 (5′HS‐2) of the human β‐globin locus control region. This activation depends on the ‐ 70 GATA and ‐ 100 CCACC or Sp1 binding sites. When a longer ‐ 714 to + 78 fragment of the PBGD promoter is used, the ‐ 70 GATA mutant still displays erythroid‐specific activity and is cis‐activated by the 5′HS‐2 enhancer, while the ‐ 100 CCACC mutant is completely inactive in the absence or in the presence of the 5′HS‐2 enhancer. Thus, the ‐ 100 CCACC binding site is indispensable for the correct activity and sensitivity of the human PBGD promoter to the 5′HS‐2 enhancer, whereas the ‐ 70 GATA binding site can functionally be replaced by upstream cis‐acting elements.
Structural studies of the O‐antigenic polysaccharide of Fusobacterium necrophorum
Authors: HERMANSSON Kerstin , PERRY Malcolm B. , ALTMAN Eleonora , BRISSON Jean‐Robert , GARCIA Manuel M.
Published: March 2005

The O‐specific polysaccharide component of the lipopolysaccharide produced by Fusobacterium necrophorum is of the teichoic acid type, with repeating units connected by phosphoric diester linkages. Dephosphorylation of the polysaccharide by treatment with aqueous hydrogen fluoride yielded a carbohydrate composed of a trisaccharide linked to an acidic component. This product, and the polysaccharide, were investigated by chemical methods and 1H‐, 13C‐, 31P‐ and 15N‐NMR spectroscopy and the former also by fast‐atom‐bombardment mass spectrometry. It is proposed that the polysaccharide is composed of repeating units having the following structure, in which Fuc represents fucose (6‐deoxy‐galactose), Am represents an acetamidino group and Sug 2,4‐diamino‐2,4,6‐trideoxy‐d‐glucose (‘bacillosamine’) acetylated at the 2‐position and acylated with a (S)‐3‐hydroxybutanoic acid at the 4‐position. The acid was identified as a 2‐amino‐2‐deoxy‐2‐C‐methylpentonic acid (2‐amino‐2‐methyl‐3,4,5‐trihydroxypentanoic acid). The configuration of this acid remains to be determined.

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pH‐induced structural transitions of bovine serum albumin
Authors: SADLER Peter J. , TUCKER Alan
Published: March 2005

1. Titration curves have been constructed for all the resolvable resonances of defatted bovine serum albumin [pH meter readings in 2H2O solution (pH*) 2–10], including complete curves for six His residues and partial curves for a further four. Three His residues have unusually low pKa values (less than 5.5).

2. Abrupt discontinuities in the titration curves of nearly all resonances were observed near pH 4.3 (N to F transition), together with a marked loss of chemical shift dispersion at low pH*. No such loss of dispersion occurs in the high pH region previously associated with the N to B transition (approximately pH 8). The NMR data provide strong evidence that the N to F transition is not localised in domain III as had been previously suggested.

3. A localised unfolding of the N‐terminal region occurs below pH* 4.5.
Proton slippage in cytochrome c oxidase of Paracoccus denitrificans
Authors: STEVERDING Dietmar , KÖHNKE Detlef , LUDWIG Bernd , KADENBACH Bernhard
Published: March 2005

Isolated cytochrome c oxidase from Paracoccus denitrificans, containing either two or three subunits, was reconstituted into liposomes and the membrane potential was measured at different rates of respiration using a triphenylmethylphosponium bromide electrode. Both enzymes revealed a non‐linear increase of the membrane potential with increasing respiratory rates. The ratios of the respiratory rates of the two proton pumps decreased with increasing membrane potential, suggesting slippage of proton pumping, as has been shown before with two cytochrome c oxidases from bovine heart, differing in H+/e− stoichiometries due to chemical modification [Steverding, D. & Kadenbach, B. (1991) J. Biol. Chem. 266, 8097–8101]. The data suggest that slippage of proton pumping represents an intrinsic property of cytochrome c oxidase associated with the two catalytic subunits, I and II.
Taking enzyme kinetics out of control; putting control into regulation
Authors: HOFMEYR Jan‐Hendrik S. , CORNISH‐BOWDEN Athel , ROHWER Johann M.
Published: March 2005

The matrix formulation of metabolic control analysis, which states that multiplying the elasticity matrix for any system by the corresponding control matrix yields an identity matrix, can be transformed into a statement that multiplying a matrix expressing internal regulatory properties by a matrix expressing external regulatory properties also yields an identity matrix. This transformation supplies the formal basis for metabolic regulation analysis, and provides the key to determining the control structure of a system without the need to know the exact changes in enzyme activities that are made to measure control coefficients.
Substrate interactions between trypanothione reductase and N1‐glutathionylspermidine disulphide at 0.28‐nm resolution
Authors: BAILEY Sue , SMITH Keith , FAIRLAMB Alan H. , HUNTER William N.
Published: March 2005

The enzyme trypanothione reductase has been identified as a prime target for the rational design of inhibitors which may have clinical use in the treatment of tropical diseases caused by the genera Trypanosoma and Leishmania. To aid the design or identification of new inhibitors of this enzyme we have elucidated the structural detail of a trypanothione reductase complexed with one of the naturally occurring substrates, N1‐glutathionylspermidine disulphide, by single‐crystal X‐ray diffraction methods at 0.28‐nm resolution. The model for the Crithidia fasciculata enzyme‐substrate complex has an R‐factor of 14.8% and root‐mean‐square deviations of 0.0015 nm and 3.3° on bond lengths and angles respectively. Hydrogen bonding and van der Waals interactions between the enzyme and substrate are dominated by the amino acid side chains. The substrate binds in a rigid active site such that one glutathione moiety is in a V‐shape, the other in an extended conformation. One spermidine moiety binds closely to a hydrophobic patch in the active site formed by a tryptophan and a methionine. Distances between the methionine Sδ and the terminal N of this spermidine suggest that a hydrogen bond may supplement the hydrophobic interactions in this part of the active site.
The structure of the gene encoding chicken ribosomal protein L37a
Authors: MACHIDA Maki , TOKU Seikichi , KENMOCHI Naoya , TANAKA Tatsuo
Published: March 2005

The nucleotide sequence of the gene encoding chicken ribosomal protein L37a was determined. The gene was 2626 bp long and distributed in four exons and three introns. The transcription initiation site is located at a cytidine residue in a tract of 16 pyrimidines flanked by (G + C)‐rich regions. Neither canonical TATA nor the CAAT box was found in the 5′‐flanking region. The region from nucleotides –78 to –35 was shown to be important for promoter activity and also to be a binding site for nuclear proteins. This region contains two copies of the sequence CATTCCGG that is common to many ribosomal protein genes of higher eukaryotes.
Characterization of the promoter region of the rat hepatocyte‐growth‐factor/scatter‐factor gene
Authors: OKAJIMA Ai , MIYAZAWA Keiji , KITAMURA Naomi
Published: March 2005

Hepatocyte growth factor/scatter factor (HGF/SF) is a potent mitogen for hepatocytes in primary culture. In response to liver damage, the levels of HGF/SF mRNA change in various tissues. In this study, we isolated a genomic DNA fragment containing the promoter region of the rat HGF/SF gene and analyzed transcription‐initiation sites and their utilization in response to acute liver injury. Rat HGF/SF‐mRNA synthesis starts from at least three sites in the liver, spleen and kidney. One of these sites is preferentially utilized in the liver and spleen in response to acute liver injury. In the 5′ flanking region, several cytokine‐related sequence elements that might be involved in the regulation of HGF/SF‐gene expression are located near the transcription‐initiation sites. The effects of cytokines related to these sequence elements on the production of HGF/SF mRNA were examined using a cell culture system. Transforming growth factor‐β1 (TGF‐β1) inhibits the production of HGF/SF mRNA by Shay granulocytic sarcoma‐derived cells. The TGF‐β1‐inhibitory element, one of the sequence elements present in the promoter sequence, may mediate the inhibition of HGF/SF‐gene expression by TGF‐β1.
Molecular cloning, developmental pattern and tissue expression of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase of the cockroach Blattella germanica
Authors: MARTINEZ‐GONZALEZ José , BUESA Carlos , PIULACHS María‐Dolors , BELLES Xavier , HEGARDT Fausto G.
Published: March 2005

In insects, 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase (HMG‐CoA) synthesizes mevalonate for the production of nonsterol isoprenoids, which are essential for growth and differentiation. To understand the regulation and developmental role of HMG‐CoA reductase, we have cloned a full‐length HMG‐CoA reductase cDNA from the cockroach Blattella germanica. This cDNA clone was isolated using as a probe a partial cDNA of B. germanica HMG‐CoA reductase, amplified using the polymerase chain reaction. The composite 3433‐bp cDNA sequence contains an open reading frame encoding a polypeptide of 856 amino acids (Mr 93165). The C‐terminal region is more similar to hamster HMG‐CoA reductase than is the Drosophila melanogaster enzyme (79% and 69% conserved residues, respectively), and the potential transmembrane domains at the N‐terminal region are structurally conservative with both enzymes. The C‐terminal region of the B. germanica protein has been expressed as a fusion protein in Escherichia coli and exhibits HMG‐CoA reductase activity. Analysis of B. germanica HMG‐CoA reductase mRNA levels, reveals a 3.6‐kb transcript, that is overexpressed in 4‐day‐old embryos. Northern‐blot analysis of RNA samples from different adult female tissues shows high HMG‐CoA reductase mRNA levels in the ovary and lower levels in brain and muscle.
Synergism between protein‐kinase C and cAMP‐dependent pathways in the expression of the interleukin‐1β gene is mediated via the activator‐protein‐1 (AP‐1) enhancer activity
Authors: SERKKOLA Elina , HURME Mikko
Published: March 2005

In many different cell types treatment with phorbol esters (e.g. 4β‐phorbol 12‐myristate 13‐acetate, PMA) leads to the activation of protein‐kinase C (PKC) and subsequently to the activation of the activator‐protein‐1(AP‐1)‐responsive gene expression. We have previously reported that a structural analog of cAMP (dibutyryl cAMP, Bt2cAMP) or agents elevating the endogenous cAMP levels strongly enhanced the PMA‐induced interleukin‐1β(IL‐1β)‐gene expression in human myeloid leukemia cells (THP‐1, HL‐60). We have now examined the role of AP‐1 in the regulation of the IL‐1β gene expression by PKC and cAMP in THP‐1 cells. AP‐1 is a complex composed of products of the jun and fos gene families. Our studies show that Bt2cAMP enhances the PMA‐induced c‐fos and jun‐B expression, but inhibits c‐jun expression. Electrophoretic mobility‐shift assay revealed that Bt2cAMP also increased the PMA‐induced AP‐1 DNA‐binding activity. The functional role of the increased AP‐1 DNA‐binding activity was studied by transfecting THP‐1 cells with reporter constructs containing AP‐1 sites [Col‐TREx5/TK‐CAT and IL‐1β‐X‐CAT, which contains the putative 12‐O‐tetradecanoyl‐phorbol‐13‐acetate(TPA)‐responsive element of the IL‐1β gene]. Transient transfection assay demonstrated that Bt2cAMP similarily increased the PMA‐induced transcription from both of these reporter constructs. Taken together, these results suggest that cAMP increases the PMA‐induced AP‐1 activity which then leads to increased IL‐1β expression.
Oxygenation of lipoproteins by mammalian lipoxygenases
Authors: BELKNER Jutta , WIESNER Rainer , RATHMAN Jörg , BARNETT Jim , SIGAL Elliott , KÜHN Hartmut
Published: March 2005

Oxidative modification converts low‐density lipoprotein (LDL) into its atherogenic form and appears to be a necessary precondition for LDL uptake by macrophages during foam cell formation. Cellular lipoxygenases have been implicated in this process. We studied the interaction of purified mammalian lipoxygenases with human LDL in vitro and found that the arachidonate 15‐lipoxygenases of rabbit and man are capable of oxygenating lipoproteins as indicated by oxygen uptake and by the formation of thiobarbituric‐acid‐reactive substances. Furthermore, oxygenated polyenoic fatty acids, such as 13‐hydro(pero)xy‐9Z,11E‐octadecadienoic acid and 15‐hydro(pero)xy‐5,8,11,13(Z,Z,Z,E)‐eicosatetraenoic acid were detected in the lipid compartment of various lipoproteins classes after lipoxygenase treatment. More than 90% of the oxygenated polyenoic fatty acids were found in the ester‐lipid fraction, particularly in the cholesterol esters, whereas only small amounts of free hydro(pero)xy polyenoic fatty acids were detected. Lipoxygenase‐catalyzed oxygenation of LDL is not restricted to the lipid compartment but also leads to a cooxidative modification of the apoproteins as indicated by changes in the electrophoretic mobility and by the formation of carbonyl derivatives of amino acid side chains. The possible biological significance of lipoxygenase‐induced oxidative modification of lipoproteins in the pathogenesis of atherosclerosis is discussed.
Purification and sequence determination of guanylate kinase from pig brain
Authors: ZSCHOCKE Peter D. , SCHILTZ Emile , SCHULZ Georg E.
Published: March 2005

A purification procedure for guanylate kinase from pig brain has been developed consisting of ammonium sulfate precipitation and heptane extraction of the crude extract, hydrophobic‐interaction chromatography, affinity chromatography and chromatofocussing. From 1.75 kg pig brain, 1.2 mg enzyme was isolated with a yield of 18% and a purity of about 90%. For sequence determination, the protein was cleaved with trypsin, cyanogen bromide and endoproteinase Glu‐C. Some of the isolated peptides were subcleaved with chymotrypsin, thermolysin or trifluoroacetic acid. The blocked N‐terminus was analyzed by mass spectrometry and by amino acid analysis of a tryptic peptide, while the C‐terminus was found in a tryptic and a chymotryptic peptide and confirmed by a carboxypeptidase Y digestion. The sequence contains 197 amino acids with a Mr of 21831, one tryptophan and one cysteine residue. It has been compared to those of the homologous enzymes of yeast and Escherichia coli, as well as to proteins from sequence data banks that show similarities. The sequence is discussed in the light of the known spatial structure of yeast guanylate kinase.
The 5′ ends of RNA oligonucleotides in Escherichia coli and mRNA degradation
Authors: CANNISTRARO Vincent J. , KENNELL David
Published: March 2005

The 5′ ends of RNA oligonucleotides in Escherichia coli were identified to assess the contributions of specific endoribonucleases to the cleaving of bulk mRNA. About 60% of the total 5′ ends have a 5′ OH, and 40% a phosphate. Of those oligonucleotides with a 5′‐OH end, 55% of the larger‐sized molecules started with 5′‐OH‐A. With decreasing size there was a progressive decrease in its relative abundance, reaching 33% for the mononucleotide pool, close to its content in E. coli mRNA. In a mutant lacking RNase I* (a form of RNase I), the fraction starting with 5′‐OH‐A was even higher; 65–70% for oligonucleotides of any size, as well as the mononucleotides, whereas only 3–5% started with 5′‐OH‐U. Oligonucleotides with a 5′‐P end were analyzed after pulse‐labeling growing cells with 32Pi. Virtually all of them had a 5′‐ppp‐purine end which would result from transcription initiations, and there were four‐times more G than A starts. The fraction of 5′ ends with a monophosphate (5′‐pN) was too low to measure.

The known degradative enzymes of E. coli (RNases I, I*, M and R) release a 5′‐OH oligonucleotide upon cleavage, whereas known processing endoribonucleases, e.g. RNases E, H, P and III, generate 5′‐P oligonucleotides. Among these enzymes, RNase M is the only one known to enrich for 5′‐OH‐A ends, since its preference is for pyrimidine‐A bonds [Cannistraro, V. J. & Kennell, D. (1989) Eur. J. Biochem. 181, 363–370]. It also gives a very low level of 5′‐OH‐U ends. These results are consistent with generalizations derived from our previous studies [Cannistraro, V. J., Subbaro, M. N. & Kennell, D. (1986) J. Mol. Biol. 192, 257–274] and suggest that RNase M is a primary endoribonuclease for mRNA degradation in E. coli. The results also indicate that RNase I* contributes a smaller fraction of cleavages to larger RNA oligonucleotides and accounts for most of the degradation of the very small oligonucleotides and almost all degradation of dinucleotide to mononucleotide.
Stimulation of HIT‐T15 insulinoma cells by glyceraldehyde does not require its metabolism
Authors: ELLIOTT Austin C. , TREBILCOCK Richard , YATES Allen P. , BEST Leonard
Published: March 2005

The addition of the triose d‐glyceraldehyde (5–20 mM) to HIT‐T15 hamster insulinoma cells caused a rapid, marked depolarisation of the plasma membrane accompanied by a pronounced intracellular acidification, an increase in the cytosolic free calcium concentration [Ca2+]i and enhanced secretion of insulin. d‐glyceraldehyde did not reduce the rate of efflux of 86Rb+ from loaded perifused cells. All of the above effects of d‐glyceraldehyde were also observed in response to l‐glyceraldehyde. The changes in membrane potential and intracellular pH (pHi) caused by d‐glyceraldehyde were unaffected by the glycolytic inhibitor iodoacetate, by K+‐channel blockers (tolbutamide and tetraethylammonium), or by inhibitors of the transport of lactate (α‐fluorocinnamate), alanine (methylaminoisobutyrate) or glucose (phloretin, phlorrizin). The glyceraldehyde‐induced depolarisation and acidification were also observed in the absence of extracellular Ca2+ or Na+. The increase in [Ca2+]i evoked by d‐glyceraldehyde was reversed by removal of Ca2+ from the medium. The formation of lactate by HIT‐T15 cells was not significantly increased by addition of 10 mM d‐glyceraldehyde or l‐glyceraldehyde. In contrast, 10 mM glucose caused an approximately fourfold rise in lactate production. The oxidation of d‐glyceraldehyde by HIT‐T15 cells was also extremely modest compared to glucose oxidation by these cells. These results suggest that the stimulation of HIT‐T15 cells by either d‐glyceraldehyde l‐glyceraldehyde does not require metabolism of the triose within the cell and may not involve closure of nucleotide‐sensitive K+ channels. We propose that the electrogenic transport of glyceraldehyde across the plasma membrane, possibly via H+ cotransport, might lead to depolarisation and hence to Ca2+ entry into the cell.
Expression of the rat growth‐hormone gene is under the influence of a cell‐type‐specific silencer element
Authors: GUÉRIN Sylvain L. , ANZIVINO Matthew J. , ROY René J. , MOORE David D.
Published: March 2005

We have previously shown that a cell‐type‐specific negative‐regulatory element, or silencer, acts to specifically restrict rat‐growth‐hormone(rGH)‐promoter activity to pituitary cells. Here we report a detailed characterization of this element. The activity of the silencer is dependent on its position relative to the promoter. The negative regulatory effect can be diminished by cotransfection with a high‐copy‐number, silencer‐containing competitor plasmid, suggesting that the function of the element is mediated by specific binding of a trans‐acting negative‐regulatory factor. The minimal region required for silencer function is contained between positions −309 and −266 relative to the start of the rGH mRNA. The specific interaction of a nuclear protein from non‐pituitary cells with this rGH DNA segment was shown by DNasel as well as dimethylsulfate methylation‐interference footprinting. A detailed examination of the DNA‐binding site for that protein clearly suggest that it belongs to the NF1 family of transcription factors.
Overproduction, solubilization, purification and DNA‐binding properties of AmpR from Citrobacter freundii
Authors: BISHOP Russell E. , WEINER Joel H.
Published: March 2005

AmpR belongs to the LysR family of prokaryotic DNA‐binding transcriptional regulators and controls induction of the enterobacterial ampCβ‐lactamase gene. The ampR gene of Citrobacter freundii was deregulated by employing the polymerase chain reaction to introduce an efficient ribosome‐binding sequence and suitable restriction enzyme sites for cloning into a chemically inducible tac‐promoter expression vector. When induced in Escherichia coli, the modified ampR gene rapidly overproduced the AmpR protein as an insoluble aggregate. The AmpR protein could be solubilized with 1.32 M guanidine/HCl and remained soluble when dialyzed against 0.5 M NaCl. The solubility properties of AmpR were exploited to selectively precipitate and resolubilize the protein in a nearly homogenous state. AmpR was then purified by a single gel‐filtration chromatography step which demonstrated that AmpR exists in solution as a monodisperse homodimeric protein. Several milligrams of purified AmpR could be obtained routinely from a 1‐l culture of induced bacteria. A DNA‐binding assay buffer containing 300 mM potassium glutamate and 30% glycerol was found to stabilize AmpR and used to demonstrate sequence‐specific DNA‐binding. Additionally, purified AmpR binds a half‐operator DNA with an inverted‐repeat sequence which competes with binding by the wild‐type operator. These findings are discussed in terms of the helix‐turn‐helix DNA‐binding motif, whereby AmpR is proposed to interact with its wild‐type operator as a dimer of dimers.