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J C Wallace

Publications and source records attributed to J C Wallace.

At least 37 records · Page 2Linked to original sources
PubMed2000

The antibiotic and anticancer active aurein peptides from the Australian Bell Frogs Litoria aurea and Litoria raniformis the solution structure of aurein 1.2.

Seventeen aurein peptides are present in the secretion from the granular dorsal glands of the Green and Golden Bell Frog Litoria aurea, and 16 from the corresponding secretion of the related Southern Bell Frog L. raniformis. Ten of these peptides are common to both species. Thirteen of the aurein peptides show wide-spectrum antibiotic and anticancer activity. These peptides are named in three groups (aureins 1-3) according to their sequences. Amongst the more active peptides are aurein 1.2 (GLFDIIKKIAESF-NH2), aurein 2.2 (GLFDIVKKVVGALGSL-NH2) and aurein 3.1 (GLFDIVKKIAGHIAGSI-NH2). Both L. aurea and L. raniformis have endoproteases that deactivate the major membrane-active aurein peptides by removing residues from both the N- and C-termini of the peptides. The most abundant degradation products have two residues missing from the N-terminal end of the peptide. The solution structure of the basic peptide, aurein 1.2, has been determined by NMR spectroscopy to be an amphipathic alpha-helix with well-defined hydrophilic and hydrophobic regions. Certain of the aurein peptides (e.g. aureins 1.2 and 3.1) show anticancer activity in the NCI test regime, with LC50 values in the 10-5-10-4 M range. The aurein 1 peptides have only 13 amino-acid residues: these are the smallest antibiotic and anticancer active peptides yet reported from an anuran. The longer aurein 4 and 5 peptides, e.g. aurein 4.1 (GLIQTIKEKLKELAGGLVTGIQS-OH) and aurein 5. 1 (GLLDIVTGLLGNLIVDVLKPKTPAS-OH) show neither antibacterial nor anticancer activity.

Amino Acid Sequence↗
PubMed2000

Double base lesions in DNA X-irradiated in the presence or absence of oxygen.

Previously, double lesions in which two adjacent bases are modified were identified in DNA oligomers exposed in solution to ionizing radiation. However, the formation of such lesions in polymer DNA had not been demonstrated. Using reference oligomer containing a specific double lesion and employing liquid chromatography-mass spectrometry (LC-MS), it was possible to show directly that double lesions are formed in irradiated calf thymus DNA. The double lesion in which a pyrimidine base is degraded to a formamido remnant and an adjacent guanine base is oxidized to 8-oxoguanine was detected in DNA X-irradiated in oxygenated aqueous solution. The double lesion in which the methyl carbon atom of a thymine base is covalently linked to carbon at the 8-position of an adjacent guanine base was detected in DNA irradiated in a deoxygenated environment.

Animals↗
PubMed1999

Functional expression, purification, and characterization of recombinant human pyruvate carboxylase.

The cDNA-encoding human pyruvate carboxylase (hPC) has been assembled and cloned into a very high efficiency mammalian expression vector and the construct transfected into 293T kidney cells. Stable clones expressing very high levels of hPC were produced and used as a source of the enzyme. Purification of the recombinant hPC was performed by selective precipitation with 40% ammonium sulfate followed by a single step avidin affinity chromatography, with an overall yield of 20%. Recombinant hPC purified by this method yielded a single band on SDS-PAGE with a specific activity of 20 U/mg. Kinetic analysis demonstrated that the recombinant human PC has the same properties as the native enzyme isolated from liver autopsy. This is the first report of production and purification of recombinant PC.

Cells, Cultured↗
PubMed1999

Insulin-like growth factor I and its binding proteins: a study of the binding interface using B-domain analogues.

The biological activity of the insulin-like growth factors (IGF-I and IGF-II) is regulated by six IGF binding proteins (IGFBPs 1-6). To examine the surface of IGF-I that associates with the IGFBPs, we created a series of six IGF-I analogues, [His(4)]-, [Gln(9)]-, [Lys(9)]-, [Ser(16)]-, [Gln(9),Ser(16)]-, and [Lys(9),Ser(16)]IGF-I, that contained substitutions for residues Thr(4), Glu(9), or Phe(16). Substitution of Ser for Phe(16) did not affect secondary structure but significantly decreased the affinity for all IGFBPs by between 14-fold and >330-fold, indicating that Phe(16) is functionally important for IGFBP association. While His(4) or Gln(9) substitutions had little effect on IGFBP affinity, changing the negative charge of Glu(9) to a positive Lys(9) selectively decreased the affinities of IGFBP-2 and -6 by 140- and 30-fold, respectively. Furthermore, the effects of mutations to both residues 9 and 16 appear to be additive. The analogues are biologically active in rat L6 myoblasts and they retain native structure as assessed by their far-UV circular dichroism (CD) profiles. We propose that Phe(16) and adjacent hydrophobic residues (Leu(5) and Leu(54)) form a functional binding pocket for IGFBP association.

Amino Acid Sequence↗
PubMed1999

Biotin protein ligase from Saccharomyces cerevisiae. The N-terminal domain is required for complete activity.

Catalytically active biotin protein ligase from Saccharomyces cerevisiae (EC 6.3.4.15) was overexpressed in Escherichia coli and purified to near homogeneity in three steps. Kinetic analysis demonstrated that the substrates ATP, biotin, and the biotin-accepting protein bind in an ordered manner in the reaction mechanism. Treatment with any of three proteases of differing specificity in vitro revealed that the sequence between residues 240 and 260 was extremely sensitive to proteolysis, suggesting that it forms an exposed linker between an N-terminal 27-kDa domain and the C-terminal 50-kDa domain containing the active site. The protease susceptibility of this linker region was considerably reduced in the presence of ATP and biotin. A second protease-sensitive sequence, located in the presumptive catalytic site, was protected against digestion by the substrates. Expression of N-terminally truncated variants of the yeast enzyme failed to complement E. coli strains defective in biotin protein ligase activity. In vitro assays performed with purified N-terminally truncated enzyme revealed that removal of the N-terminal domain reduced BPL activity by greater than 3500-fold. Our data indicate that both the N-terminal domain and the C-terminal domain containing the active site are necessary for complete catalytic function.

Bacterial Proteins↗
PubMed1999

The biotin domain peptide from the biotin carboxyl carrier protein of Escherichia coli acetyl-CoA carboxylase causes a marked increase in the catalytic efficiency of biotin carboxylase and carboxyltransferase relative to free biotin.

Acetyl-CoA carboxylase catalyzes the first committed step in the biosynthesis of long-chain fatty acids. The Escherichia coli form of the enzyme consists of a biotin carboxylase activity, a biotin carboxyl carrier protein, and a carboxyltransferase activity. The C-terminal 87 amino acids of the biotin carboxyl carrier protein (BCCP87) form a domain that can be independently expressed, biotinylated, and purified (Chapman-Smith, A., Turner, D. L., Cronan, J. E., Morris, T. W., and Wallace, J. C. (1994) Biochem. J. 302, 881-887). The ability of the biotinylated form of this 87-residue protein (holoBCCP87) to act as a substrate for biotin carboxylase and carboxyltransferase was assessed and compared with the results with free biotin. In the case of biotin carboxylase holoBCCP87 was an excellent substrate with a K(m) of 0.16 +/- 0.05 mM and V(max) of 1000.8 +/- 182.0 min(-1). The V/K or catalytic efficiency of biotin carboxylase with holoBCCP87 as substrate was 8000-fold greater than with biotin as substrate. Stimulation of the ATP synthesis reaction of biotin carboxylase where carbamyl phosphate reacted with ADP by holoBCCP87 was 5-fold greater than with an equivalent amount of biotin. The interaction of holoBCCP87 with carboxyltransferase was characterized in the reverse direction where malonyl-CoA reacted with holoBCCP87 to form acetyl-CoA and carboxyholoBCCP87. The K(m) for holoBCCP87 was 0.45 +/- 0.07 mM while the V(max) was 2031.8 +/- 231.0 min(-1). The V/K or catalytic efficiency of carboxyltransferase with holoBCCP87 as substrate is 2000-fold greater than with biotin as substrate.

Acetyl-CoA Carboxylase↗
PubMed1999

Sequence-tagged connectors: a sequence approach to mapping and scanning the human genome.

The sequence-tagged connector (STC) strategy proposes to generate sequence tags densely scattered (every 3.3 kilobases) across the human genome by arraying 450,000 bacterial artificial chromosomes (BACs) with randomly cleaved inserts, sequencing both ends of each, and preparing a restriction enzyme fingerprint of each. The STC resource, containing end sequences, fingerprints, and arrayed BACs, creates a map where the interrelationships of the individual BAC clones are resolved through their STCs as overlapping BAC clones are sequenced. Once a seed or initiation BAC clone is sequenced, the minimum overlapping 5' and 3' BAC clones can be identified computationally and sequenced. By reiterating this "sequence-then-map by computer analysis against the STC database" strategy, a minimum tiling path of clones can be sequenced at a rate that is primarily limited by the sequencing throughput of individual genome centers. As of February 1999, we had deposited, together with The Institute for Genomic Research (TIGR), into GenBank 314,000 STCs ( approximately 135 megabases), or 4.5% of human genomic DNA. This genome survey reveals numerous genes, genome-wide repeats, simple sequence repeats (potential genetic markers), and CpG islands (potential gene initiation sites). It also illustrates the power of the STC strategy for creating minimum tiling paths of BAC clones for large-scale genomic sequencing. Because the STC resource permits the easy integration of genetic, physical, gene, and sequence maps for chromosomes, it will be a powerful tool for the initial analysis of the human genome and other complex genomes.

Chromosome Mapping↗
PubMed1999

Assay for reactive oxygen species-induced DNA damage: measurement of the formamido and thymine glycol lesions.

A 32P-postlabeling assay has been developed for the simultaneous detection of the thymine glycol lesion and the formamido remnant of pyrimidine bases in DNA exposed to reactive oxygen species (ROS). The formamido lesion is a principal lesion produced in X-irradiated DNA oligomers when oxygen is available to mediate the damage process. Production of the well-known thymine glycol lesion is less dependent on the concentration of oxygen. These two lesions have the common property that they make the phosphoester bond 3' to the modified nucleoside resistant to hydrolysis by nuclease P1. Our assay uses 32P-postlabeling to measure these lesions in the form of modified dimers obtained from DNA by nuclease P1 digestion. Appropriate carriers and internal standards have been chemically synthesized to improve the reliability and accuracy of the assay. The measurements were accomplished on 1-microgram samples of DNA.

Acid Phosphatase↗
PubMed1999

Structure, function and regulation of pyruvate carboxylase.

Pyruvate carboxylase (PC; EC 6.4.1.1), a member of the biotin-dependent enzyme family, catalyses the ATP-dependent carboxylation of pyruvate to oxaloacetate. PC has been found in a wide variety of prokaryotes and eukaryotes. In mammals, PC plays a crucial role in gluconeogenesis and lipogenesis, in the biosynthesis of neurotransmitter substances, and in glucose-induced insulin secretion by pancreatic islets. The reaction catalysed by PC and the physical properties of the enzyme have been studied extensively. Although no high-resolution three-dimensional structure has yet been determined by X-ray crystallography, structural studies of PC have been conducted by electron microscopy, by limited proteolysis, and by cloning and sequencing of genes and cDNA encoding the enzyme. Most well characterized forms of active PC consist of four identical subunits arranged in a tetrahedron-like structure. Each subunit contains three functional domains: the biotin carboxylation domain, the transcarboxylation domain and the biotin carboxyl carrier domain. Different physiological conditions, including diabetes, hyperthyroidism, genetic obesity and postnatal development, increase the level of PC expression through transcriptional and translational mechanisms, whereas insulin inhibits PC expression. Glucocorticoids, glucagon and catecholamines cause an increase in PC activity or in the rate of pyruvate carboxylation in the short term. Molecular defects of PC in humans have recently been associated with four point mutations within the structural region of the PC gene, namely Val145-->Ala, Arg451-->Cys, Ala610-->Thr and Met743-->Thr.

Animals↗
PubMed1999

Solution structures of apo and holo biotinyl domains from acetyl coenzyme A carboxylase of Escherichia coli determined by triple-resonance nuclear magnetic resonance spectroscopy.

A subgene encoding the 87 C-terminal amino acids of the biotinyl carboxy carrier protein (BCCP) from the acetyl CoA carboxylase of Escherichia coli was overexpressed and the apoprotein biotinylated in vitro. The structures of both the apo and holo forms of the biotinyl domain were determined by means of multidimensional NMR spectroscopy. That of the holo domain was well-defined, except for the 10 N-terminal residues, which form part of the flexible linker between the biotinyl and subunit-binding domains of BCCP. In agreement with X-ray crystallographic studies [Athappilly, F. K., and Hendrickson, W. A. (1995) Structure 3, 1407-1419], the structure comprises a flattened beta-barrel composed of two four-stranded beta-sheets with a 2-fold axis of quasi-symmetry and the biotinyl-lysine residue displayed in an exposed beta-turn on the side of the protein opposite from the N- and C-terminal residues. The biotin group is immobilized on the protein surface, with the ureido ring held down by interactions with a protruding polypeptide "thumb" formed by residues 94-101. However, at the site of carboxylation, no evidence could be found in solution for the predicted hydrogen bond between the main chain O of Thr94 and the ureido HN1'. The structure of the apo domain is essentially identical, although the packing of side chains is more favorable in the holo domain, and this may be reflected in differences in the dynamics of the two forms. The thumb region appears to be lacking in almost all other biotinyl domain sequences, and it may be that the immobilization of the biotinyl-lysine residue in the biotinyl domain of BCCP is an unusual requirement, needed for the catalytic reaction of acetyl CoA carboxylase.

Acetyl-CoA Carboxylase↗
PubMed1999

Molecular recognition in a post-translational modification of exceptional specificity. Mutants of the biotinylated domain of acetyl-CoA carboxylase defective in recognition by biotin protein ligase.

We have used localized mutagenesis of the biotin domain of the Escherichia coli biotin carboxyl carrier protein coupled with a genetic selection to identify regions of the domain having a role in interactions with the modifying enzyme, biotin protein ligase. We purified several singly substituted mutant biotin domains that showed reduced biotinylation in vivo and characterized these proteins in vitro. This approach has allowed us to distinguish putative biotin protein ligase interaction mutations from structurally defective proteins. Two mutant proteins with glutamate to lysine substitutions (at residues 119 or 147) behaved as authentic ligase interaction mutants. The E119K protein was virtually inactive as a substrate for biotin protein ligase, whereas the E147K protein could be biotinylated, albeit poorly. Neither substitution affected the overall structure of the domain, assayed by disulfide dimer formation and trypsin resistance. Substitutions of the highly conserved glycine residues at positions 133 and 143 or at a key hydrophobic core residue, Val-146, gave structurally unstable proteins.

Acetyl-CoA Carboxylase↗
PubMed1999

The citropin peptides from the skin glands of the Australian Blue Mountains tree frog Litoria citropa. Part 2: sequence determination using electrospray mass spectrometry.

A combination of electrospray mass spectrometry, Lys-C digest/mass spectrometry and automated Edman sequencing provides the amino acid sequences of nineteen citropin peptides isolated from the granular dorsal and submental glands of the Blue Mountains tree frog Litoria citropa. Citropin 1.1 [Gly Leu Phe Asp Val Ile Lys Lys Val Ala Ser Val Ile Gly Gly Leu (NH(2))] and citropin 1.2 [Gly Leu Phe Asp Ile Ile Lys Lys Val Ala Ser Val Val Gly Gly Leu (NH(2))] are the two major skin peptides: both show significant wide-spectrum antibacterial activity.

Amino Acid Sequence↗
PubMed1999

Host defence peptides from the skin glands of the Australian blue mountains tree-frog Litoria citropa. Solution structure of the antibacterial peptide citropin 1.1.

Nineteen citropin peptides are present in the secretion from the granular dorsal glands of the Blue Mountains tree-frog Litoria citropa; 15 of these peptides are also present in the secretion from the submental gland. Two major peptides, citropin 1.1 (GLFDVIKKVASVIGGL-NH2), citropin 1.2 (GLFDIIKKVASVVGGL-NH2) and a minor peptide, citropin 1.3 (GLFDIIKKVASVIGGL-NH2) are wide-spectrum antibacterial peptides. The amphibian has an endoprotease which deactivates these membrane-active peptides by removing residues from the N-terminal end: loss of three residues gives the most abundant degradation products. The solution structure of the basic peptide citropin 1.1 has been determined by NMR spectroscopy [in a solvent mixture of trifluoroethanol/water (1 : 1)] to be an amphipathic alpha-helix with well-defined hydrophobic and hydrophilic regions. The additional four peptides produced by the dorsal glands are structurally related to the antibacterial citropin 1 peptides but contain three more residues at their C-terminus [e.g. citropin 1.1.3 (GLFDVIKKVASVIGLASP-OH)]. These peptides show minimal antibacterial activity; their role in the amphibian skin is not known.

Amino Acid Sequence↗
PubMed1999

An overall perspective of X-ray damage to a DNA oligomer mediated by reactive oxygen species.

The products produced by X irradiation of an oxygenated aqueous solution containing d(CpApTpG) were analyzed by NMR spectroscopy and mass spectrometry. Thirteen different base modifications were detected, including a novel product formed by the addition of oxygen to guanine. Seven different strand break products were identified, including strands having 5'-phosphoryl groups, 3'-phosphoryl groups and groups having 3'-phosphoglycolates as termini. The products produced in largest yield contained base modifications: Pyrimidine bases degraded to a formamido moiety, the 8-oxo-7,8-dihydroguanine (8-oxoguanine) lesion, and double base lesions in which both the 8-oxo-7,8-dihydroguanine lesion and a formamido remnant are present.

DNA↗
PubMed1998

Regulation of rat pyruvate carboxylase gene expression by alternate promoters during development, in genetically obese rats and in insulin-secreting cells. Multiple transcripts with 5'-end heterogeneity modulate translation.

A previous study on the gene structure of rat pyruvate carboxylase revealed that two tissue-specific promoters are responsible for the production of multiple transcripts with 5'-end heterogeneity (Jitrapakdee, S., Booker, G. W., Cassady, A. I., and Wallace, J. C. (1997) J. Biol. Chem. 272, 20522-20530). Here we report transcription and translation regulation of pyruvate carboxylase (PC) expression during development and in genetically obese rats. The abundance of PC mRNAs was low in fetal liver but increased by 2-4-fold within 7 days after birth, concomitant with an 8-fold increase in the amount of immunoreactive PC and its activity and then decreased during the weaning period. Reverse transcriptase polymerase chain reaction analysis indicated that the proximal promoter was activated during the suckling period and reduced in activity at weaning. In genetically obese Zucker rats, adipose PC was 4-5-fold increased, concomitant with a 5-6-fold increase in mRNA level. Reverse transcriptase-polymerase chain reaction analysis also showed that the proximal promoter was activated in the hyperlipogenic condition. Conversely, transcription of the proximal promoter was not detectable in various liver cell lines, suggesting that this promoter was not functional under cell culture conditions. In rat pancreatic islets and insulinoma cells, only transcripts D and E, generated from the distal promoter of the PC gene, were expressed. Glucose increased PC transcripts from the distal promoter when the insulinoma cells were maintained in 10 mM glucose. We conclude that the proximal promoter of the rat PC gene plays a major role in gluconeogenesis and lipogenesis, whereas the distal promoter is necessary for anaplerosis. In vitro translation and in vivo polysome profile analysis indicated that transcripts C and E were translated with similar translational efficiencies that are substantially greater than that of transcript D, suggesting that 5'-untranslated regions play a role in translational control.

Aging↗
PubMed1998

Development of a bicistronic vector driven by the human polypeptide chain elongation factor 1alpha promoter for creation of stable mammalian cell lines that express very high levels of recombinant proteins.

We have developed a new vector for the stable expression of recombinant protein in mammalian cells. In this vector, designated pEFIRES-P, both the recombinant cDNA and the puromycin resistant gene (pac) are transcribed as a single message driven by the strong human polypeptide chain elongation factor 1alpha promoter. The presence of an internal ribosome entry site ensures any clones that are resistant to puromycin also express high levels of recombinant protein encoded by cDNA cloned upstream from the pac gene. We report the use of this vector to generate stable cell lines expressing human pyruvate carboxylase and show that following transfection and clonal selection, all twenty colonies that conferred high dose resistance to puromycin also expressed very high levels of functional pyruvate carboxylase. Use of pEFIRES-P should permit the rapid and efficient generation of stable cell lines for the characterisation of recombinant proteins.

Base Sequence↗