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J C Wallace

Publications and source records attributed to J C Wallace.

At least 91 records · Page 5Linked to original sources
PubMed1995

Continuous 14 day infusion of IGF-II increases the growth of normal female rats, but exhibits a lower potency than IGF-I.

The effects of continuous 14 day infusion of recombinant human IGF-I (104 or 260 micrograms/day) or IGF-II (104, 260 or 650 micrograms/day) via s.c. implanted osmotic pumps were compared in young female rats in order to establish the relative efficacies of these two growth factors. Significant increase in body weight gain and feed conversion efficiency were achieved by 260 micrograms/day of IGF-I or 650 micrograms/day of IGF-II. These treatments were associated with increased nitrogen retention and increases in the fractional weights of kidneys, spleen, total gut and individual gut regions. There was an increase in the size of villi and muscularis lining the jejunum, suggesting an increased absorptive capacity of the gut. However there was no significant change in the amount of faecal nitrogen excretion when expressed as a percentage of nitrogen intake. Interestingly, IGF-II was at least as potent as IGF-I in increasing the depth of jejunal crypts. Infusion of equivalent doses of either IGF-I or IGF-II resulted in similar increases in circulating concentrations of the respective peptides, though IGF-II infusion dose-dependently decreased plasma IGF-I concentrations from those of the controls. Plasma IGF-binding protein levels were increased by both IGF-I and IGF-II treatments, though IGF-I elicited greater responses. In summary, IGF-II can promote the growth of young female rats, although generally less potently than IGF-I.

Animals↗
PubMed1995

Circulating insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs) and tissue mRNA levels of IGFBP-2 and IGFBP-4 in the ovine fetus.

The IGF-binding proteins (IGFBPs) are a family of at least six structurally related proteins, which bind the IGFs and modulate their actions, including the regulation of pre- and postnatal growth. In this study we have examined the relationship between circulating and tissue mRNA levels of IGFBPs and related this to circulating IGFs in the fetal sheep over the gestational period when rapid growth and development occurs. Circulating IGFBP-2, as measured by Western ligand blot (WLB), increases between early and mid gestation, remains high, then declines throughout late gestation (P = 0.0002). Circulating IGFBP-3 increases throughout gestation, as measured by WLB or RIA (P = 0.04 and P = 0.0001 respectively), as does circulating IGFBP-4 (P = 0.004). These ontogenic changes in circulating IGFBPs-2 and -4 are paralleled by changes in liver mRNA for these proteins and, for IGFBP-2, by those in kidney IGFBP-2 mRNA also. This suggests that liver and kidney may be the primary contributors to circulating IGFBP-2 and the liver to circulating IGFBP-4, IGFBP-2 mRNA is present in the heart and lung in early gestation but barely detectable in these tissues after approximately 60 days gestation. IGFBP-4 mRNA is also present in the heart in early but not late gestation, but is abundant in the lung throughout gestation. These results demonstrate tissue specific and developmental regulation of IGFBPs-2 and -4 at the mRNA level. To assess any role the circulating IGFs may play in mediating these changes in IGFBPs, or vice versa, both plasma IGF-I and IGF-II were measured by RIA. Circulating IGF-I increases as gestation progresses (P = 0.0001), while circulating IGF-II increases between early and mid gestation, remains high (P = 0.01), then declines. Circulating IGF-I is positively correlated with fetal weight (r = 0.66, P = 0.03), circulating IGFBP-3 (r = 0.54, P = 0.01) and IGFBP-4 (r = 0.52, P = 0.01). Circulating IGF-II positively correlates with circulating IGFBP-2 (r = 0.48, P = 0.02) throughout gestation and at 1 day postnatally. These relationships are consistent with circulating IGF-I influencing IGFBPs-3 and -4, and similarly, IGF-II determining IGFBP-2, or vice versa. Alternatively, these correlations may reflect coordinate regulation of IGF and IGFBP by a common factor.

Animals↗
PubMed1995

Long R3 insulin-like growth factor-I (IGF-I) infusion stimulates organ growth but reduces plasma IGF-I, IGF-II and IGF binding protein concentrations in the guinea pig.

We have tested whether an animal with substantial amounts of both IGF-I and IGF-II in circulation, such as the guinea pig, would respond to chronic IGF infusion in the same manner as the adult rat, which has negligible amounts of IGF-II in blood. Female guinea pigs of 350 g body weight were continuously infused for 7 days with recombinant guinea pig IGF-I or -II (120 or 360 micrograms/day) or long R3 IGF-I (LR3IGF-I) (120 micrograms/day), an analogue which has much reduced affinities for IGF binding proteins. IGF-I or IGF-II infusion led to substantial increases in plasma IGF-I or IGF-II respectively in comparison with vehicle-infused animals. Nevertheless, body weight gain, feed intake, feed conversion efficiency and carcass composition were not significantly affected by any treatment (significance was deemed to be P < 0.05). Amongst the tissues examined only the fractional weight (g/kg body weight) of the adrenals was increased, and that only by the higher dose (360 micrograms/day) of IGF-I. However, the fractional weight of adrenals, gut, kidneys and spleen were significantly increased by LR3IGF-I, but again overall growth was not stimulated. A possible explanation for the lack of IGF-I effects is that total circulating IGF concentrations were not increased by these treatments. IGF-II significantly raised total IGF concentrations at the higher dose only. Plasma IGF-I was reduced by IGF-II infusion, as was plasma IGF-II by IGF-I infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Glands↗
PubMed1995

The radiation chemistry of d(CpGpTpA) in the presence of oxygen.

This report extends our investigation of double base lesions produced by irradiation of DNA model compounds. Studies of d(CpGpTpA) X-irradiated in oxygenated aqueous solution show that two of the five principal products are oligomers having adjacent bases damaged. In these oligomers guanine is hydroxylated at the 8-position and an adjacent pyrimidine is degraded to a formamido remnant.

DNA Damage↗
PubMed1995

Free radical-induced double base lesions.

Evidence is presented for the formation of products in irradiated dinucleoside monophosphates in which both bases are damaged. The dinucleoside monophosphates d(GpT), d(GpC), d(TpG) and d(CpG) were X-irradiated in oxygenated aqueous solution. Product identification was by NMR spectroscopy. In products containing double base lesions, guanine is converted to 8-hydroxyguanine and the pyrimidine base is degraded to a formamido remnant.

Chromatography, High Pressure Liquid↗
PubMed1994

A method for the determination of the cellular phosphorylation potential and glycolytic intermediates in yeast.

A method is described for rapidly quenching metabolism in yeast and extracting metabolites for analysis. The saponin digitonin is used to permeabilize the yeast cell membrane, in conjunction with perchloric acid (PCA) to quench metabolism and extract metabolites. Using this digitonin-PCA quench and extraction procedure, we have determined ATP concentrations in Saccharomyces cerevisiae in the range 2.89-3.39 mM, [ATP]/[ADP] ratios of 4.5-6.6, and phosphorylation potentials of 48.7-49.9 kJ.mol-1. A direct comparison with the currently accepted freeze/grind PCA/thaw procedure for extraction of yeast cell metabolites shows that essentially the same values are obtained by both techniques. The digitonin-PCA quench extraction method, used in conjunction with automated enzymatic analyses of metabolites, allows rapid extraction and analysis of large numbers of samples and metabolites and hence permits detailed investigations of intermediary metabolism in yeast.

Acetates↗
PubMed1994

Expression, biotinylation and purification of a biotin-domain peptide from the biotin carboxy carrier protein of Escherichia coli acetyl-CoA carboxylase.

A protein segment consisting of the C-terminal 87 residues of the biotin carboxy carrier protein from Escherichia coli acetyl-CoA carboxylase was overexpressed in E. coli. The expressed biotin-domain peptide can be fully biotinylated by coexpression with a plasmid that overproduces E. coli biotin ligase. The extent of biotinylation was limited in vivo, but could be taken to completion in cell lysates on addition of ATP and biotin. We used the coexpression of biotin ligase and acceptor protein to label the biotin-domain peptide in vitro with [3H]biotin, which greatly facilitated development of a purification procedure. The apo (unbiotinylated) form of the protein was prepared by induction of biotin-domain expression in a strain lacking the biotin-ligase-overproduction plasmid. The apo domain could be separated from the biotinylated protein by ion-exchange chromatography or non-denaturing PAGE, and was converted into the biotinylated form of the peptide on addition of purified biotin ligase. The identify of the purified biotin-domain peptide was confirmed by N-terminal sequence analysis, amino acid analysis and m.s. The domain was readily produced and purified in sufficient quantities for n.m.r. structural analysis.

Acetyl-CoA Carboxylase↗
PubMed1994

Regulation of pyruvate carboxylase isozyme (PYC1, PYC2) gene expression in Saccharomyces cerevisiae during fermentative and nonfermentative growth.

In Saccharomyces cerevisiae there are two isoenzymes of pyruvate carboxylase (Pyc) encoded by separate genes, designated PYC1 and PYC2. In the wild type yeast, the expression of both genes is influenced by both the growth phase and the type of carbon source, indicating discrete regulatory mechanisms and metabolic roles for PYC1 and PYC2. On glucose minimal medium PYC1 and PYC2 are differentially regulated as shown by a constant level of PYC1 expression throughout the main growth phase compared to a high level of PYC2 expression only in the early growth phase. On ethanol minimal medium, the growth-related pattern of PYC1 and PYC2 expression was similar as shown by a 3.6-fold decline from early to mid log phase. PYC1 expression, however, was activated 10-fold above PYC2 mRNA levels during this period of growth. To further investigate the roles of the two PYC genes we determined the growth phenotypes and expression levels of PYC in pyc1 and pyc2 single null mutants. During fermentative growth, the lack of either PYC gene had little effect on the level and pattern of expression of the other PYC gene, indicating further their separate regulation. In comparison to the pyc2 null, the pyc1 null strain showed a 3- to 4-fold lower level of Pyc activity and Pyc protein concentration. Moreover, the pyc1 null showed a strong requirement for L-aspartate for efficient growth, indicating the importance of PYC1 expression for the synthesis of C4 intermediates. DV6.2 (PYC1, pyc2 delta) showed a 3.2-fold higher level of activity on ethanol minimal medium when compared to growth on glucose minimal medium, and supported growth in the absence of L-aspartate. The pyc1 null, MW21.3 (pyc1 delta, PYC2), on the other hand, did not support growth on ethanol in the absence of aspartate. This study represents the first report on the characterisation of expression of the PYC genes in yeast throughout growth. Their metabolic roles for both fermentative and gluconeogenic growth are considered.

Blotting, Southern↗
PubMed1994

Isolation and characterization of ovine IGFBP-4: protein purification and cDNA sequence.

Three different molecular mass forms of IGF-binding proteins (IGFBPs) were purified from ovine plasma by IGF-I affinity chromatography and reverse-phase HPLC: a 46 kDa doublet and 29 kDa and 24 kDa forms. Amino-terminal sequence analysis confirmed that these proteins were ovine (o)IGFBP-3 (46 kDa) and two molecular size variants of oIGFBP-4. oIGFBP-3 and the 29 kDa form of oIGFBP-4 were shown to be N-glycosylated. Isoelectric points were determined to be at approximately pH 6 for oIGFBP-3 and at pH 7 and pH 7.5 for the 29 and 24 kDa forms of oIGFBP-4 respectively. The two different molecular mass variants of oIGFBP-4 had similar IGF-binding properties. Compared with human IGFBP-3 and oIGFBP-3, the two variants of oIGFBP-4 exhibited lower relative binding to amino-terminally modified IGF-I analogues in a competitive IGF-binding assay. The full protein sequence of oIGFBP-4, as deduced from the cDNA sequence, showed a high degree of identity with rat (90%), human (96%) and bovine (98%) IGFBP-4. The cDNA sequence also showed homology over regions of the 3' non-coding sequence, particularly in comparison with bovine IGFBP-4 (96%). Northern analysis of mRNA for oIGFBP-4 indicated a 2.6 kb major transcript and two minor transcripts of approximately 2.1 and 1.8 kb. oIGFBP-4 mRNA transcripts were detected in adult ewe liver > kidney > lung >> heart and also in several fetal tissues, thus suggesting tissue-specific and developmental regulation. The availability of purified oIGFBP-4 and oIGFBP-3 as well as DNA probes for oIGFBP-4 will enable further study of the properties and functions of these proteins, as well as the establishment of specific assays for these IGFBPs.

Amino Acid Sequence↗
PubMed1993

Insulin-like growth factor (IGF)-II binding to IGF-binding proteins and IGF receptors is modified by deletion of the N-terminal hexapeptide or substitution of arginine for glutamate-6 in IGF-II.

Recombinant insulin-like growth factor-II (IGF-II) and two structural analogues, des(1-6)IGF-II and [Arg6]-IGF-II, were produced to investigate the role of N-terminal residues in binding to IGF-binding proteins (IGFBPs) and hence the biological properties of the modified peptides. The growth factors were modelled on two previously characterized variants of IGF-I, des(1-3)IGF-I and [Arg3]-IGF-I, which both show substantially decreased binding to IGFBPs and were expressed as fusion proteins in Escherichia coli. The biological activities of the corresponding analogues of IGF-I and IGF-II were compared in rat L6 myoblasts and H35B hepatoma cells. In the L6-myoblast protein-synthesis assay, the IGF-II analogues, des(1-6)IGF-II and [Arg6]-IGF-II, were slightly more potent than IGF-II but about 10-fold less potent than IGF-I and 100-fold less potent than the respective IGF-I analogues, des(1-3)IGF-I and [Arg3]IGF-I. In H35 hepatoma cells the anabolic response measured was the inhibition of protein breakdown, and the potency order was insulin >>> [Arg3]-IGF-I > des(1-3)IGF-I > [Arg6]-IGF-II > des(1-6)IGF-II > IGF-I > IGF-II. Binding of the IGFs and their analogues to the type 1 IGF receptor in L6 myoblasts and to the insulin receptor in H35 hepatoma cells did not fully explain the observed anabolic potency differences. Moreover, binding of all four analogues to the IGFBPs secreted by L6 myoblasts and H35B hepatoma cells was greatly decreased compared with the parent IGF. We conclude that the observed anabolic response to each IGF was determined by their relative binding to the competing cell receptor and IGFBP binding sites present.

Amino Acid Sequence↗
PubMed1993

Solution structure of nucleic acid photoadduct, deoxy-m5HO6-uridylyl(5-5)(3'-5')deoxyuridine by NMR and restrained molecular dynamics.

Sensitized UV-B irradiation (sunlamps) of the dinucleoside monophosphate, d-TpF (F = fluorouracil), produces the usual cyclobutane-type photodimer and an additional defluorinated 5-5 photoadduct, d-T5p5U. In d-T5p5U, the original C5 = C6 structure is modified such that the C5 (d-T5p-) is covalently bonded with the C5 (-p5U) (where the fluorine had been) and the C6 (d-T5p-) acquires an OH group. 2D NOE data and the results of J-coupling analysis are used as constraints to refine structures of d-T5p5U in restrained molecular dynamics calculations. The structures obtained show the most probable chiralities of the C5 and C6 atoms of the Thy-portion to be 5R and 6R, respectively. The orientation of the CH3- and uracil-groups are pseudo-axial and pseudo-equatorial, respectively, with respect to the C5 atom. Glycosidic angles are high-anti and anti for the d-T5p- and the -p5U residue, respectively. C3'-endo like sugar puckering is predominant in the d-T5p- residue while C2'-endo like puckering is predominant at the -p5U residue.

Dinucleoside Phosphates↗
PubMed1993

The existence of multiple tetrameric conformers of chicken liver pyruvate carboxylase and their roles in dilution inactivation.

The time-dependent loss of enzymic activity and tetrameric structure of chicken liver pyruvate carboxylase (EC 6.4.1.1) after dilution below 2 units/ml was apparently monophasic and first-order. When examined over a range of initial enzyme concentrations, both activity and tetrameric structure decayed to equilibrium levels which were dependent on the initial concentration. The observed rate constants for the loss of enzymic activity (i) showed no apparent dependence on the initial enzyme concentration, and (ii) were of similar magnitude to the corresponding rate constants of dissociation. Computer simulations of the most likely kinetic model suggest that the predominant form of the dissociated enzyme is the monomer. Dilution of pyruvate carboxylase in the presence of the allosteric activator acetyl-CoA largely prevented the subsequent dissociation of the tetrameric molecule. In addition, acetyl-CoA was able to cause a degree of activation and reassociation when added after dilution inactivation had been allowed to occur. Electron-microscopic observation showed the treatment with avidin before dilution markedly decreased the degree of dissociation of the enzyme tetramer. This structure-stabilizing effect of avidin was dependent on preincubation of the concentrated enzyme solution with acetyl-CoA. We propose that, over a range of protein concentrations, the tetrameric enzyme exists in two forms that are in equilibrium, and that acetyl-CoA alters the equilibrium to favour the more compact form.

Acetyl Coenzyme A↗
PubMed1993

Substituent effects on the puckering mode of the cyclobutane ring and the glycosyl bond of cis-syn photodimers.

The cyclobutane ring (CB) puckering of a cis-syn DNA photodimer (cis-syn d-T[p]T) differs from that of a cis-syn RNA photodimer (cis-syn r-U[p]U) [J.-K. Kim and J.L. Alderfer (1992) Journal of Biomolecular Structure and Dynamics, Vol. 9, p. 1705]. In cis-syn d-T[p]T, interconversion of the CB ring between CB+ and CB- is observed, while in cis-syn r-U[p]U only CB- is observed. In the CB+ conformation, the two thymine rings of the dimer are twisted in a right-handed fashion, as are the bases in B-form DNA. In case of CB- they are twisted in a left-handed fashion. The C5 (base) and/or C2' (sugar) substituents apparently affect the CB ring flexibility in cis-syn d-T[p]T and cis-syn r-U[p]U. To study the effects of the C5 substituent on CB ring flexibility, two-dimensional nuclear Overhauser effect (NOE) and 31P-nmr experiments were performed on cis-syn d-T[p]U, cis-syn d-U[p]T, and cis-syn d-U[p]U photodimers to investigate the CB puckering mode and overall molecular conformation and dynamics. The NOE results indicate the 5-methyl group in the photodimer induces conformational flexibility of the CB ring. In cis-syn d-T[p]U and cis-syn d-U[p]T, both CB+ and CB- puckering modes are observed. This indicates interconversion between two modes takes place as observed in cis-syn d-T[p]T. In the case of cis-syn d-U[p]U, only the puckering CB- mode is observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclobutanes↗
PubMed1993

Modification of maize-seed-protein quality.

The storage proteins of maize are a group of alcohol-soluble polypeptides called zeins. These proteins are synthesized in the developing endosperm, where they form protein bodies within the rough endoplasmic reticulum. Because they account for more than half of the total seed protein, zeins are the primary determinants of the amino acid composition of the seed. All of the zeins are devoid of lysine an essential amino acid for monogastric animals. We have modified the genes encoding zeins so that they encode proteins that contain lysine and tryptophan. Analysis of the synthesis and processing of these modified zein proteins indicates that the addition of lysine and tryptophan does not interfere with their association into protein bodies.

Amino Acid Sequence↗
PubMed1993

UV irradiation of nucleic acids: characterization of photoproducts of thymidylyl-(3'-->5')-2'-deoxy-5-fluorouridine.

The acetone-sensitized irradiation using UV-B (ultraviolet light, 280-320 nm; sunlamps) of thymidylyl(3'-->5')deoxyfluorouridine monophosphate produces two main photoproducts. The distribution of these photoproducts is dependent on the pH of the irradiation solution. At pH 6, the cis-syn cyclobutane-type photodimer is the major product, whereas at high pH (8-10) a photoadduct is the major product. These photoproducts have been identified and structurally characterized by H-1 and C-13 NMR spectroscopy. The photoadduct arises from defluorination of the 5-fluorouracil moiety. The structure of the photoadduct maintains the sugar-phosphate backbone of the starting material (d-TpF), and contains a saturated thymine moiety with an added Thy(C6-hydroxyl) and a Thy(C5)-(C5)Ura covalent bond.

Chromatography, High Pressure Liquid↗
PubMed1993

Plasma clearance and tissue distribution of labelled insulin-like growth factor-I (IGF-I) and an analogue LR3IGF-I in pregnant rats.

To determine whether the changes in insulin-like growth factor-binding proteins (IGFBPs) and IGF-I levels during pregnancy in rats affect the clearance of IGFs from the circulation, we have measured pharmacokinetic parameters and the tissue distribution of radiolabelled IGF-I and LR3IGF-I, a potent analogue which has a markedly reduced affinity of binding to the IGFBPs. Intravenous boluses of radiolabelled growth factors were administered to catheterized virgin and age-matched pregnant rats on day 18 of gestation when plasma IGFBPs, in particular IGFBP-3, are dramatically reduced. IGF-I was cleared more rapidly from plasma in pregnant rats compared with the virgins; metabolic clearance rate (MCR) = 2.88 +/- 0.12 (S.E.M.) and 0.90 +/- 0.05 ml/min per kg respectively. Although LR3IGF-I was cleared more rapidly than IGF-I from plasma, similar clearance rates for the analogue were obtained in both pregnant and virgin animals, MCR = 9.19 +/- 0.15 and 9.84 +/- 0.28 ml/min per kg respectively. In virgin rat plasma, labelled IGF-I was mainly associated with the 150 kDa complex, whereas in pregnant rat plasma IGF-I was predominantly associated with lower M(r) IGFBPs (approximately 30-50 kDa). The majority of LR3IGF-I was detected as free peptide. A larger proportion of the tracer was detected as small M(r) breakdown products in plasma from rats under conditions of reduced binding to IGFBPs, e.g. during pregnancy or when LR3IGF-I was the labelled tracer, suggesting greater rates of IGF degradation. More LR3IGF-I tracer was detected in kidneys, ovaries and adrenals of virgin rats and in the ovaries and adrenals of pregnant rats, compared with IGF-I tracer. IGF-I radioactivity was greater than LR3IGF-I in caecum, brain, liver and heart in virgin rats and in kidneys, caecum, brain, liver, heart, placenta, fetus and fetal plasma of the pregnant rats. These results show that the reduction in IGFBP during pregnancy dramatically increased the clearance of IGF-I from the circulation towards that obtained with LR3IGF-I. The observation that less LR3IGF-I was detected in placenta, fetus and fetal plasma compared with IGF-I raises the possibility that the ability to bind IGFBPs during pregnancy may enhance IGF uptake by the conceptus.

Adrenal Glands↗
PubMed1993

Evolution of insulin-like growth factor binding proteins.

In order to understand more about the origins and evolutionary relationships of insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) we have commenced characterization of the IGFBPs in non-mammalian sera. We show by both in vitro labelling with subsequent analytical gel filtration and by ligand blotting that IGFBPs are present in lamprey serum, indicating that the IGFBPs are indeed, very ancient proteins. Furthermore we present evidence that the major serum IGFBP in lamprey is significantly smaller than the high molecular weight IGFBP3 complex in rat serum. In addition, we show that IGFBPs occur widely throughout vertebrates as we detect IGFBPs in ligand blots of serum from marsupials, monotremes, reptiles, birds and turtles.

Animals↗