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Biomedical subjects

J G Robertson

Publications and source records attributed to J G Robertson.

At least 19 recordsLinked to original sources
PubMed2001

Hepatocyte growth factor and macrophage-stimulating protein are upregulated during excisional wound repair in rats.

Hepatocyte growth factor (HGF) and macrophage-stimulating protein (MSP) are structurally related molecules that stimulate epithelial cell proliferation and migration. MSP also acts directly as a chemoattractant for resident macrophages. These activities are integral to the wound repair processes of inflammation, epithelialization and tissue remodelling. To begin to examine the involvement of HGF and MSP in healing of cutaneous wounds we have mapped the temporal expression of these two molecules and their receptors, MET and RON respectively, in adult rat excisional wounds. Four 2x2-cm full-thickness excisional wounds were created on the dorsum of 18 rats, and biopsies were taken through the wounds at 3, 5, 7, 14, 21, and 28 days postwounding. These biopsies were analyzed using immunofluorescent staining and in situ hybridization (ISH). The number of cells staining positively for HGF and MET significantly increased in response to wounding. HGF staining and mRNA peaked at 7 days postwounding whereas MET was upregulated earlier, peaking after 3 days. Both HGF and MET protein were observed in fibroblasts of the dermis and in the newly forming granulation tissue. ISH studies also revealed that fibroblasts at the wound edges and within the newly forming granulation tissue also expressed HGF and c-met mRNA. Immunofluorescent staining revealed both MSP and RON within the wound, with maximum staining occurring between 7 and 21 days for both the ligand and receptor. In addition, MSP co-localized with a small subset of ED1-positive cells (monocytes). In contrast, ED2-positive cells (macrophages) did not co-localize with MSP. Thus, increased expression of HGF, MSP and their receptors MET and RON respectively was observed in response to wounding. Furthermore, MSP co-localization with a subset of monocytes may confirm a role for MSP in the activation of mature macrophages, which may be important in tissue remodelling.

Animals↗
PubMed2000

4-Thiazolidinones: novel inhibitors of the bacterial enzyme MurB.

4-Thiazolidinones were synthesized and evaluated for their ability to inhibit the bacterial enzyme MurB. Selected 4-thiazolidinones displayed activity against the enzyme in vitro. This activity, coupled with the design principles of the thiazolidinones, supports the postulate that 4-thiazolidinones may be recognized as diphosphate mimics by a biological selector.

Bacteria↗
PubMed2000

Mitogenic whey extract stimulates wound repair activity in vitro and promotes healing of rat incisional wounds.

The ability of single growth factors to promote healing of normal and compromised wounds has been well described, but wound healing is a process requiring the coordinated action of multiple growth factors. Only the synergistic effect on wound healing of combinations containing at most two individual growth factors has been reported. We sought to assess the ability of a novel milk-derived growth factor-enriched preparation ¿mitogenic bovine whey extract (MBWE), which contains six known growth factors, to promote repair processes in organotypic in vitro models and incisional wounds in vivo. MBWE stimulated the contraction of fibroblast-populated collagen lattices in a dose-dependent fashion and promoted the closure of excisional wounds in embryonic day 17 fetal rat skin. Application of MBWE increased incisional wound strength in normal animals on days 3, 5, 7, and 10 and reversed the decrease in wound strength observed following steroid treatment. Wound histology showed increased fibroblast numbers in wounds from normal and steroid-compromised animals. These data suggest the mixture of factors present in bovine milk exerts a direct action on the cells of cutaneous wound repair to enhance both normal and compromised healing.

3T3 Cells↗
PubMed1999

Clearance of IGFs and insulin from wounds: effect of IGF-binding protein interactions.

We have examined the role binding proteins have in regulating the clearance of exogenous growth factors from wounds. Hunt-Schilling chambers were subcutaneously implanted in rats, and the clearance of insulin-like growth factor (IGF) I from the chamber wound fluid was compared with IGF-II, LR3-IGF-I, which binds poorly to IGF-binding proteins (IGFBP), or insulin. Elimination rate constants of the slow phase of the decay curves did not differ between IGF-I and IGF-II. However, LR3-IGF-I and insulin were cleared more rapidly from wound fluid than IGF-I so that the half-lives for IGF-I, IGF-II, LR3-IGF-I, and insulin were 872, 861, 563, and 324 min, respectively. In wound fluid, minimal degradation of the IGFs occurred, whereas insulin was degraded considerably. The increased clearance of LR3-IGF-I and insulin equated with a reduced association with wound fluid IGFBPs, and increased amounts of radioactivity of these peptides were detected in the circulation and urine. These results show that this model of wound repair may be of use in examining the kinetics of growth factors and other bioactive molecules in extravascular spaces and support the hypothesis that IGFBPs can be significant regulators of IGF bioavailability in vivo.

Animals↗
PubMed1997

Characterization of NADP+ binding to perdeuterated MurB: backbone atom NMR assignments and chemical-shift changes.

Backbone-atom resonances have been assigned for both the substrate-free and the NADP+-complexed forms of UDP-N-acetylenolpyruvylglucosamine reductase (MurB), a monomeric, 347-residue (38.5 kDa) flavoenzyme essential for bacterial cell-wall biosynthesis. NMR studies were performed using perdeuterated, uniformly 13C/15N-labeled samples of MurB. In the case of substrate-free MurB, one or more backbone atoms have been assigned for 334 residues (96%). The assigned backbone atoms include 309 1HN and 15N atoms (94%), 315 13CO atoms (91%), 331 13C(alpha) atoms (95%), and 297 13C(beta) atoms (93%). For NADP+-complexed MurB, one or more backbone atoms have been assigned for 313 residues (90%); these include 283 1HN and 15N atoms (86%), 305 13CO atoms (88%), 310 13C(alpha) atoms (89%), and 269 13C(beta) atoms (84%). The strategies used for obtaining resonance assignments are described in detail. Information on the secondary structure in solution for both the substrate-free and NADP+-complexed forms of the enzyme has been derived both from 13C(alpha) and 13C(beta) chemical-shift deviations from random-coil values and from 1HN-1HN NOEs. These data are compared to X-ray crystallographic structures of substrate-free MurB and MurB complexed with the UDP-N-acetylglucosamine enolpyruvate (UNAGEP) substrate. NADP+ binding induces significant chemical-shift changes in residues both within the known UNAGEP and FAD binding pockets and within regions known to undergo conformational changes upon UNAGEP binding. The NMR data indicate that NADP+ and UNAGEP utilize the same binding pocket and, furthermore, that the binding of NADP+ induces structural changes in MurB. Finally, many of the residues within the UNAGEP/NADP+ binding pocket were difficult to assign due to dynamic processes which weaken and/or broaden the respective resonances. Overall, our results are consistent with MurB having a flexible active site.

Amino Acid Sequence↗
PubMed1997

Spectroscopic properties of Escherichia coli UDP-N-acetylenolpyruvylglucosamine reductase.

Purified uridine diphosphate N-acetylenolpyruvylglucosamine reductase (E.C. 1.1.1.158) was analyzed by circular dichroism (CD) and UV-visible spectroscopy to establish the spectral properties of its tightly bound flavin adenine dinucleotide (FAD) cofactor. The polypeptide backbone displayed a single circular dichroic minimum at 208 nm and a single maximum at 193 nm. The CD spectrum of bound flavin exhibited a single major negative Cotton peak at 364 nm and two minor negative Cotton peaks at 464 and 495 nm. The protein was reversibly unfolded in 9.8 M urea and refolded in buffer in the presence of excess FAD. The refolded enzyme incorporated FAD and catalyzed full activity. The bound FAD displayed an absorption maximum at 464 nm with an extinction coefficient of epsilon 464 = 11700 M-1 cm-1. Anaerobic reduction with dithionite was complete at 1 equiv. Anaerobic reduction with nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), also was essentially complete at 1 equiv and produced a long-wavelength absorbance band characteristic of an FAD-pyridine nucleotide charge transfer complex. Photochemical bleaching in the presence of ethylenediaminetetraacetic acid (EDTA) followed exponential kinetics. None of the anaerobic reductive titrations produced a spectral intermediate characteristic of a flavin semiquinone, and all reduced enzyme species could be fully reoxidized by oxygen, with full recovery of catalytic activity. Photochemically reduced enzyme was reoxidized by titration with either NADP+ or uridine diphospho N-acetylglucosamine enolpyruvate (UNAGEP). Reoxidation by NADP+ reached a chemical equilibrium, whereas reoxidation by UNAGEP was stoichiometric. Binding of NADP+ or UNAGEP to the oxidized form of the enzyme produced a dead-end complex that could be titrated by following a 10-nm red shift in the absorption spectrum of the bound FAD. The Kd of NADP+ for oxidized enzyme was 0.7 +/- 0.3 microM and the Kd of UNAGEP was 2.7 +/- 0.3 microM. Solvent deuterium isotope effects on binding were observed for both NADP+ and UNAGEP, depending on the pH. At pH 8.5, the HKd/DKd was 2.2 for NADP+ and 3.9 for UNAGEP. No spectral changes were observed in the presence of a 40-fold excess of uridine diphospho N-acetylmuramic acid (UNAM) either aerobically or anaerobically. These studies have identified spectral signals for five steps in the kinetic mechanism, have indicated that product formation is essentially irreversible, and have indicated that hydrogen bonding or protonation contributes significantly to ground-state complex formation with the physiological substrate.

Anaerobiosis↗
PubMed1996

Structural studies of Escherichia coli UDP-N-acetylmuramate:L-alanine ligase.

Uridine diphosphate N-acetylmuramate:L-alanine ligase (EC 6.3.2.8, UNAM:L-Ala ligase or MurC gene product) adds the first amino acid to the sugar moiety of the peptidoglycan precursor, catalyzing one of the essential steps in cell wall biosynthesis for both gram-positive and gram-negative bacteria. Here, we report our studies on the secondary and quaternary structures of UNAM:L-Ala ligase from Escherichia coli. The molecular weight of the purified recombinant enzyme determined by electrospray ionization mass spectrometry agreed well with the molecular weight deduced from the DNA sequence. Through sedimentation equilibrium analysis, we show that the enzyme exists in equilibrium between monomeric and dimeric forms and that the dissociation constant of the dimer, Kd, was determined to be 1.1 +/- 0.4 microM at 37 degrees C and 0.58 +/- 0.30 microM at 4 degrees C. A very similar Kd value was also obtained at 37 degrees C by gel filtration chromatography. The secondary structure of the enzyme was characterized by circular dichroism spectroscopy. No change in the secondary structure was observed between the monomeric and dimeric forms of the enzyme. The activity assays at enzyme concentrations both below and above the determined Kd value lead to the conclusion that the enzyme is active both as dimers and as monomers and that the specific activity is independent of the oligomerization state.

Centrifugation, Isopycnic↗
PubMed1996

Insulin-like growth factor I (IGF-I) and IGF-Binding proteins in rat wound fluid.

Insulin-like growth factors (IGFs) play an important role in tissue repair, including healing of dermal and epidermal injury. In this study we have measured changes in the IGF:IGF-binding protein (IGFBP) profile of rat wound fluid (WF) collected after sc implantation of Hunt-Schilling chambers for 21 days. WF IGF-I levels 1 day after implantation were equivalent to plasma levels, then fell during the first 7 days before recovering to approximately two thirds of plasma levels by day 21. Western ligand blots of whole WF revealed a profile qualitatively similar to that found in plasma, although the intensity of the IGFBP-3 band was significantly less than that in plasma. Neutral gel chromatography of pooled day 14 WF, after in vitro incubation with [125I]IGF-I, separated the radioligand into three distinct regions of 150, 40, and 7.5 kDa. However, compared to plasma recovery of[125I]IGF-I in the 150-kDa region in WF was reduced, and that in the 40-kDa region was increased. Ligand blotting of the WF-derived neutral gel fractions revealed IGFBP-3 within the 150-kDa complex. Incubation of WF with plasma (1:1, vol/vol) resulted in a progressive decline in the intensity of the plasma IGFBP-3 band. Protease inhibitors, including EDTA, antipain, or aprotonin, inhibited this process. We have described the changes over time in WF IGF-I concentrations, characterized the IGFBP profile, and demonstrated the presence of IGFBP-3 proteolytic activity in WF. The latter may play a role in the regulation of IGF bioavailability during the repair process.

Animals↗
PubMed1995

Determination of subunit dissociation constants in native and inactivated CTP synthetase by sedimentation equilibrium.

Sedimentation equilibrium was used to correlate changes in aggregation state with active site modification of Escherichia coli CTP synthetase. The native enzyme equilibrated between monomers, dimers, and tetramers in the absence of substrates. At enzyme concentrations above 5 microM, tetramers represented 40% of the species in solution. Inactivation by 6-diazo-5-oxonorleucine (DON) or thiourea dioxide reduced the amount of tetramer to below detectable limits. However, inactivated enzyme still equilibrated between monomers and dimers. Simultaneous analysis of multispeed data at three protein concentrations yielded estimates of the dissociation constants for the monomer-dimer and dimer-tetramer equilibria. For multiple data sets of native enzyme, K1,2 was between 1 and 2 microM, and K2,4 was between 1 and 18 microM. For DON inactivated enzyme, K1,2 was 3-4 microM, and for thiourea dioxide inactivated enzyme, K1,2 was approximately 1 microM. The values for K1,2 are consistent with previously published studies by gel filtration, demonstrating that the enzyme dissociates to monomers in very dilute solution (Anderson, 1983). However, the sedimentation equilibrium experiments are the first to show that the enzyme forms tetramers in the absence of nucleotides. This result implies the presence of stable conformations in the native enzyme capable of dynamic equilibrium between monomers, dimers, and tetramers. The results presented here illustrate the sensitivity of sedimentation equilibrium for measuring the aggregation state of equilibrating enzyme species and demonstrate that active site modifications disrupt the quaternary structure of CTP synthetase.

Animals↗
PubMed1995

Steady-state kinetic mechanism of Escherichia coli UDP-N-acetylenolpyruvylglucosamine reductase.

The Escherichia coli MurB gene encoding UDP-N-acetylenolpyruvylglucosamine reductase was expressed to a level of approximately 100 mg/L as a fusion construct with maltose binding protein. Rapid affinity purification, proteolysis, and anion exchange chromatography yielded homogeneous enzyme containing 1 mol/mol bound FAD. Enzyme was maximally activated by K+, NH4+, and Rb+ at cation concentrations between 10 and 50 mM. Steady-state enzyme kinetics at pH 8.0 and 37 degrees C revealed weak and strong substrate inhibition by NADPH and UDP-N-acetylenolpyruvylglucosamine, respectively, where the KiS were 910 microM and 73 microM. Substrate inhibition was pH dependent for both substrates. Initial velocity measurements as a function of both substrates produced patterns consistent with a ping pong bi bi double competitive substrate inhibition mechanism. Data at pH 8.0 yielded kinetic constants corresponding to Km,UNAGEP = 24 +/- 3 microM, Ki,UNAGEP = 73 +/- 19 microM, Km,NADPH = 17 +/- 3 microM, Ki,NADPH = 910 +/- 670 microM, and kcat = 62 +/- 3 s-1. A slow anaerobic exchange reaction with thio-NADP+ provided evidence for release of NADP+ in the absence of UNAGEP. Alternate reduced nicotinamide dinucleotides, including NHXDPH, 3'-NADPH, and alpha-NADPH, were substrates, whereas NADH was not. Several nucleotides, including ADP and UDP, were weak inhibitors of the enzyme with inhibition constants between 5 and 97 mM. Various analogs of NADP+, including 3'-NADP+, thio-NADP+, APADP+, NEthDP+, and NHXDP+, were inhibitors of the enzyme with respect to NADPH and yielded inhibition constants in the range of 110-1100 microM. Analogs without the 2'- or 3'-phosphate of NADPH or NADP+ were not substrates or inhibitors. Double inhibition experiments with varied APADP+ and UNAG produced inhibition patterns consistent with mutually exclusive inhibitor binding. The data suggest that NADPH and UNAGEP share a subsite that prevents both molecules from binding at once.

Anaerobiosis↗
PubMed1995

Dimerization of native and C-terminally proteolyzed p56lck tyrosine kinase.

Recombinant p56lck tyrosine kinase was purified to near homogeneity from a baculovirus/insect cell expression system. Treatment with thrombin proteolytically removed the C-terminal 54 amino acids from p56lck. Processed enzyme migrated on sodium dodecyl sulfate (SDS) gels with a M(r) approximately 6,000 lower than intact enzyme. Analytical ultracentrifugation of intact and processed p56lck gave M(r)'s of 62,600 and 56,200, respectively, confirming that the thrombin treated enzyme existed in solution as a processed polypeptide and that there was no anomalous migration in SDS gels due to thrombin treatment. Simultaneous multispeed analysis of sedimentation equilibrium data demonstrated that both intact and processed enzyme can dimerize with a weak binding constant in the range of 200-300 microM. Purified intact p56lck incorporated 2 mol of [32P]P(i) per mole of enzyme. Purified processed p56lck incorporated only 1 mol of [32P]P(i) per mole of enzyme. The loss of 1 mol of [32P]P(i) per mole of enzyme after thrombin deletion of the C-terminus demonstrates that p56lck undergoes autophosphorylation at the C-terminus. The data are consistent with autophosphorylation at tyrosine 505, which has previously been thought to be a regulatory phosphorylation site, but which now must also be considered as an autophosphorylation site.

Amino Acid Sequence↗
PubMed1994

Complete assignment of disulfide bonds in bovine dopamine beta-hydroxylase.

Peptide mapping, chemical sequencing, microbore HPLC/electrospray ionization mass spectrometry (LC/ESI/MS), and matrix-assisted laser desorption mass spectrometry (MALDI/MS) were used to identify the sites of intra- and intermolecular disulfide linkages in bovine dopamine beta-hydroxylase. The enzyme contains 14 cysteines and seven disulfides per monomer. Edman sequencing of tryptic and peptic peptides determined linkages at positions Cys140-Cys582, Cys218-Cys269, Cys255-Cys281, Cys452-Cys474, Cys514-Cys514, and Cys516-Cys516, where cysteines at positions 514 and 516 on one monomer disulfide pair with their homologs on a second monomer. These linkages were confirmed by LC/ESI/MS and MALDI/MS. Further analysis by LC/ESI/MS and MALDI/MS identified linkages at positions Cys376-Cys489 and Cys380-Cys551. Cysteines 140 and 582 form a disulfide linkage that folds the C-terminus back in proximity to the N-terminus. The remaining intramolecular disulfides occur along two separate internal regions of the protein. The density of histidine residues in these two regions suggests binding sites for two Cu2+ atoms per monomer. In addition, previously identified amino acids that react with mechanism-based inactivators occur in these two regions. We propose that these five internal disulfide bonds define two Cu2+ binding domains that make up the active site of a dopamine beta-hydroxylase monomer. Considering previous data on the location of glycosylation sites, mechanism-based inactivation sites, and the disulfide linkages presented here, the data suggest an overall topology were the N- and C-termini are in close proximity and are solvent exposed and where the Cu2+ binding sites are buried in two interior domains stabilized by five disulfide bonds.

Amino Acid Sequence↗
PubMed1994

Child support orders: a perspective on reform.

This article presents a brief historical account of child support reform in the United States during this century. Reform in this area primarily reflects a shift from judicial discretion to administrative regularity. The two predominant types of child support guidelines in use today, income shares and percentage of income, are described and compared. The authors then present information on some of the current issues with regard to child support guideline reform. Finally, a Child Support Assurance system, which would provide a publicly guaranteed minimum benefit award to custodial parents under special circumstances, is proposed. Further discussion of child support reform is presented in the Overview and Analysis section of this journal issue.

Child↗
PubMed1993

Characterization of metal ion activation and inhibition of CTP synthetase.

A reinvestigation of the metal ion activation and specificity of CTP synthetase was begun in order to separate effects due to binding of free metal ions and binding of nucleotide-complexed ions. Apo-CTP synthetase was prepared by dialysis against 5 mM EDTA. Analysis of apo-enzyme by atomic absorption spectroscopy revealed that all bound metal ions could be removed. Thus, apo-enzyme contained no detectable amounts of Mg2+, Mn2+, Cu2+, Zn2+, Co2+, Ni2+, or Fe2+. The half-saturation value of Mg(2+)-dependent enzyme activation was approximately 2.6 mM at a total concentration of 2 mM nucleotides (ATP plus UTP). These data suggest that the enzyme requires more Mg2+ for full catalytic activity than required simply to complex the nucleotide substrates. Analysis of velocity versus [Mg2+]free demonstrated that activity depends on [Mg2+]free. The half-saturation values for [Mg2+]free were 660 and 280 microM in the NH4(+)- and glutamine-dependent assays, respectively. The half-saturation values for [Mn2+]free and [Co2+]free were approximately 2.6 and 3.8 microM in the NH4(+)-dependent assay and 2.8 and 4.7 microM in the glutamine-dependent assay. These results are consistent with the presence of a separate binding site for free metal ion on the enzyme. Over the range of 0.1-10 mM, neither Cu2+, Zn2+, Ni2+, nor Ca2+ activated the enzyme. Also, both Cu2+ and Zn2+ were effective inhibitors of CTP synthetase in the absence of dithiothreitol at concentrations < 50 microM. Inhibition by Zn2+ was reversed by EDTA, whereas inhibition by Cu2+ was not. In the presence of dithiothreitol, Zn2+, Co2+ and Ni2+ inhibited the enzyme at less than 200 microM metal ion.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1992

Inactivation and covalent modification of CTP synthetase by thiourea dioxide.

Thiourea dioxide was used in chemical modification studies to identify functionally important amino acids in Escherichia coli CTP synthetase. Incubation at pH 8.0 in the absence of substrates led to rapid, time dependent, and irreversible inactivation of the enzyme. The second-order rate constant for inactivation was 0.18 M-1 s-1. Inactivation also occurred in the absence of oxygen and in the presence of catalase, thereby ruling out mixed-function oxidation/reduction as the mode of amino acid modification. Saturating concentrations of the substrates ATP and UTP, and the allosteric activator GTP prevented inactivation by thiourea dioxide, whereas saturating concentrations of glutamine (a substrate) did not. The concentration dependence of nucleotide protection revealed cooperative behavior with respect to individual nucleotides and with respect to various combinations of nucleotides. Mixtures of nucleotides afforded greater protection against inactivation than single nucleotides alone, and a combination of the substrates ATP and UTP provided the most protection. The Hill coefficient for nucleotide protection was approximately 2 for ATP, UTP, and GTP. In the presence of 1:1 ratios of ATP:UTP, ATP:GTP, and UTP:GTP, the Hill coefficient was approximately 4 in each case. Fluorescence and circular dichroism measurements indicated that modification by thiourea dioxide causes detectable changes in the structure of the protein. Modification with [14C]thiourea dioxide demonstrated that complete inactivation correlates with incorporation of 3 mol of [14C]thiourea dioxide per mole of CTP synthetase monomer. The specificity of thiourea dioxide for lysine residues indicates that one or more lysines are most likely involved in CTP synthetase activity. The data further indicate that nucleotide binding prevents access to these functionally important residues.

Binding Sites↗
PubMed1991

The effect of modification on the susceptibility of collagen to proteolysis: I. Chemical modification of amino acid side chains.

A series of chemically modified collagens were subjected to proteolysis by lysozomal cathepsins, pepsin and trypsin. Modifications of the collagens included acetylation, succinylation, methylation and borohydride reduction. Changes in the integrity of the materials were also monitored by differential scanning calorimetry (DSC). All modified collagens were implanted intramuscularly to assess their relative biodegradation rates in vivo. Methylation of the collagen showed extensive denaturation as confirmed by DSC, pepsin solublization to small fragments and by increased susceptibility to trypsin. However, methylation and succinylation made little difference to hydrolysis by cathepsins. Acetylation and borohydride reduction gave increased resistance to cathepsins as well as to pepsin, this latter also being found with the succinylated substrate. In-vivo implantation data showed both succinylation and methylation increased the rate of biodegradation but that the other modifications did not affect the rate of breakdown when compared with control unmodified collagen. The results of this study showed that chemical modification of collagen can alter in vivo degradation rates and could aid in designing collagen-based prostheses.

Acetylation↗