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Biomedical subjects

J D Gregory

Publications and source records attributed to J D Gregory.

At least 19 recordsLinked to original sources
PubMed1997

Effect of proteoglycan removal on solute mobility in articular cartilage.

Transport of nutrients, cytokines, pharmacologic agents, and matrix components through articular cartilage is critical for the viability and structural integrity of the tissue. To understand the role of the extracellular matrix in regulating this process, we measured the diffusivity of three uncharged solutes of different molecular size (glucose, MW 180; inulin, MW 5000; dextran, MW 70,000) into intact cartilage and cartilage that had its proteoglycan (PG) component removed. Solute diffusivity was measured by performing transient (nonsteady state) one-dimensional diffusion tests using radiolabelled solutes. Compared to intact cartilage, the diffusivity of glucose was unchanged after PG removal, inulin was unchanged but dextran increased by 1.7 times after 71% PG removal, and both inulin and dextran increased by 1.6 and 2.0 times, respectively, after 93% PG removal. The diffusivities of inulin and dextran were inversely proportional to the PG content. While no change was found in the tissue's bulk fluid content, PG depletion resulted in an increase in fluid content in the upper regions of the tissue and a decrease in the lower regions. These results indicate that in intact tissue small uncharged solutes have free mobility through the inter-molecular and intra-molecular PG volumes, larger molecules have limited intra-molecular mobility, and very large molecules are excluded from the intra-molecular space.

Animals↗
PubMed1995

Kinetic, binding, and NMR studies of perdeuterated yeast phosphoglycerate kinase and its interactions with substrates.

Perdeuterated yeast phosphoglycerate kinase (2HPGK) was prepared from yeast cells grown in 99.9% 2H2O and an acid hydrolysate from deuterated algal cells. Kinetic and binding studies suggested that perdeuterated enzyme was similar to the isotopically normal PGK. The use of 2HPGK not only eliminated the spectral overlap between the enzyme and substrate nuclear Overhauser effect (NOE) cross-peaks, but also permitted observation of weak transfer NOE cross-peaks between the substrate protons that are greater than 4 A apart. Intensity of NOE cross-peaks was used to determine the interproton distances of enzyme-bound Mg(II)dATP. These distances were then used in model building studies to determine the conformation of Mg(II)dATP. The average conformation of enzyme-bound dATP is anti with O4'/C2' endo ribose pucker and trans, gauche about the C4'-C5' bond. Although many spin diffusion pathways were eliminated by protein deuteration, spin diffusion was still observed between the protons of the substrate at mixing times longer than 25 ms.

Binding Sites↗
PubMed1994

Substrate activity of Rh(III)ATP with phosphoglycerate kinase and the role of the metal ion in catalysis.

An exchange-inert Rh(H2O)4ATP complex showed a one-turnover substrate activity with yeast phosphoglycerate kinase. Transfer of the phosphoryl group between ATP and 3-phosphoglycerate occurs with both substrates in the coordination sphere of the metal ion. Because of the slow ligand exchange rates of Rh3+, the reaction product 1,3-diphosphoglycerate (1,3-dPGA) remained coordinated to the metal ion. During the course of the reaction, the enzyme was inactivated, suggesting that the metal ion is coordinated to a protein side chain. Thus the product Rh(H2O)nADP.1,3-dPGA remained bound to the enzyme even after removal of excess substrate. These results suggested that the metal ion may not only act as an electron sink to activate the electrophile, but it may also help to optimally align both substrates for phosphoryl transfer by coordination to both substrates. It is therefore likely that entry of 3-phospho-D-glycerate into the coordination sphere of metal of a metal-ATP complex may start the proposed hinge-bending motion of yeast phosphoglycerate kinase to form a "closed" active site between the two substrate binding domains of the enzyme.

Adenosine Triphosphate↗
PubMed1993

Arrangement of substrates at the active site of yeast phosphoglycerate kinase. Effect of sulfate ion.

beta, gamma-Bidentate CrATP, a stable exchange-inert metal-nucleotide analog, was used as a probe of the conformation of the substrates at the active site of yeast phosphoglycerate kinase (PGK). The paramagnetic effects of Cr3+ on the longitudinal relaxation rates (1/T1p) of the 1H, 31P, and 13C nuclei of 3-phospho-D-glycerate (3PGA) were examined to determine the distances between enzyme-bound CrATP and the nuclei of 3PGA in the ternary PGK.CrATP.3PGA complex. Kinetic studies indicated that Ki(CrATP) was dependent on the concentration of 3PGA that was abolished in the presence of activator sulfate ion. Similarly, NMR studies showed that the environment of CrATP bound at the active site was altered by the presence of sulfate ion. The determined metal-to-nucleus distances show the close proximity of the substrates at the active site of PGK, which is consistent with a hinge-bending mechanism of the enzyme for bringing the two substrates together. Additionally, the presence of an activating concentration of sulfate was observed to cause significant changes in both the relative Cr(3+)-to-3PGA distances and the overall conformation of 3PGA when compared to the findings in the absence of sulfate. This is consistent with the active site being in a "less closed" configuration when sulfate is present. Under both conditions, models can be constructed that allow for direct transfer of a phosphoryl group between the bound substrates.

Adenosine Triphosphate↗
PubMed1993

Proteoglycans in cultures of skin fibroblasts in recessive dystrophic epidermolysis bullosa.

The proteoglycans of cultured fibroblasts from the skin of three patients with recessive dystrophic epidermolysis bullosa and three normals were compared after labeling with [35S]sulfate and [3H]leucine. The behavior in gel chromatography of the intact proteoglycans and several properties of their component glycosaminoglycans (size, content of iduronic acid, and content of 4- and 6-sulfate) showed no statistical differences. In addition, the binding of intact proteoglycans and of their constituent proteins and glycosaminoglycans to type I collagen were measured by affinity chromatography. No differences were found that could account for the skin lesions in recessive dystrophic epidermolysis bullosa.

Adolescent↗
PubMed1992

Inactivation of yeast phosphoglycerate kinase by Cr-ATP complexes and its implications on the conformation of the enzyme active site.

Exchange-inert beta, gamma-bidentate Cr(H2O)x(NH3)y ATP complexes inactivate yeast phosphoglycerate kinase (PGK) by forming a coordination complex at the enzyme active site. The observed inactivation rates ranged from 0.019 min-1 to 0.118 min-1 for Cr(NH3)4ATP and Cr(H2O)4ATP, respectively. Incorporation of one mol of Cr-ATP to the enzyme was sufficient for complete inactivation of the enzyme. The presence of Mg-ATP protected the enzyme against inactivation by Cr-ATP. The other substrate 3-phosphoglycerate (3-PGA), when present, reduced the observed inactivation rates. The reduction of the k(obs) by 3-PGA was proportional to the number of NH3 ligands present in the coordination sphere of Cr3+ in the Cr-ATP complex, suggesting that in the ternary enzyme-Cr-ATP-3-PGA complex 3-PGA may be coordinated to the metal ion. When the effector sulfate ion was present, the presence of 3-PGA did not cause any further effects on the observed inactivation rates. This suggests that bound substrates are in a different arrangement at the active site when sulfate is present and therefore 3-PGA may not need to displace a ligand from Cr3+. Additionally, PGK exhibited a stereoselectivity for the binding of Cr(H2O)4ATP. delta diastereomer of Cr(H2O)4ATP yielded an order of magnitude smaller Ki value compared to the value observed with the lambda isomer. The recovery of enzyme activity was observed over a period of a few hours upon removal of excess Cr-ATP. The presence of substrates and/or effector ion sulfate did not alter the observed reactivation rate. There was no difference in the reactivation rates of the enzyme which was inactivated with Cr(H2O)4ATP or Cr(NH3)4ATP with and without 3-PGA. Increasing the ligand exchange rates of Cr3+ of Cr-ATP by increasing the pH value of the recovery medium from 5.9 to 6.8 increased the rate of recovery by a factor of 8. The pH dependence of the reactivation indicated that one hydroxyl group is involved in the recovery of the enzyme activity in enzyme CrATP and enzyme.CrATP.3-PGA complexes.

Adenosine Triphosphate↗
PubMed1991

Arrangement of the substrates at the active site of brain pyridoxal kinase.

The distances between enzyme-bound paramagnetic CrATP (a stable, beta, gamma-bidentate complex of Cr3+ and ATP) at the active site of sheep brain pyridoxal kinase and the protons of bound inhibitor 4-dPyr (4-deoxypyridoxine) were determined in the ternary enzyme-CrATP.4-dPyr complex by measuring the paramagnetic effects of Cr3+ on the longitudinal relaxation rates (1/T1p) of the protons of 4-dPyr. The correlation time for the Cr(3+)-4-dPyr dipolar interaction on the enzyme was estimated as 1.59 ns by the frequency dependence of 1/T1p of water protons. Temperature dependence of 1/T1p values indicated the fast exchange of 4-dPyr from the paramagnetic enzyme.CrATP.4-dPyr complex; hence the measured 1/T1p values can be used for metalnucleus distance determinations. The distances from the Cr3+ of the enzyme-bound CrATP to the 2-methyl (7.19 A), 4-methyl (7.18 A), and H6 proton (6.18 A) of the 4-dPyr are too great to permit a direct coordination of any group from 4-dPyr. However, these distances can be built into a model in which phosphorus of the gamma-phosphoryl group of ATP is 4 A away from the oxygen atom of the 5-CH2OH group of the 4-dPyr. This suggests that phosphorylation of pyridoxal can occur via direct transfer of the phosphoryl group between the bound substrates at the active site of pyridoxal kinase.

Adenosine Triphosphate↗
PubMed1990

Examination of corneal proteoglycans and glycosaminoglycans by rotary shadowing and electron microscopy.

Proteoglycans (PGs) from cornea and their relevant glycosaminoglycan (GAG) chains, dermatan sulphate (DS) and keratin sulphate (KS), were examined by electron microscopy following rotary shadowing, and compared with hyaluronan (HA), chondroitin sulphate (CS), alginate, heparin, heparan sulphate (HS) and methyl cellulose. Corneal DS PG had the tadpole shape previously seen in scleral DS FG, and the images from corneal KS PG could be interpreted similarly, although the GAG (KS) chains were very much fainter than those of DS PG GAG. Isolated GAG (KS, DS, CS, HA, etc.) examined in the same way showed images that decreased very significantly in clarity and contrast, in the sequence HA greater than DS greater than CS greater than KS. The presence of secondary and tertiary structures in the GAGs may be at least partly responsible for these variations. HA appeared to be double stranded, and DS frequently self-aggregated, KS and HS showed tendencies to coil into globular shapes. It is concluded that it is unsafe to assume the absence of GAGs, based on these techniques, and quantitative measurements of length may be subject to error. The results on corneal DS PG confirm and extend the hypothesis that PGs specifically associated with collagen fibrils are tadpole shaped.

Alginates↗
PubMed1990

Biochemical analyses of proteoglycans in rabbit corneal scars.

Macromolecules from normal rabbit cornea and cornea containing a 2-mm diameter button of scar tissue were biosynthetically labeled with 35S-sulfate and 3H-glucosamine in vivo and in organ culture. Labeled macromolecules, including proteoglycans (PGs) extracted from the normal cornea, scar tissue, and corneal tissue adjacent to the scar with guanidine hydrochloride were chromatographed on DEAE-Sepharose CL-6B columns and eluted with increasing concentrations of NaCl. The elution pattern of corneal macromolecules synthesized in vitro was remarkably similar to that in vivo. In another experiment, corneas having 2-, 4-, and 8-week-old scars were labeled in organ culture and also extracted. Scars synthesized PGs with lower sulfation than those of adjacent corneal tissue. Although PG synthesis in scar decreased with wound age, the synthesis in adjacent cornea remained the same. In a third experiment, PGs extracted from pools of unlabeled 2- and 4-week-old scars, adjacent corneal tissue, and normal corneas were chromatographed on ion-exchange columns and analyzed chemically. The quantity of PGs in scar and adjacent cornea increased with healing time. The ratios of keratan sulfate PG to dermatan sulfate PG in normal cornea, scar, and adjacent cornea was 2.3, 0.6, and 1.5, respectively. The PGs from adjacent corneal tissue had a higher charge density than those from scar. The predominant adjacent-cornea dermatan sulfate PG had a higher charge density than that in normal cornea. The authors conclude that cornea adjacent to the healing wound synthesized PGs measurably different fro those in scar and normal cornea.

Animals↗
PubMed1989

Secondary structure of a core protein from pig skin proteodermatan sulfate: CD and Fourier transform IR spectroscopic studies in solution.

The secondary structure of a 38 kDa core protein from pig skin proteodermatan sulfate (PDS), was investigated in solution using CD and Fourier transform (FT) ir spectroscopy. Both techniques generally have provided complementary data on the secondary structures of proteins. CD spectral analysis has shown that the core protein contains 60% beta-turn and alpha-helical structures, the rest being "unordered" structure. FT ir data do not permit calculation of quantitative contributions of substructures, at the present time, to the overall secondary structure of the core protein. CD spectrum of the intact PDS is similar to the core protein CD spectrum.

Animals↗
PubMed1988

Organ culture of rabbit cornea: morphological analyses.

Tissues maintained in vitro often undergo changes in the pattern of protein synthesis that result in the deposition of macromolecules quantitatively or qualitatively unrelated to those normally synthesized. In these preliminary studies, we modified published techniques to maintain adult and neonate rabbit corneas in vitro for 24 to 48 h. Measurements of corneal wet weight, and histologic and ultrastructural analyses were made to determine the success of maintaining rabbit corneas in culture. The results show that rabbit corneas can maintain normal corneal hydration and tissue structure for at least 48 h when incubated in Coon's modification of Ham's F12, 5% fetal or newborn calf serum, 2 mM glutamine, and 2% chondroitin sulfate or 2% 50 kDa dextran sulfate at 37 degrees C in a 5% CO2/air atmosphere. In addition, we confirmed previous observations that corneal explants must have a 1 to 2 mm rim of limbal sclera, and the organ placed in the culture dish with the epithelial side down to guard against damage and insure endothelial functioning. Normal ultrastructure of neonate rabbit cornea is also maintained when organ-cultured with these procedures. Moreover, neonate corneas continue to synthesize collagen in culture.

Animals↗
PubMed1988

Monoclonal antibodies to heparan sulfate proteoglycan: development and application to the study of normal tissue and pathologic human kidney biopsies.

Monoclonal antibodies (mAbs) (4F2 and 7E12) were prepared against heparan sulfate proteoglycan (HSPG) isolated from bovine glomeruli. Enzyme linked immunosorbent assays (ELISA) and immunoblotting demonstrated that the mABs reacted with HSPG. Indirect immunofluorescence (IF) showed that the mAbs stained renal basement membranes (BMs) and BMs in other organs of normal bovine and human tissues in patterns typical of HSPG. Immunoinhibition studies, and immunoblotting of heparan lyase digested HSPG, indicated that the mAbs recognize HSPG core protein. In kidney biopsies from patients with acute poststreptococcal GN, intact linear glomerular BM (GBM) staining for HSPG was noted despite markedly widened capillary loops. In membranous and in diffuse proliferative lupus GN, loss of HSPG staining was demonstrated at sites of immunodeposition of IgG or C3, while increased staining for HSPG was noted in areas of newly formed GBM. Extensive loss of HSPG was seen in areas of glomerular sclerosis and necrosis. In biopsies from patients with minimal change glomerulonephritis (GN) and mesangioproliferative lupus GN, a normal linear GBM distribution of HSPG was noted. The findings are discussed in the context of current knowledge regarding the pathogenesis of glomerular injury. MAbs to BM HSPG should prove useful for future immunochemical studies, and for the study of diseases of the basement membrane.

Animals↗
PubMed1988

Developmental changes in proteoglycans of rabbit corneal stroma.

Proteoglycans have been extracted from rabbit corneal stromas at developmental stages from fetal to adult. Ion exchange fractionation and gel chromatography show that proteodermatan sulfates decrease in sulfation and relative amount and proteokeratan sulfates increase in sulfation and relative amount during development. There are small increases in size of all of the proteoglycans up to 2 weeks after birth, and the final adult composition is achieved by 8 weeks.

Animals↗
PubMed1986

Ocular distribution of keratan sulfates during pre- and postnatal development in rabbits.

Twenty-six monoclonal antibodies (MAbs) developed against rabbit corneal proteokeratan sulfate (PKS), were used to evaluate immunohistochemically the ocular distribution of PKS during prenatal and early postnatal development in rabbits. These MAbs were directed against epitopes located in the keratan sulfate (KS) chains of the proteoglycan (SundarRaj et al., 1985). Staining of cryostat sections of the eyes was carried out using an indirect peroxidase-conjugated technique. Only one of the MAbs reacted with the presumptive corneal region at day 13 or 16 of fetal development. By day 20, more MAbs reacted with the corneal stroma. There were distinct differences, however, in the distribution of the epitopes recognized by the various MAbs. A few of them stained only the posterior region of the cornea, whereas others showed a decreasing staining gradient from the posterior to the anterior region. By day 24, all of the MAbs reacted with the corneal stroma, but some reacted also with the limbal region and with the conjunctival stromal matrix. One MAb also reacted with the conjunctival epithelial layer, but only at this stage of development. Conjunctival staining was more intense at day 28 of fetal development and at day 2 postnatally. KS was not detectable in the conjunctiva of adult rabbits with any of the MABs. These results suggest that although KS synthesis starts at very early stages of fetal development, there are progressive changes in its antigenic structure in specific regions of the cornea and conjunctiva during corneal development.

Animals↗
PubMed1985

Sera from patients with poststreptococcal glomerulonephritis contain antibodies to glomerular heparan sulfate proteoglycan.

Antibodies, found in human sera from patients with poststreptococcal glomerulonephritis, against proteoglycans (PG) derived from bovine and human glomeruli were investigated. PG were isolated by 4 M guanidine-HCl extraction of whole glomeruli, followed by DEAE-Sepharose CL-6B ion exchange chromatography. The anionic fractions were further purified by chromatography on Sepharose CL-4B. Biochemical analysis of the two resulting peaks revealed the presence of high molecular weight anionic material containing protein, uronic acid, glucosamine, and galactosamine. Enzymatic and chemical susceptibilities indicated the presence of heparan sulfate PG and a galactosamine-containing PG. Immunologic studies revealed the presence of anti-PG antibodies to both PG peaks of the Sepharose CL-4B column in glomerulonephritis sera. Inhibition studies using an ELISA demonstrated that heparan sulfate was a major antigenic determinant. Cross-reactivity with both mammalian and streptococcal hyaluronate was noted. Inhibition studies also indicated the presence of a second antigenic site containing N-acetylgalactosamine, possibly representing chondroitin or dermatan sulfate PG.

Acute Disease↗
PubMed1985

Monoclonal antibodies to proteokeratan sulfate of rabbit corneal stroma.

Hybridomas were developed that secreted monoclonal antibodies against two proteokeratan sulfates (PKS) from rabbit corneal stroma. A total of 28 antibodies were isolated, all of the IgG type with kappa light chains. All were found to react with both PKSs. As judged by immunohistochemical staining, none of them reacted with scleral or conjunctival components, nor with sections of skin, but all reacted with nasal cartilage. When tested by enzyme-linked immunosorbent assays, against components of the proteoglycans, all of the antibodies reacted with keratan sulfate-peptide (isolated from papain digests of PKS or of cartilage proteoglycan), and all but two reacted with oligosaccharide-containing protein cores (prepared by keratanase treatment of PKS). Reactivity with cores was probably due to residual portions of the keratan sulfate chains since the endogenous oligosaccharide-peptides (non-sulfated, non-keratan sulfate oligosaccharides isolated after papain digestion of PKS) were not active. None of the antibodies reacted with protein cores made by removal of carbohydrate by hydrolysis with trifluoromethanesulfonic acid. All except one reacted with fragments of keratan sulfate (made by keratanase digestion). These observations are evidence for structural requirements at three different levels of completeness of the antigen for recognition among the various antibodies. In addition, none of the antibodies reacted immunohistochemically with macular dystrophic corneas, confirming the finding of others that the defect lies in the keratan sulfate portion of the proteoglycans.

Animals↗
PubMed1984

Dermatophytes identified at the Australian National Reference Laboratory in Medical Mycology 1966-1982.

Over a 17 yr period from 1966 to 1982, 4354 dermatophytes were identified at the Australian National Reference Laboratory in Medical Mycology. The most frequently identified species was Trichophyton rubrum, accounting for 35.3% of identifications, followed by Trichophyton mentagrophytes (26.5%), Trichophyton tonsurans (12.8%), Epidermophyton floccosum (10.7%) and Microsporum canis (8.4%). Specimens taken from the feet were the most common source of the Trichophyton rubrum isolates, followed by specimens from the groin. The highest incidence of T. rubrum and other Trichophyton infections was found in males in the age group 21-30 yr. Microsporum species were most commonly isolated from children aged 10 yr or less. In 1.5% of specimens, more than one fungal species was isolated. In most of these instances a yeast (predominantly Candida species) was found in addition to a Trichophyton species.

Adolescent↗