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L H Tang

Publications and source records attributed to L H Tang.

90 records · Page 5Linked to original sources
PubMed1987

Thermal and solvent stability of proteoglycan aggregates by quasielastic laser light-scattering.

The dissociation behavior of several species of proteoglycan aggregates (PGA) has been studied quantitatively by monitoring the changes in particle size by dynamic laser light-scattering. Firstly, studies of the thermal dissociation of reconstituted PGA from bovine nasal septum and bovine fetal epiphysus are described. The effect of link protein in stabilizing reconstituted PGA against changes in temperature has been demonstrated. It was also confirmed that, upon prolonged heating at 70 degrees, the dissociation of link-containing, reconstituted PGA is effectively irreversible. Secondly, the dissociation characteristics of native PGA isolated from chick chondrocyte and rat chondrosarcoma cell-cultures were investigated in aqueous solvents containing increasing concentrations of guanidine hydrochloride. It was found that the more densely packed, native, aggregate structure has a higher susceptibility to thermal disruption than the reconstituted, link-containing material. Considerable loss of subunits from the native aggregates occurs at temperature where the link protein retains its activity. The dissociation of native PGA is irreversible, in the sense that reconstituted PGA always exhibits smaller sizes than the native PGA, reflecting a smaller degree of aggregation of the subunits.

Animals↗
PubMed1985

Proteoglycans from bovine nasal and articular cartilages. Fractionation of the link proteins by wheat germ agglutinin affinity chromatography.

Two forms of link protein, 46 and 51 kDa, are present in proteoglycan aggregates from both bovine nasal and bovine articular cartilages. Studies reported here show that the link proteins bind to concanavalin A, Lens culinaris agglutinin, Ricinus communis agglutinin, soybean agglutinin, and wheat germ agglutinin lectins. When the link proteins are eluted from these lectins with appropriate competing sugars, the 46- and the 51-kDa link proteins elute together and no separation is achieved. However, when the link proteins bound to wheat germ agglutinin are eluted with a 0 to 4 M guanidine hydrochloride linear gradient, a good separation of the 46- and 51-kDa link proteins is achieved. Wheat germ agglutinin affinity chromatography has been used on a preparative scale to isolate the 51-kDa link protein from mature bovine articular cartilage to homogeneity, in amounts sufficient to examine its effect on proteoglycan aggregate size and stability in sedimentation velocity studies. Proteoglycan aggregates were reassembled from proteoglycan monomers and hyaluronate in the absence of link protein, in the presence of both 46- and 51-kDa link proteins, and in the presence of the individual 51-kDa link protein. The sizes of the aggregates were compared in terms of sedimentation coefficients (s(0)20). The stability of the aggregates was compared in terms of the per cent aggregate present at pH 7 and 5. At pH 7, the sedimentation coefficients (s(0)20) of link-free aggregates, aggregates formed with both link proteins, and aggregates formed with 51-kDa link protein were 72, 93, and 112 S, respectively. Thus, the 51-kDa link protein has a pronounced effect on aggregate size. The link-free aggregate was grossly unstable, and only 36% aggregate was present at pH 5. The aggregate formed with both link proteins was effectively stabilized against dissociation and 79% aggregate was present at pH 5. The aggregate formed with 51-kDa link protein was not effectively stabilized against dissociation, and only 60% aggregate was present at pH 5. Thus, despite its pronounced effect on aggregate size, the 51-kDa link protein does not effectively stabilize the proteoglycan aggregate against dissociation. These results suggest that the 51-kDa link protein may selectively increase aggregate size, while the 46-kDa link protein may be required to effectively stabilize the proteoglycan aggregate against dissociation.

Animals↗
PubMed1985

Isolation of dermatan sulfate proteoglycans from mature bovine articular cartilages.

Two species of dermatan sulfate proteoglycans, called DS-PGI and DS-PGII, have been isolated from mature bovine articular cartilages. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis at low ionic strength in 0.01 M phosphate the dermatan sulfate proteoglycans appeared as a single polydisperse species whose molecular weight ranged from 80,000 to 140,000. The dermatan sulfate proteoglycans eluted as a single peak on Sepharose CL-4B chromatography in 4 M guanidine hydrochloride and showed no tendency to separate into two components. Following chondroitinase AC and ABC digestion, a core protein was obtained whose molecular weight was 45,000. However, what appeared to be a single dermatan sulfate proteoglycan was consistently separated into two species of distinctly different mobilities by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at high ionic strength in 0.375 M Tris. The molecular weight of the smaller species (DS-PGII) ranged from 87,000 to 120,000. The molecular weight of the larger species (DS-PGI) ranged from 165,000 to 285,000. DS-PGI self-associates in 0.375 M Tris, while DS-PGII does not. This phenomenon was exploited to separate DS-PGI and DS-PGII by preparative electrophoresis on 5 to 20% gradient slab gels. The immunological identities of the individual species, DS-PGI and DS-PGII, were examined by enzyme-linked immunosorbent assay using polyclonal antiserum to cartilage-specific proteoglycan monomer from bovine articular cartilage and polyclonal and monoclonal antibodies to DS-PGII. The polyclonal antiserum to cartilage-specific proteoglycan monomer did not react with DS-PGI or DS-PGII, indicating that DS-PGI and DS-PGII possess different core proteins from cartilage-specific proteoglycan monomer. Polyclonal and monoclonal antibodies raised against the mixture of DS-PGI and DS-PGII reacted strongly with DS-PGII, but weakly or not at all with DS-PGI. These results suggest that DS-PGI and DS-PGII possess different core proteins and may represent two different species of dermatan sulfate proteoglycans.

Animals↗
PubMed1984

Cartilage link proteins. Biochemical and immunochemical studies of isolation and heterogeneity.

Native and clostripain-derived link proteins from proteoglycan aggregates were separated by gel chromatography in 4 M guanidine HCl from low-buoyant-density proteoglycan and proteoglycan hyaluronic acid-binding region after extraction from adult bovine nasal cartilage and the Swarm rat chondrosarcoma. Separations were monitored with Laurell immunoelectrophoresis using precipitating antibodies to link protein, hyaluronic acid-binding region, and low-buoyant-density proteoglycan. These immunoanalyses, with sodium dodecyl sulfate-polyacrylamide gel analyses, revealed the usefulness of this combined approach when assessing the purities and identities of these molecules. Using these isolated link proteins we provide data suggesting the presence of two immunologically detectable forms of link proteins of the same molecular weight. On immunoelectrophoresis both rat and bovine link proteins of a single molecular weight each produced two precipitin reactions with antibody to link protein. They were of similar intensity for native molecules but of different intensity for clostripain-isolated link protein. Isoelectric focusing in 6 M urea revealed that the 48,000 and 44,000 molecular weight link proteins of bovine nasal cartilage together produce a complex pattern of many bands. Link proteins of a single molecular size produced a much simpler, predominantly five-banded focusing pattern. Immunoelectrophoresis of the electrofocused clostripain rat link protein (42,000) revealed that the three major central bands each produced double precipitin reactions. Mixing of native and clostripain-derived link protein in 4 M guanidine HCl followed by dialysis to 6 M urea prior to isoelectric focusing did not change the focusing position of the individual bands. This suggests that the heterogeneity of focusing forms was due to the existence of different isoforms. The double precipitin reactions may be due to the existence of two different conformations expressing different epitopes of the kind reported previously by Thonar et al. (Thonar, E. J.-M. A., Kimura, J. H., Hascall, V. C., and Poole, A. R. (1982) J. Biol. Chem. 257, 14173-14180).

Animals↗
PubMed1984

The effect of link protein on proteoglycan aggregate structure. An electron microscopic study of the molecular architecture and dimensions of proteoglycan aggregates reassembled from the proteoglycan monomers and link proteins of bovine fetal epiphyseal cartilage.

Proteoglycan monomer and link protein were prepared from bovine fetal epiphyseal cartilage. Proteoglycan aggregates were reassembled from proteoglycan monomers and hyaluronic acid in the presence or in the absence of link protein at pH 7 and at pH 5. The proteoglycan solutions were spread on nitrocellulose films and examined by electron microscopy. At pH 7, the aggregates formed in the presence of link protein showed dramatic differences in their dimensions, compared with the link protein-free aggregates. The link protein-containing aggregates were five times longer and contained three times as many monomers per aggregates. The mean distance between monomers was twice as long and the spacing between monomers was more regular in the link protein-containing aggregates. Essentially the same differences between link protein-free and link protein-containing proteoglycan aggregates were observed at pH 5. These results show that link protein increases proteoglycan aggregate size by facilitating the binding of more monomers to hyaluronic acid and influences the spacing of monomers along hyaluronic acid chains.

Animals↗
PubMed1984

Viscoelastic properties of proteoglycan subunits and aggregates in varying solution concentrations.

Using a cone-on-plate mechanical spectrometer, we have measured the linear and non-linear rheological properties of cartilage proteoglycan solutions at concentrations similar to those found in situ. Solutions of bovine nasal cartilage proteoglycan subunits (22S) and aggregates (79S) were studied at concentrations ranging from 10 to 50 mg ml-1. We determined: (1) the complex viscoelastic shear modulus G (omega) under small amplitude (0.02 radians) oscillatory excitation at frequencies (omega) ranging from 1.0 to 20.0 Hz, (2) the non-linear shear rate (gamma) dependent apparent viscosity napp (gamma) in continuous shear, and (3) the non-linear shear rate dependent primary normal stress difference sigma 1 (gamma) in continuous shear. Both the apparent viscosity and normal stress difference were measured over four decades of shear rates ranging from 0.25 to 250 s-1. Analysis of the experimental results were performed using a variety of materially objective non-linear viscoelastic constitutive laws. We found that the non-linear, four-coefficient Oldroyd rate-type model was most effective for describing the measured flow characteristics of proteoglycan subunit and aggregate solutions. Values of the relaxation time lambda 1, retardation time lambda 2, zero shear viscosity no, and nonlinear viscosity parameter muo were computed for the aggregate and subunit solutions at all of the solute concentrations used. The four independent material coefficients showed marked dependence on the two different molecular conformations, i.e. aggregate or subunit, of proteoglycans in solution.

Animals↗
PubMed1980

Proteoglycans from bovine nasal cartilage. Immunochemical studies of link protein.

Antisera have been prepared against purified proteoglycan monomer and link protein from bovine cartilages. The immunological properties of these species have been studied by immunodiffusion, Laurell rocket immunoelectrophoresis, crossed immunoelectrophoresis, hemagglutination, and immunofluorescence. Antisera raised against either adult bovine articular or foetal bovine epiphyseal proteoglycan monomer were monospecific. They did not react with link protein, type II collagen, nor with any other molecular species isolated from bovine articular and epiphyseal cartilages. Antisera to link protein, purified from bovine nasal cartilage by dissociative density gradient centrifugation followed by chromatography under dissociative conditions on Sephacryl S-200, showed no reaction with proteoglycan monomer by immunodiffusion. However, antisera to link protein showed a weak reaction with native monomer on Laurell rocket immunoelectrophoresis (not detectable when chondroitinase ABC-treated monomer was used), and with chondroitinase ABC-treated monomer by hemagglutination assay. These traces of antibody to proteoglycan monomer were inactivated by immunoreaction with proteoglycan monomer in solution to provide a monospecific antiserum to link protein. Purified antibodies specific for proteoglycan monomer, some of which did not react with the hyaluronic acid binding region of bovine nasal monomer, were prepared from anti-link sera by affinity chromatography using chondroitinase ABC-treated bovine nasal proteoglycan monomer. Purified link protein, which contains 48,000- and 44,000-dalton subunits, contains two immunologically distinguishable species. An immunoassay for purified link protein was developed using Laurell rocket electrophoresis. A micro-method, using crossed-immunoelectrophoresis, for studying the binding of link protein to proteoglycan monomer and to hyaluronate is described. The complex of link protein bound to hyaluronate was dissociated on reaction of link protein with antibody.

Animals↗
PubMed1980

Localization of proteoglycan monomer and link protein in the matrix of bovine articular cartilage: An immunohistochemical study.

Using monospecific antisera and immunofluorescence microscopy, proteoglycan monomer (PG), and link proteins were demonstrated throughout the extracellular matrix of bovine articular cartilage. A narrow band of strong pericellular staining was usually observed for both molecules, indicating a pericellular concentration of proteoglycan monomer: this conclusion was supported by dye-binding studies. Whereas PG was evenly distributed throughout the remaining matrix, more link protein was detectable in interterritorial sites in middle and deep zones. Well-defined zones of weaker territorial staining for link protein stained strongest for chondroitin sulfate. Trypsin treatment of cartilage resulted in a loss of most of the PG staining, but some selective retention of link protein, particularly around chondrocytes in the superficial zone at and near the articular surface. This residual staining was largely removed if sections were fixed after chondroitinase treatment. After extraction of cartilage with 4M guanidine hydrochloride, only PG remained and this was concentrated in the superficial zone. These observations are shown to support the concept of aggregation of PG and link protein with hyaluronic acid (HA) in cartilage matrix, and the binding of PG and link protein to HA, which is attached to the chondrocyte surface. Culture of cartilage depleted of PG and link protein by trypsin demonstrated that individual chondrocytes can secrete both PG and link proteins and that the organization of cartilage matrix can be regenerated in part over a period of 4 days.

Animals↗
PubMed1977

Analysis of various indefinite self-associations.

Two methods have been developed for the analysis of four types of indefinite self-associations. Unlike previous treatments by others, the procedures can be applied to nonideal cases. The two methods were first tested with simulated data, and it was found that one could indeed distinguish between the four types of indefinite self-associations. For a more realistic test, sedimentation equilibrium experiments were performed on solutions of beta-lactoglobulin A at 16 degrees C in 0.15 ionic strength acetate buffer, pH 4.65. The self-association of the beta-lactoglobulin A was best described by either method as a sequential indefinite self-association having two equilibrium constants and one second virial coefficient.

Buffers↗
PubMed1976

A comparative study on bovine alpha-lactalbumin and lysozyme by nanosecond fluorometry.

Analysis of the time decay of fluorescence anisotropy of 1-dimethylaminoaphthalene-5-sulfonyl (DNS) and fluorescamine derivatives of bovine alpha-lactalbumin and lysozyme reveals that no significant differences in mean rotational relaxation times are present. While fluorescamine molecules appear to orient randomly on these proteins, DNS is bound with a preferential orientation. Other fluorescence characteristics of the labels are also cited.

Animals↗
PubMedDate not supplied

Small GTP-binding proteins in parietal cells: candidate modulators of parietal cell membrane dynamics.

The stimulated fusion of intracellular H/K-ATPase-containing tubulovesicles with a target canalicular membrane surface is central to the process of acid secretion. A super-family of small GTP-binding proteins (smGTPBPs) has been implicated in many aspects of intracellular dynamics and vesicle membrane trafficking. We have investigated the presence of smGTPBPs in isolated rabbit parietal cells. Parietal cells possess a number of smGTPBP species with molecular masses of 18-28 kDa. One 23 kDa smGTPBP has been localized to tubulovesicles and identified immunochemically as rab2. Rab2 redistributes during stimulation in concert with the movement of the H/K-ATPase. The results demonstrate that specific smGTPBPs are associated with the parietal cell secretory apparatus. Small GTP-binding proteins are important candidate regulators of parietal secretory membrane dynamics.

Animals↗
PubMedDate not supplied

Autoregulation of enterochromaffin-like cell histamine secretion via the histamine 3 receptor subtype.

INTRODUCTION: The neuroendocrine histamine-secreting cell of the gastric fundus, the enferochromaffin-like cell, is the principal regulator of parietal cell acid secretion. We have proposed that histamine may regulate its own synthesis and release via an autocrine mechanism. The purpose of this study was to evaluate the role of the histamine receptor subtypes H1, H2 and H3 in the regulation of this phenomenon. METHODS: Purified ECL cells were isolated by pronase digestion and EDTA exposure of the rat stomach, followed by particle size and density separation using counterflow elutriation and Nycodenz gradient centrifugation, 24-hr cultured cells were pretreated for 30 min with the agents; H1 receptor agonist (2-[(3-trimethyl)-diphenyl] histamine) (TMPH), H1 receptor antagonist (terfenadine); H2 receptor agonist (dimaprit) or antagonist (cimetidine or loxitidine); or H3 receptor agonist (imetit) or antagonist (thioperamide) (all tested, 10(-10)-10(-6) M). Gastrin was then used to stimulate histamine secretion. Histamine secretion was quantified by specific enzyme-immunoassay. RESULTS: Basal histamine secretion was 2.7 +/- 0.14 nmol/10(3) cells. Gastrin-stimulated (10 nM) levels were 4.6 +/- 0.4 nmol/10(3) cells (p < .01). TMPH inhibited both basal and gastrin driven histamine secretion with a maximal effect (34 percent) (1.78 +/- 0.08 nmol/10(3) cells) and an IC50 of > 5 x 10(-7) M. H1 receptor antagonism did not alter histamine secretion alone or in combination with gastrin. Neither H2 receptor stimulation nor antagonism had any effect on histamine secretion alone or in combination with gastrin. Gastrin-induced histamine secretion was dose-dependently inhibited by imetit (H3 agonist) with a maximal effect (2.4 +/- 0.6 nmol/10(3) cells) (p < .05) and an IC50 of 10(-9) M. Conversely, Thioperamide (H3 antagonist) dose-dependently augmented gastrin-stimulated histamine secretion with a maximum effect (5.7 +/- 0.5 nmol/10(3) cells) (p < .05) at 10(-8) M and an EC50 of 7 x 10(-10) M. CONCLUSION: These data are consistent with the presence of an H3 receptor on the ECL cell which modulates gastrin-stimulated histamine secretion. Our observations support the proposal that a histamine-mediated short-loop autocrine regulatory mechanism of ECL cell secretion exists.

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