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Biomedical subjects

D J Klein

Publications and source records attributed to D J Klein.

At least 37 records · Page 2Linked to original sources
PubMed1996

Prospective randomized trial of vitrectomy or observation for stage 2 macular holes. Vitrectomy for Macular Hole Study Group.

PURPOSE: To determine the risks and benefits of vitrectomy surgery in eyes with stage 2 macular holes. METHODS: A multicentered, controlled, randomized clinical trial was performed with participation of 16 community and university-based ophthalmology clinics. Thirty-six eyes with stage 2 macular holes and 12 months of follow-up were studied. Pars plana vitrectomy with separation of the posterior hyaloid membrane and intraocular injection of perfluoropropane (C3F8) was followed by postoperative face-down positioning for two weeks. This protocol was compared with observation alone. Outcome variables included anatomic closure of the macular hole, macular hole size, and four standardized measures of vision. RESULTS: At 12 months, 15 (71%) of 21 eyes randomly assigned to observation progressed to stages 3 or 4, compared with three (20%) of 15 eyes randomly assigned to surgery (P < .006). Compared with eyes randomly assigned to observation, eyes randomly assigned to surgery had significantly smaller hole diameters (P < .01) and significantly better visual acuity outcomes, as measured by the Word Reading (P = .02) and Potential Acuity Meter (P = .002) charts. No significant differences were found for the Early Treatment Diabetic Retinopathy Study chart and Contrast Sensitivity test. CONCLUSION: Compared with observation alone, surgical intervention in stage 2 macular holes resulted in a significantly lower incidence of hole enlargement and appeared to be associated with better outcome in some measures of visual acuity.

Aged↗
PubMed1995

Laminin decreases PRL and IGFBP-1 expression during in vitro decidualization of human endometrial stromal cells.

The expression of laminin, a major constituent of endometrial cell basement membranes, is increased during differentiation of human endometrial stromal cells (decidualization). To determine whether laminin plays a role in decidualization, we studied the effects of laminin substrate on the synthesis and release of prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1), two major secretory proteins of decidualized stromal cells. Endometrial stromal cells were plated on laminin as well as several other extracellular matrix (ECM) proteins (types 1 and IV collagen or fibronectin) and on plastic, and cultured in media containing medroxyprogesterone acetate (MPA) and estradiol. Cells cultured on plastic or ECM proteins displayed similar morphological changes indicative of decidualization. However, the release of PRL and IGFBP-1 from cells cultured on plastic and ECM proteins (types 1 and IV collagen and fibronection) was approximately 2.1-fold and 2.8-fold greater respectively, than from cells cultured on laminin. The decrease in PRL and IGFBP-1 expression in cells cultured on laminin was not due to differences in initial cell attachment efficiency or final DNA content. In addition, laminin had no effect on the content of laminin protein or fibronectin mRNA levels, indicating that the effects of laminin on PRL and IGFBP-1 were specific. PGE2 stimulated the release of PRL and IGFBP-1 from cells cultured on laminin to levels comparable to those from cells cultured on plastic or other ECM proteins. This indicates that the decrease in PRL and IGFBP-1 release by laminin was not due to a generalized unresponsiveness. In contrast to the effects of laminin during decidualization, PRL expression was not altered by laminin in terminally differentiated decidual cells isolated at term. Our results support a role for laminin in selectively regulating PRL and IGFBP-1 gene expression during in vitro decidualization of human endometrial stromal cells.

Carrier Proteins↗
PubMed1995

Proteoglycan synthesis by bovine myocardial endothelial cells is increased by long-term exposure to high concentrations of glucose.

The role of the metabolic milieu in control of proteoglycan synthesis was investigated using bovine myocardial endothelial cells (BMEC) grown for six to eight passages in media containing either 5.6 or 25 mM glucose. Macromolecular Na[35S]sulfate incorporation into proteoglycans was increased by exposure to 25 mM when compared with 5.6 mM glucose (7.05 +/- 0.40 [SD] vs. 3.5 +/- 0.50 x 10(-4) dpm/microgram DNA). In contrast, [3H]leucine incorporation was unaffected by glucose (11.27 +/- 0.85 vs. 9.88 +/- 1.23 x 10(-5) dpm/microgram DNA). The distribution of isotopes between media and cell layer fractions was not different in the two conditions. Addition of 19.4 mM mannitol to 5.6 mM glucose containing media had no effect on isotope incorporation. The HPLC-DEAE and Sepharose CL-6B elution profiles of media 35S-proteoglycans synthesized under each condition were similar. A Sepharose CL-4B Kav 0.08 heparan sulfate proteoglycan accounted for 20% of the total 35S-incorporation. Perlecan domain III mRNA was identified by Northern analysis and domain 1 by the polymerase chain reaction (PCR) in total BMEC RNA. A mixture of chondroitin/dermatan sulfate proteoglycans accounted for 67% of 35S-incorporation. They eluted from Sepharose CL-6B at Kav 0 and 0.22. Two [3H]leucine labeled core proteins of 135 and 50 kD were identified in each of these 35S-proteoglycan peaks. Biglycan but not decorin mRNAs were detected by Northern analysis and by PCR. These data demonstrate that prolonged exposure to high glucose concentrations in vitro stimulate the accumulation of [35S]sulfate into microvascular endothelial cell proteoglycans without significant alterations in their overall hydrodynamic or charge related properties. Modulation of proteoglycan synthesis by glucose may participate in the pathogenesis of the small vessel complications of diabetes.

Animals↗
PubMed1994

Insulin-like growth factor-binding protein-3 (IGFBP-3) concentration in rat Sertoli cell-conditioned medium is regulated by a pathway involving association of IGFBP-3 with cell surface proteoglycans.

Rat Sertoli cells in culture produce insulin-like growth factor-I (IGF-I), and IGF-binding protein-3 (IGFBP-3) is the predominant IGFBP. Previous studies suggested that the concentration of IGFBP-3 in Sertoli cell-conditioned medium is regulated by a clearance pathway involving association of IGFBP-3 with the Sertoli cell surface. The purpose of these studies was to characterize further the nature of the interaction of IGFBP-3 with the cell surface and to examine how this interaction might be regulating IGFBP-3 concentrations in conditioned medium. Recombinant nonglycosylated human IGFBP-3 was added to cultured Sertoli cells derived from 15-day-old rats, and the change in concentration over time in the medium was measured by [125I]IGF-I ligand blot analysis and Western blot analysis using an antiserum specific for human IGFBP-3. Chloroquine, an inhibitor of lysosomal enzymes involved in internalization of proteins and reduction of temperature, inhibited IGFBP-3 reduction in Sertoli cell medium. Soluble heparin, at a half-maximal concentration of approximately 5 microgram/ml, inhibited the decrease in IGFBP-3 in the medium. In addition, soluble heparin decreased the amount of IGFBP-3 detectable on the cell surface, as determined by performing ligand blots on plasma membrane preparations. Finally, pretreatment of Sertoli cells with sodium chlorate, an inhibitor of cell surface proteoglycan sulfation, also retarded the decrease in recombinant IGFBP-3. Taken together, these data suggest that an important mechanism influencing the concentration of IGFBP-3 3 in Sertoli cell-conditioned medium is a pathway involving IGFBP-3 interaction with the cell surface proteoglycans.

Animals↗
PubMed1993

Alterations in glomerular proteoglycan metabolism in experimental non-insulin dependent diabetes mellitus.

Glomerular proteoglycans (PG) are important in modulating extracellular matrix assembly and glomerular permselectivity. In the obese Zucker rat, an experimental model of non-insulin dependent diabetes mellitus, expansion of the mesangial matrix, and microalbuminuria occur before the development of overt renal disease. The in vivo incorporation of (35S)sulfate into glomerular PG in 12-wk-old obese Zucker rats at the onset of microalbuminuria was compared with that of 12-wk-old lean Zucker rats. Specific (35S)sulfate incorporation into glomerular PG over 8 h was increased by 57% in obese rats compared with lean rats, suggesting increased PG synthesis. However, at variance with the observation in experimental models of insulin-dependent diabetes mellitus, the proportion of total glomerular (35S)PG released by heparin treatment was unchanged. Heparan sulfate (HS)-PG constituted over 60% of radiolabeled de novo synthesized glomerular PG. Similar proportions of HS-PG were extracted from the glomeruli of obese and lean rats. Isolated glomeruli spontaneously released two HS-PG, which constituted approximately 30% of total glomerular (35S)PG. On the basis of their chromatographic and electrophoretic patterns, these PG were similar in obese and lean rats. Heparin treatment of isolated glomeruli released an additional HS-PG, which appeared to be derived primarily from the glomerular extracellular matrix compartment and not from the detergent soluble cell fraction. Heparin-releasable HS-PG from both the lean and obese Zucker rats eluted at a KAV of 0.31 from Sepharose CL-6B chromatographic columns, indicating a hydrodynamic size similar to that reported for glomerular basement membrane HS-PG. However, gel electrophoresis demonstrated faster migration of the HS-PG released by heparin from the glomeruli of obese Zucker rats, suggesting increased electronegativity. Thus, early in the course of nephropathy in the obese Zucker rat, there is increased glomerular PG synthesis with no change in the proportions of the constitutively releasable and heparin-releasable HS-PG. Whether electrophoretic abnormalities of the heparin-releasable HS-PG observed in the obese rats contribute to the development of albuminuria and/or mesangial matrix expansion remains to be established.

Animals↗
PubMed1992

Treatment of diabetes mellitus.

New treatment strategies for subjects with Type 1 (insulin-dependent) and Type 2 (non-insulin-dependent) diabetes mellitus are being developed. Pilot studies utilising insulin itself have been reported to prevent Type 1 diabetes in subjects likely, by immunogenetic and physiologic criteria, to develop clinically overt disease, while the results of nicoti-namide trials in these subjects remain preliminary. Immunotherapy with cyclosporin A and azathioprine can slow disease progression and may produce long-term remissions when given within two months of onset of clinically overt disease. In subjects with established disease, familial clustering of diabetic nephropathy may be related to concomitant susceptibility to hypertension and elevated rates of Na/H countertransport. Treatment of hypertension associated with the nephropathy appears to slow renal deterioration. Whether reversal of the metabolic consequences of insulin deficiency or resistance also prevents chronic diabetic complications has not been firmly established. In the presence of a reduced Beta-cell mass, moderate hyperglycaemia may itself contribute to decreased muscle glucose uptake but not glycogen synthesis in Type 1 and Type 2 diabetes. Reversal of chronic hyperglycaemia by pancreas and islet cell transplantation, vanadate, and sulphonylureas are discussed as alternate strategies to insulin treatment in establishing normoglycaemia and furthering our understanding of insulin action and secretion at the cellular level. There remains a need to develop more sensitive biochemical and genetic markers to identify subjects at increased risk for developing chronic diabetic complications.

Animals↗
PubMed1992

A cell surface chondroitin sulfate proteoglycan, immunologically related to CD44, is involved in type I collagen-mediated melanoma cell motility and invasion.

The metastatic spread of tumor cells occurs through a complex series of events, one of which involves the adhesion of tumor cells to extracellular matrix (ECM) components. Multiple interactions between cell surface receptors of an adherent tumor cell and the surrounding ECM contribute to cell motility and invasion. The current studies evaluate the role of a cell surface chondroitin sulfate proteoglycan (CSPG) in the adhesion, motility, and invasive behavior of a highly metastatic mouse melanoma cell line (K1735 M4) on type I collagen matrices. By blocking mouse melanoma cell production of CSPG with p-nitrophenyl beta-D-xylopyranoside (beta-D-xyloside), a compound that uncouples chondroitin sulfate from CSPG core protein synthesis, we observed a corresponding decrease in melanoma cell motility on type I collagen and invasive behavior into type I collagen gels. Melanoma cell motility on type I collagen could also be inhibited by removing cell surface chondroitin sulfate with chondroitinase. In contrast, type I collagen-mediated melanoma cell adhesion and spreading were not affected by either beta-D-xyloside or chondroitinase treatments. These results suggest that mouse melanoma CSPG is not a primary cell adhesion receptor, but may play a role in melanoma cell motility and invasion at the level of cellular translocation. Furthermore, purified mouse melanoma cell surface CSPG was shown, by affinity chromatography and in solid phase binding assays, to bind to type I collagen and this interaction was shown to be mediated, at least in part, by chondroitin sulfate. Additionally we have determined that mouse melanoma CSPG is composed of a 110-kD core protein that is recognized by anti-CD44 antibodies on Western blots. Collectively, our data suggests that interactions between a cell surface CD44-related CSPG and type I collagen in the ECM may play an important role in mouse melanoma cell motility and invasion, and that the chondroitin sulfate portion of the proteoglycan seems to be a critical component in mediating this effect.

Animals↗
PubMed1992

Cell surface phosphatidylinositol-anchored heparan sulfate proteoglycan initiates mouse melanoma cell adhesion to a fibronectin-derived, heparin-binding synthetic peptide.

Cell surface heparan sulfate proteoglycan (HSPG) from metastatic mouse melanoma cells initiates cell adhesion to the synthetic peptide FN-C/H II, a heparin-binding peptide from the 33-kD A chain-derived fragment of fibronectin. Mouse melanoma cell adhesion to FN-C/H II was sensitive to soluble heparin and pretreatment of mouse melanoma cells with heparitinase. In contrast, cell adhesion to the fibronectin synthetic peptide CS1 is mediated through an alpha 4 beta 1 integrin and was resistant to heparin or heparitinase treatment. Mouse melanoma cell HSPG was metabolically labeled with [35S]sulfate and extracted with detergent. After HPLC-DEAE purification, 35S-HSPG eluted from a dissociative CL-4B column with a Kav approximately 0.45, while 35S-heparan sulfate (HS) chains eluted with a Kav approximately 0.62. The HSPG contained a major 63-kD core protein after heparitinase digestion. Polyclonal antibodies generated against HSPG purified from mouse melanoma cells grown in vivo also identified a 63-kD core protein. This HSPG is an integral plasma membrane component by virtue of its binding to Octyl Sepharose affinity columns and that anti-HSPG antibody staining exhibited a cell surface localization. The HSPG is anchored to the cell surface through phosphatidylinositol (PI) linkages, as evidenced in part by the ability of PI-specific phospholipase C to eliminate binding of the detergent-extracted HSPG to Octyl Sepharose. Furthermore, the mouse melanoma HSPG core protein could be metabolically labeled with 3H-ethanolamine. The involvement of mouse melanoma cell surface HSPG in cell adhesion to fibronectin was also demonstrated by the ability of anti-HSPG antibodies and anti-HSPG IgG Fab monomers to inhibit mouse melanoma cell adhesion to FN-C/H II. 35S-HSPG and 35S-HS bind to FN-C/H II affinity columns and require 0.25 M NaCl for elution. However, heparitinase-treated 125I-labeled HSPG failed to bind FN-C/H II, suggesting that HS, and not HSPG core protein, binds FN-C/H II. These data support the hypothesis that a phosphatidylinositol-anchored HSPG on mouse melanoma cells (MPIHP-63) initiates recognition to FN-C/H II, and implicate PI-associated signal transduction pathways in mediating melanoma cell adhesion to this defined ligand.

Amino Acid Sequence↗
PubMed1991

Pancreatic endocrine function in cystic fibrosis.

To characterize pancreatic endocrine secretion and to examine interrelationships among alterations in alpha, beta, and pancreatic polypeptide cell function in patients with cystic fibrosis (CF), we studied 19 patients with exocrine insufficiency (EXO), including 9 receiving insulin therapy (EXO-IT); 10 patients with no exocrine insufficiency (NEXO); and 10 normal control subjects. First-phase C-peptide response to intravenously administered glucose was significantly impaired in CF patients with exocrine insufficiency (EXO-IT = 0.02 +/- 0.01; EXO = 0.11 +/- 0.02; NEXO = 0.25 +/- 0.05; control subjects = 0.30 +/- 0.04 nmol/L). Lowering fasting glucose levels with exogenous insulin administration in EXO-IT did not improve beta cell responsivity to glucose. The C-peptide response to arginine was less impaired (EXO-IT = 0.12 +/- 0.02; EXO = 0.15 +/- 0.02; NEXO = 0.23 +/- 0.06; control subjects = 0.28 +/- 0.04 nmol/L). Alpha cell function, measured as peak glucagon secretion in response to hypoglycemia, was diminished in EXO but not NEXO (EXO-IT = 21 +/- 10; EXO = 62 +/- 19; NEXO = 123 +/- 29; control subjects = 109 +/- 12 ng/L). Despite diminished glucagon response, EXO patients recovered normally from hypoglycemia. Peak pancreatic polypeptide response to hypoglycemia distinguished CF patients with exocrine insufficiency from those without exocrine insufficiency (EXO-IT = 3 +/- 2; EXO = 3 +/- 1; NEXO = 226 +/- 68; control subjects = 273 +/- 100 pmol/L). Thus CF patients with exocrine disease have less alpha, beta, and pancreatic polypeptide cell function than CF patients without exocrine disease. These data suggest either that exocrine disease causes endocrine dysfunction in CF or that a common pathogenic process simultaneously and independently impairs exocrine and endocrine function.

Adult↗
PubMed1991

Effects of IGF-I and glucose on protein and proteoglycan synthesis by human fetal mesangial cells in culture.

Abnormalities in proteoglycan metabolism have been implicated in the pathogenesis of diabetic nephropathy. Whether hyperglycemia plays a direct role in these events is unknown. To evaluate the effects of high glucose concentrations and insulinlike growth factor I (IGF-I) on kidney proteoglycan and protein metabolism, we incubated quiescent, subconfluent human fetal mesangial cells for 24 h in serum-free media containing either physiological (5.6-mM) or elevated (25-mM) glucose concentrations with or without 1.3 x 10(-9) M IGF-I. In the presence of physiological glucose concentrations, IGF-I stimulated incorporation of [3H]leucine into protein and [35S]sulfate or [3H]glucosamine into proteoglycans. High glucose concentrations significantly amplified IGF-I-mediated stimulation of protein synthesis but totally abolished IGF-I-induced proteoglycan synthesis. The hydrodynamic size and proportions of heparan-35SO4 and chondroitin/dermatan-35SO4 proteoglycans in all experimental media were the same. However, high glucose concentrations decreased the iduronic acid content of dermatan-35SO4. In separate experiments, quiescent cells were cultured for 7 days in media supplemented with 2% fetal calf serum. IGF-I had no effect on mesangial cell proliferation, but as cells reached confluence, high glucose concentrations significantly inhibited cell proliferation. This inhibition was not mimicked by isosmolar concentrations of mannitol. After 7 days, uptake of radioactive precursors into proteoglycans and proteins over 24 h was similar under all culture conditions. However, IGF-I decreased the ratio of [35S]sulfate to [3H]glucosamine in proteoglycans and their glycosaminoglycan side chains. This difference persisted in disaccharides derived by chondroitin ABC lyase digestion of dermatan-35SO4.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Division↗
PubMed1991

An overview of role of matrix components.

The glomerular filtration barrier consists of complex matrix constituents interposed between the glomerular endothelial and epithelial cells, which constitute the lining of the glomerular capillaries. The matrix components include collagen and noncollagenous molecules such as laminin and proteoglycans. This review describes herein the metabolic consequences of diabetes mellitus to which increased permeability of the filtration barrier may be attributed by altering matrix molecules.

Animals↗
PubMed1990

Proteoglycans synthesized by human glomerular mesangial cells in culture.

Human fetal kidney mesangial cells were cultured for 24 h in the presence of 3H-amino acids and [35S] sulfate and chased for 24 h in nonradioactive medium. Incubation medium and cell layer proteoglycans were purified twice by high performance liquid chromatography-DEAE chromatography followed by gel filtration chromatography. The major medium 35S-macromolecules were chondroitin/dermatan-35SO4 proteoglycans. A small, Sepharose CL-6B Kav 0.14 dermatan-35SO4 proteoglycan was detected in the labeling medium and was released into both the early (time 0-0.5 h) and late (6-24 h) chase media. It contained 38 kDa 4-sulfated 35S-GAGs with a high content of iduronic acid and a 45-kDa protein core. A protein core of similar molecular weight was detected in the culture medium by Western analysis using antibodies to biglycan or proteoglycan-I (Fisher, L. W., Termine, J. D., and Young, M. F. (1989) J. Biol. Chem. 264, 4571-4576). This 35S-proteoglycan was not detected in the cell layer. However, a small dermatan-35SO4 with little or no protein core was present in the intracellular compartment. A large, Sepharose CL-6B excluded chondroitin-35SO4 proteoglycan was released into the culture medium and was detected between 6 and 24 h in chase medium. It eluted near the void volume of both associative and dissociative Sepharose CL-4B columns. It contained 30-kDa 4- and 6-sulfated 35S-GAGs and a 253-kDa protein core. A chondroitin-35SO4 proteoglycan with similar sized 35S-GAGs was detected in both the detergent-soluble and insoluble cell layer compartments. A Sepharose CL-6B Kav 0.11 heparin-35SO4 proteoglycan with a 220-kDa protein core and 38-kDa 35S-GAGs was rapidly released from the cell layer. This proteoglycan was larger than that previously described in isolated rat glomeruli or glomerular basement membranes, but had a core protein similar in size to one previously detected in these tissues. A larger heparan-35SO4 proteoglycan with larger 35S-GAGs was present in the detergent-insoluble cell layer compartment. The proteoglycans released by glomerular mesangial cells in culture resembled those synthesized by aortic smooth muscle cells in culture or extracted from aorta, supporting the notion that these cells are of vascular origin.

Blotting, Western↗
PubMed1990

Partial characterization of proteoglycans synthesized by human glomerular epithelial cells in culture.

Confluent adult and fetal human glomerular epithelial cells were incubated for 24 h in the presence of [3H]-amino acids and [35S]sulfate. Two heparan-35SO4 proteoglycans were released into the culture medium. These 35S-labeled proteoglycans eluted as a single peak from anion exchange chromatographic columns, but were separable by gel filtration on Sepharose CL-6B columns. The larger heparan-35SO4 proteoglycan eluted with the column void volume and at a Kav of 0.26 from Sepharose CL-4B columns. The most abundant medium heparan-35SO4 proteoglycan was a high buoyant density proteoglycan similar in hydrodynamic size (Sepharose CL-6B Kav 0.23) to those previously described in glomerular basement membranes and isolated glomeruli. Heparan-35SO4 chains from both proteoglycans were 36 kDa. A smaller proportion of Sepharose CL-6B excluded dermatan-35SO4 proteoglycan was also synthesized by these cells. The predominant protein cores of both medium heparan-35SO4 proteoglycans were approximately 230 and 180 kDa. A hybrid chondroitin/dermatan-heparan-35SO4 proteoglycan with an 80-kDa protein core copurified with the smaller medium heparan-35SO4 proteoglycan. This 35S-labeled proteoglycan appeared as a diffuse, chondroitinase ABC sensitive 155-kDa fluorographic band in sodium dodecyl sulfate-polyacrylamide gels after the Sepharose CL-6B Kav 0.23 35S-labeled proteoglycan fraction was digested with heparitinase. The heparitinase generated heparan sulfate proteoglycan protein cores and the 155-kDa hybrid proteoglycan fragment had molecular weights similar to those previously identified in rat glomerular basement membrane and glomeruli using antibodies against a basement membrane tumor proteoglycan precursor (Klein et al. J. Cell Biol. 106, 963-970, 1988). Thus, human glomerular epithelial cells in culture are capable of synthesizing, processing, and releasing heparan sulfate proteoglycans which are similar to those synthesized in vivo and found in the glomerular basement membrane. These proteoglycans may belong to a family of related basement membrane proteoglycans.

Adult↗
PubMed1990

RGD-independent cell adhesion to the carboxy-terminal heparin-binding fragment of fibronectin involves heparin-dependent and -independent activities.

Cell adhesion to extracellular matrix components such as fibronectin has a complex basis, involving multiple determinants on the molecule that react with discrete cell surface macromolecules. Our previous results have demonstrated that normal and transformed cells adhere and spread on a 33-kD heparin binding fragment that originates from the carboxy-terminal end of particular isoforms (A-chains) of human fibronectin. This fragment promotes melanoma adhesion and spreading in an arginyl-glycyl-aspartyl-serine (RGDS) independent manner, suggesting that cell adhesion to this region of fibronectin is independent of the typical RGD/integrin-mediated binding. Two synthetic peptides from this region of fibronectin were recently identified that bound [3H]heparin in a solid-phase assay and promoted the adhesion and spreading of melanoma cells (McCarthy, J. B., M. K. Chelberg, D. J. Mickelson, and L. T. Furcht. 1988. Biochemistry. 27:1380-1388). The current studies further define the cell adhesion and heparin binding properties of one of these synthetic peptides. This peptide, termed peptide I, has the sequence YEKPGSP-PREVVPRPRPGV and represents residues 1906-1924 of human plasma fibronectin. In addition to promoting RGD-independent melanoma adhesion and spreading in a concentration-dependent manner, this peptide significantly inhibited cell adhesion to the 33-kD fragment or intact fibronectin. Polyclonal antibodies generated against peptide I also significantly inhibited cell adhesion to the peptide, to the 33-kD fragment, but had minimal effect on melanoma adhesion to fibronectin. Anti-peptide I antibodies also partially inhibited [3H]heparin binding to fibronectin, suggesting that peptide I represents a major heparin binding domain on the intact molecule. The cell adhesion activity of another peptide from the 33-kD fragment, termed CS1 (Humphries, M. J., A. Komoriya, S. K. Akiyama, K. Olden, and K. M. Yamada. 1987. J. Biol. Chem., 262:6886-6892) was contrasted with peptide I. Whereas both peptides promoted RGD-independent cell adhesion, peptide CS1 failed to bind heparin, and exogenous peptide CS1 failed to inhibit peptide I-mediated cell adhesion. The results demonstrate a role for distinct heparin-dependent and -independent cell adhesion determinants on the 33-kD fragment, neither of which are related to the RGD-dependent integrin interaction with fibronectin.

Amino Acid Sequence↗
PubMed1989

Chondroitin sulfate proteoglycan synthesis and reutilization of beta-D-xyloside-initiated chondroitin/dermatan sulfate glycosaminoglycans in fetal kidney branching morphogenesis.

Branching morphogenesis and chondroitin sulfate proteoglycan synthesis by explanted fetal mouse kidneys were previously shown to be inhibited by p-nitrophenyl beta-D-xylopyranoside (beta-D-xyloside) while glomerular development and heparan sulfate proteoglycan synthesis were unaffected. The metabolic fate of fetal kidney explant proteoglycans was investigated to determine whether or not recovery of proteoglycan synthesis and morphogenesis occur after exposure to beta-D-xyloside. Chondroitin sulfate proteoglycan synthesis resumed within 4 hr of removal of beta-D-xyloside and was enhanced once beta-D-xyloside-initiated chondroitin/dermatan-35SO4 glycosaminoglycans (GAGs) were released from the tissue. Radioactivity incorporated into beta-D-xyloside-initiated chondroitin/dermatan-35SO4 GAGs during labeling in the presence of beta-D-xyloside was reutilized in the synthesis of chondroitin-35SO4 proteoglycan during a 24-hr chase in nonradioactive medium without beta-D-xyloside. Further, highly purified beta-D-xyloside-initiated chondroitin/dermatan-35SO4 GAGs were taken up by kidneys more avidly than was free [35S]sulfate. These 35S-GAGs were degraded and reutilized in the synthesis of chondroitin-35SO4 proteoglycan. Ureteric bud branching resumed 48 hr after beta-D-xyloside was removed from the incubation medium. These findings support the idea that both chondroitin sulfate proteoglycan synthesis and proteoglycan processing may be involved in branching morphogenesis.

Animals↗
PubMed1989

Release of glomerular heparan-35SO4 proteoglycan by heparin from glomeruli of streptozocin-induced diabetic rats.

Abnormalities in the incorporation of heparan sulfate proteoglycan into the glomerular basement membrane have been implicated in the pathogenesis of various proteinuric states, including diabetes mellitus. To understand further the interactions between proteoglycans and glomerular extracellular matrices, glomeruli were isolated from normal and streptozocin-induced diabetic rats after in vivo exposure to 35S-labeled sulfate and were treated with heparin in vitro. Heparin treatment released a unique heparan sulfate proteoglycan from glomerular cell surface or extracellular matrix proteoglycan receptors. Another, smaller heparan sulfate proteoglycan was the most abundant proteoglycan released into medium and was released constitutively in medium with or without added heparin. While the two heparin-extracted proteoglycans copurified on anion-exchange and gel-filtration chromatographic columns, they were resolved by composite 0.6% agarose--1.8% polyacrylamide gel electrophoresis. Glomeruli from diabetic rats contained decreased proportions of the heparin-releasable heparan sulfate proteoglycan and more constitutively released heparan sulfate proteoglycan. The apparent molecular weight and intrinsic charge of the heparin-released proteoglycan mixture and the apparent molecular weight and sulfation pattern of their 35S-labeled glycosaminoglycan chains after nitrous acid deaminative cleavage were similar in the two groups. A brief trypsin digestion of heparin-treated glomeruli released proportionately less integral membrane and extracellular matrix 35S-labeled proteoglycans and 35S-labeled glycopeptides from diabetic glomeruli than form control glomeruli. Elution of these 35S-labeled macromolecules from anion-exchange columns and migration in agarose-polyacrylamide gels were similar in the two groups. Abnormalities in proteoglycan-matrix interactions or proteoglycan processing may account for changes in the proportions of heparin- and trypsin-extracted proteoglycan compartments in diabetes.

Animals↗
PubMed1988

Diabetes mellitus associated with cystic fibrosis.

The prevalence of overt diabetes mellitus and carbohydrate intolerance was studied in 448 patients with cystic fibrosis (CF). Insulin-dependent diabetes (IDDM) developed in 7.6% of patients (13 male and 21 female). Survival was significantly lower (P less than 0.01) in the IDDM-CF group, with fewer than 25% surviving to age 30 years, whereas nearly 60% of the nondiabetic CF population reached this age. A significant deterioration in CF clinical status, based on NIH score, became apparent 2 years before onset of overt IDDM (P less than 0.05 at 2 years prior, P less than 0.01 at IDDM diagnosis). Total glycosylated hemoglobin (HbA1) was significantly (P less than 0.001) higher for the total CF population (7.3% +/- 1.2%) than for the general non-CF population (6.5% +/- 0.7%), and in the IDDM-CF group (P less than 0.05) compared with normoglycemic CF control patients. Female patients had a higher mean HbA1 after 12 years of age than their male counterparts did (P less than 0.02). HBA1 did not predict the development of IDDM, but there was a weak inverse relationship between HbA1 and both NIH clinical score (r = -0.41, P less than 0.02) and standard pulmonary function tests (forced vital capacity, r = -0.25, P less than 0.01) in the general CF population. Therefore, impaired carbohydrate tolerance in CF is associated with progressive clinical deterioration.

Adolescent↗