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J Winer

Publications and source records attributed to J Winer.

At least 37 records · Page 2Linked to original sources
PubMed1992

The vascular endothelial growth factor proteins: identification of biologically relevant regions by neutralizing monoclonal antibodies.

Angiogenesis plays critical roles in organ development during embryonic and fetal life, wound healing and in a variety of pathological conditions. Vascular endothelial growth factor (VEGF) is a secreted growth factor specific for vascular endothelial cells which induces angiogenesis in vivo. To gain a better understanding of the physiological role of VEGF, we have generated and characterized four murine monoclonal antibodies (mAbs) using the 165 amino acid species of recombinant human VEGF as immunogen. These mAbs (A3.13.1, A4.6.1, B4.3.1 and B2.6.2) belong to IgG1 isotype and have high affinities for VEGF (dissociation constants range from 2.2 x 10(-9) to 4 x 10(-10) M). Two different epitopes were detected with these mAbs. One epitope is recognized by mAbs A3.13.1 and B2.6.2, and the other recognized by mAbs A4.6.1 and B4.3.1. The epitope recognized by mAb A4.6.1 appears to be continuous while mAb B2.6.2 recognizes a discontinuous epitope. MAb A4.6.1 recognized three species of VEGF generated by alternative splicing, VEGF121, VEGF165 and VEGF189 while mAb B2.6.2 binds only VEGF165 and VEGF189. Results using an in vitro bovine adrenal cortex endothelial cell proliferation assay, in in vivo vascular permeability assay and an in vivo embryonic chicken angiogenesis assay showed that mAb A4.6.1 has potent VEGF neutralizing activities. MAb A4.6.1 was shown to block the binding of VEGF to its receptor(s) suggesting the inhibitory mechanism for VEGF activities. These well-defined mAbs should be very powerful tools to understand the structure-function relationship of various domains of VEGF and may have therapeutic potential.

Adrenal Cortex↗
PubMed1991

The vascular endothelial growth factor family of polypeptides.

Vascular endothelial growth factor (VEGF) was identified as a heparin-binding polypeptide mitogen with a target cell specificity restricted to vascular endothelial cells. Molecular cloning reveals the existence of four species of VEGF having 121, 165, 189, and 206 amino acids. These have strikingly different secretion patterns, which suggests multiple physiological roles for this family of polypeptides. The two shorter forms are efficiently secreted, while the longer ones are mostly cell-associated. Alternative splicing of mRNA, rather that transcription from different genes, is the mechanism for their generation. In situ hybridization reveals that the VEGF mRNA is widely distributed in most tissues and organs and expressed at particularly high levels in areas of active vascular proliferation, like the ovarian corpus luteum. Ligand autoradiography on rat tissue sections demonstrates that VEGF binding sites are associated with vascular endothelial cells of both fenestrated and non-fenestrated capillaries and with the endothelium of large vessels, while no displaceable binding is evident on non-endothelial cell types. These findings support the hypothesis that VEGF plays a highly specific role in the maintenance and in the induction of growth of vascular endothelial cells.

Endothelial Growth Factors↗
PubMed1991

Association between glycoconjugate antibodies and Campylobacter infection in patients with Guillain-Barré syndrome.

In a retrospective study, we have analysed sera from a well-characterised Guillain-Barré syndrome (GBS) patient group for antibodies that react with gangliosides. Of 95 GBS patients and 85 control patients analysed, we found that 14 (15%) of GBS patients but only one control patient had antibodies that react with the gangliosides GM1 and/or GD1b but not GM2, GD1a and GT1b using a sensitive enzyme-linked immunosorbent assay (ELISA). This pattern of reactivity suggests binding to the carbohydrate structure Gal(beta 1-3)GalNAc which is shared between some glycolipids and glycoproteins. Similar antibodies have been found previously in a subpopulation of patients with lower motor neuron disease. In the present study, the predominant immunoglobulin class of these anti-glycoconjugate antibodies was IgG rather than IgM. A correlation was found between the presence of these antibodies and prognosis in terms of disability at 3 and 12 months after presentation. Patients with anti-glycoconjugate antibodies also had a higher incidence of previous Campylobacter infections than the rest of the patient group, although the significance of this remains to be determined.

Antibodies↗
PubMed1991

The vascular endothelial growth factor family: identification of a fourth molecular species and characterization of alternative splicing of RNA.

Vascular endothelial growth factor (VEGF) was recently identified as a secreted, direct-acting mitogen specific for vascular endothelial cells and capable of stimulating angiogenesis in vivo. Molecular cloning revealed multiple forms of VEGF, apparently arising from alternative splicing of its RNA transcript. We have examined various human cDNA libraries by the polymerase chain reaction technique and discovered a fourth molecular form, VEGF206. This form contains a 41-amino acid insertion relative to the most abundant form, VEGF165, and includes the highly basic 24-amino acid insertion found in VEGF189. Southern blot analysis revealed that a single gene encoded these various forms, and nucleic acid sequence analysis of a portion of the VEGF gene revealed an intron/exon structure compatible with alternative splicing of RNA as a mechanism for their generation. Transient transfection of human embryonic kidney 293 cells showed that, like VEGF189, VEGF206 was predominately cell-associated and only very poorly secreted despite the presence of the signal peptide identical to that found in VEGF121 and VEGF165, both of which are efficiently exported from the cell. Vascular permeability activity was detected in the medium of 293 cells transfected with all four forms of VEGF; however, endothelial cell mitogenic activity was apparent only with VEGF121 and VEGF165. Thus, alternative splicing of VEGF RNA can produce four polypeptides with strikingly different secretion patterns, which suggests multiple physiological roles for this family of proteins.

Amino Acid Sequence↗
PubMed1991

Aortic smooth muscle cells express and secrete vascular endothelial growth factor.

We examined whether cultured bovine aortic smooth muscle (ASM) cells express VEGF. RNA blot analysis of total cellular RNA derived from ASM cells demonstrates the expression of the VEGF gene. ASM cells release in the medium a VEGF-like endothelial cell mitogen which binds to heparin-sepharose and has an apparent molecular weight of 40-45 kDa as assessed by an HPLC gel filtration column. Consistent with VEGF, this mitogen does not stimulate the proliferation of ASM cells. Immunoblot analysis of the bioactive material with an antibody specific for VEGF demonstrates the presence of a major immunoreactive band with an apparent molecular mass of 23 kDa and a minor band with a molecular mass of approximately 18 kDa, in reducing conditions. The major band has very similar apparent molecular weight as the 165 amino-acid species of human recombinant VEGF of folliculo-stellate cells derived VEGF. These data demonstrate the expression and synthesis of VEGF by cultured ASM cells and suggest that the 164 amino-acid species is the predominant molecular form of the growth factor secreted by such cells. VEGF released by ASM cells may play a paracrine role in the maintenance of the integrity of the endothelial lining or in the abnormal proliferation of the vasa vasorum which takes place in atherosclerosis.

Animals↗
PubMed1989

Plasma exchange for Guillain-Barré syndrome.

The impact of plasma exchange (PE) on the treatment of severe Guillain-Barré syndrome (GBS) was studied by comparing all the 16 patients treated with PE in a London teaching hospital between January 1985 and August 1987, with 64 historical controls drawn from a series of patients observed during a prospective study in South East England in 1983 and 1984. There were no GBS-related deaths in the PE treated group but seven in the historical controls (2P = 0.39). The median duration of ventilation was only 20 days (range 7-64) in the PE group compared with 36 days (range 14-365) in the surviving patients in the control group (2P = 0.06, 95% confidence interval of difference in medians -36 to 0 days). The PE group walked earlier without aid (median 55.5 compared with 86 days, 2P = 0.04, 95% confidence interval of difference in medians -88 to -2 days). Three months after the onset of neuropathy all PE treated patients were able to walk with support or better, whereas 19 of the surviving historical controls were unable to walk even with support (2P = 0.009). The costs of PE were offset by savings in intensive care unit and hospital expenditure.

Adult↗
PubMed1988

Structural properties of the renal parathyroid hormone receptor: hydrodynamic analysis and protease sensitivity.

The highly specific ligand [125I]bovine (b) PTH-(1-34) and a chemical cross-linking technique were used to explore structural features of the canine renal cortical PTH receptor. Membranes isolated under conditions designed to inhibit endogenous proteolysis displayed a major 85K labeled PTH receptor moiety on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cross-linked receptors were solubilized with Lubrol-PX and partially purified by affinity chromatography on wheat germ agglutinin-agarose, and their hydrodynamic properties were assessed [Stokes radius = 7.3 +/- 0.1 nm; sedimentation coefficient = 6.4 +/- 0.2S; partial specific volume = 0.758 +/- 0.01 ml/g; frictional coefficient = 1.68 +/- 0.04; mol wt (Mr) = 216,000 +/- 14,000]. Corrections for detergent binding and for the presence of carbohydrate yielded an estimated Mr of 166,000 +/- 11,000 for the solubilized PTH receptor. Thus, the renal PTH receptor is oligomeric, with a Mr approximating that expected of a homodimer of 85K subunits. Peptide-mapping experiments revealed the presence within the 85K PTH receptor subunit of at least two major regions sensitive to proteolytic attack. Both elastase and an endogenous renal protease(s) cleaved the PTH receptor to a 70K form that is fully functional with respect to high affinity, guanyl nucleotide-sensitive PTH binding. Cleavage in a second domain by elastase, S. aureus V8 protease, or chymotrypsin generated a 50K labeled PTH receptor fragment. Cleavage at this second site was prevented by prior occupancy of the receptor with [125I]bPTH-(1-34), suggesting that this domain may be functionally important. Reduction of receptor disulfide bonds with dithiothreitol and beta-mercaptoethanol released a low Mr (less than or equal to 14K) labeled PTH receptor component, similar treatment of renal membranes abolished specific PTH binding, indicating that an intact disulfide bond(s) is essential for receptor function. These results provide new insights into the structural basis of PTH receptor function.

Animals↗
PubMed1987

The canine renal parathyroid hormone receptor is a glycoprotein: characterization and partial purification.

Covalent labeling of the canine renal parathyroid hormone receptor with [125I]bPTH(1-34) reveals several major binding components that display characteristics consistent with a physiologically relevant adenylate cyclase linked receptor. Through the use of the specific glycosidases neuraminidase and endoglycosidase F and affinity chromatography on lectin-agarose gels, we show here that the receptor is a glycoprotein that contains several complex N-linked carbohydrate chains consisting of terminal sialic acid and penultimate galactose in a beta 1,4 linkage to N-acetyl-D-glucosamine. No high mannose chains or O-linked glycans appear to be present. The peptide molecular weight of the deglycosylated labeled receptor is 62,000 [or 58,000 if the mass of bPTH(1-34) is excluded]. The binding of [125I]bPTH(1-34) to the receptor is inhibited in a dose-dependent fashion by wheat-germ agglutinin, but not by either succinylated wheat-germ agglutinin or Ricinus communis lectin, suggesting that terminal sialic acid may be involved in agonist binding. A combination of lectin affinity chromatography and immunoaffinity chromatography affords a 200-fold purification of the covalently labeled receptor.

Animals↗
PubMed1987

Covalent labeling of a high-affinity, guanyl nucleotide sensitive parathyroid hormone receptor in canine renal cortex.

Putative parathyroid hormone (PTH) receptors in canine renal membranes were affinity labeled with 125I-bPTH(1-34) using the heterobifunctional cross-linking reagent N-hydroxysuccinimidyl 4-azidobenzoate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a major 85,000 molecular weight (Mr) PTH binding component, the labeling of which was inhibited by nanomolar concentrations of unlabeled PTH and by micromolar concentrations of 5'-guanylyl imidodiphosphate [Gpp-(NH)p]. Labeling was not influenced by the unrelated peptides insulin and arginine vasopressin. Minor PTH binding components of Mr 55,000 and 130,000 were also seen, and labeling of these was likewise sensitive to unlabeled PTH and to Gpp(NH)p. Omission of protease inhibitors during the isolation of plasma membranes resulted in the loss of the Mr 85,000 PTH binding species and the appearance of an Mr 70,000 form. Several minor PTH binding components also were observed. Equilibrium binding studies showed that such membranes had an affinity for PTH indistinguishable from that in membranes isolated with protease inhibitors and displaying a major Mr 85,000 PTH binding species. We conclude that the major form of the adenylate cyclase coupled PTH receptor in canine renal membranes is an Mr 85,000 protein. An endogenous enzyme, probably a lysosomal cathepsin, can cleave this form to produce an Mr 70,000 receptor that retains full functional activity with respect to high-affinity, guanyl nucleotide sensitive PTH binding. The ability to covalently label the PTH receptor in high yield represents a major step toward the structural characterization of this important detector molecule.

Animals↗
PubMed1986

Solubilization of a guanine nucleotide-sensitive parathyroid hormone-receptor complex from canine renal cortex.

Canine renal cortical PTH receptors were solubilized after occupancy of membrane-associated receptors with the agonist ligand [125I]bovine (b) PTH-(1-34). Stabilization of binding during solubilization required the use of high concentrations of BSA (optimally 5%) and appropriate detergents (0.5% 3-[(3-cholamidopropyl)dimethylammonio]2-hydroxy-1-propanesulfonate, 0.5% 3-[(3-cholamidopropanyl)dimethylammonio]1-propanesulfonate, or 0.5-1.0% digitonin). The soluble fraction (240,000 X gav supernatant) contained [125I]bPTH-(1-34) associated with macromolecular components as well as unbound [125I]bPTH-(1-34) that dissociated during solubilization. The soluble macromolecular complex had functional properties expected of a ternary complex consisting of [125I]bPTH-(1-34) receptor stimulatory guanine nucleotide-binding protein (Ns). Thus, the dissociation of labeled PTH at 30 C was slow (t1/2 = 75 min); in the presence of GTP (10(-4) M), 75% of the sites displayed rapid dissociation kinetics (t1/2 = 2.3 min). This effect was nucleotide specific, with GTP approximately equal to GTP gamma S approximately equal to GDP greater than GDP beta S greater than ITP approximately equal to guanylylimidodiphosphate much greater than GMP approximately equal to App(NH)p. ATP was ineffective. GTP produced a half-maximal response at a concentration of 200 nM. These results are consistent with the reported nucleotide specificity and affinity of purified Ns. Treatment of membranes with N-ethylmaleimide during the binding reaction rendered the solubilized complex refractory to GTP. Gel filtration chromatography (Sepharose 6B) revealed a GTP-sensitive complex that eluted in the position expected of a detergent-free spherical protein of 180,000 daltons. This complex may consist of the 60,000 to 70,000-dalton PTH-binding subunit (previously identified by photoaffinity labeling) together with Ns.

Animals↗
PubMed1982

Photoaffinity labeling of the canine renal receptor for parathyroid hormone.

Studies were undertaken to identify and characterize components of the parathyroid hormone receptor. An analog of the bovine parathyroid hormone(1-34) sequence was derivatized at the 23-tryptophan position with the photoreactive reagent 2-nitro-5-azidophenylsulfenyl chloride. 2-Nitro-5-azidophenylsulfenyl-bovine parathyroid hormone (NAPS-bPTH) analog retained full biological activity with respect to receptor binding and activation of adenylate cyclase in canine renal cortical plasma membranes. 125I-bPTH(1-34) analog was also derivatized without loss of receptor-binding activity. When 125I-NAPS-bPTH(1-34) analog was incubated with canine renal plasma membranes and then photolyzed, at least two 125I-labeled membrane components were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The incorporation of 125I radioactivity into one of these components (Mr congruent to 60,000) was inhibited when incubation and photolysis were performed in the presence of excess, unlabeled bPTH(1-34). Furthermore, photolysis of membranes in the presence of NAPS-bPTH(1-34) analog led to activation of adenylate cyclase which persisted following washing to remove noncovalently bound peptide, suggesting a functional, covalent hormone-receptor complex had been formed. We conclude that the M r congruent to 60,000 membrane component may be a part of the renal receptor for parathyroid hormone.

Adenylyl Cyclases↗
PubMed1982

The effect of renal function on changes in circulating concentrations of 1,25-dihydroxycholecalciferol after an oral dose.

1. 1,25- Dihydroxycholecalciferol [1,25-(OH)2D3] was administered orally to four normal subjects and six patients with chronic renal failure not on dialysis. The serum concentration of 1,25-(OH)2D3 was measured by radioimmunoassay in both groups from samples taken before, and at regular intervals up to 48 h after, the oral dose. 2. The plasma half-time for the disappearance of the administered 1,25-(OH)2D3 was estimated by determining the time for a 50% reduction from the peak increment of the sterol. In normal subjects the calculated value ranged from 5 to 8 h compared with 18 to 44 h in uraemic patients. 3. It appears from our studies that in uraemic subjects there is impaired ability to metabolize or excrete 1,25-(OH)2D3.

Adult↗
PubMed1976

Puberty in girls: correlation of serum levels of gonadotropins, prolactin, androgens, estrogens, and progestins with physical changes.

Twenty-seven girls aged 8 to 18 were studied in a longitudinal prospective fashion. Serum samples were collected at 6 month intervals up to 4 years and radioassayed for hormones of pituitary, ovarian, and adrenal origin. A progressive elevation of luteinizing hormone (LH), follicle-stimulating hormone/FSH), estradiol (E2), dehydroepiandrosterone (DHA), and androstenedione (delta4) occurred during puberty and continued until menarche. The onset of puberty occurred concomitantly with an elevation of estrone (E1) dehydroepiandrosterone (DHA), dehydroepiandrosterone sulfate (DHAS), and 17-hydroxyprogesterone (17-OH-P). Prolactin (Prol) and progesterone (Prog) concentrations did not change during puberty until after menarche. After menarche, levels of LH and FSH were comparable with menstruating adult females. Concentrations of E2 and Prog were lower during the second half of the cycle among most regularly menstruating subjects than expected during the luteal phase. LH and Prog levels indirectly suggest that ovulation occurs in a few girls within months after menarche.

Adolescent↗