Simple, rapid, high-purity preparation of recombinant human platelet-derived growth factor-BB.

Recombinant human platelet-derived growth factor-BB (rhPDGF-BB) is used to treat full-thickness diabetic ulcers and is being investigated for use in other chronic ulcers, non-healing wounds, and periodontal defects. A simple, novel method for expression and purification of rhPDGF-BB from Escherichia coli is now described. This method produces the dimeric protein in high yield (10-12 mg/g wet cell mass) and with a purity >95%.

Increased yield of high purity recombinant human interferon-gamma utilizing reversed phase column chromatography.

Increasing therapeutic applications for recombinant human interferon-gamma (rhIFN-gamma), an antiviral proinflammatory cytokine, has broadened interest in optimizing methods for its production and purification. We describe a reversed phase chromatography (RPC) procedure using Source-30 matrix in the purification of rhIFN-gamma from Escherichia coli that results in a higher yield than previously reported. The purified rhIFN-gamma monomer from the RPC column is refolded in Tris buffer.

Type IV collagen of the glomerular basement membrane. Evidence that the chain specificity of network assembly is encoded by the noncollagenous NC1 domains.

The ultrafiltration function of the glomerular basement membrane (GBM) of the kidney is impaired in genetic and acquired diseases that affect type IV collagen. The GBM is composed of five (alpha1 to alpha5) of the six chains of type IV collagen, organized into an alpha1.alpha2(IV) and an alpha3.

The goodpasture autoantigen. Mapping the major conformational epitope(s) of alpha3(IV) collagen to residues 17-31 and 127-141 of the NC1 domain.

The Goodpasture (GP) autoantigen has been identified as the alpha3(IV) collagen chain, one of six homologous chains designated alpha1-alpha6 that comprise type IV collagen (Hudson, B. G., Reeders, S.

Goodpasture antigen: expression of the full-length alpha3(IV) chain of collagen IV and localization of epitopes exclusively to the noncollagenous domain.

Background: Tissue injury in Goodpasture (GP) syndrome (rapidly progressive glomerular nephritis and pulmonary hemorrhage) is mediated by antibasement membrane antibodies that are targeted to the alpha3(IV) chain of type IV collagen, one of five alpha(IV) chains that occur in the glomerular basement membrane. GP antibodies are known to bind epitopes within the carboxyl terminal noncollagenous domain (NC1) of the alpha3(IV) chain, termed the GP autoantigen. Whether epitopes also exist in the 1400-residue collagenous domain is unknown because studies to date have focused solely on the NC1 domain.

Glomerular basement membrane. Identification of a novel disulfide-cross-linked network of alpha3, alpha4, and alpha5 chains of type IV collagen and its implications for the pathogenesis of Alport syndrome.

Glomerular basement membrane (GBM) plays a crucial function in the ultrafiltration of blood plasma by the kidney. This function is impaired in Alport syndrome, a hereditary disorder that is caused by mutations in the gene encoding type IV collagen, but it is not known how the mutations lead to a defective GBM. In the present study, the supramolecular organization of type IV collagen of GBM was investigated.

Seminiferous tubule basement membrane. Composition and organization of type IV collagen chains, and the linkage of alpha3(IV) and alpha5(IV) chains.

Seminiferous tubule basement membrane (STBM) plays an important role in spermatogenesis. In the present study, the composition and structural organization of type IV collagen of bovine STBM was investigated. STBM was found to be composed of all six alpha-chains of type IV collagen based upon immunocytochemical and biochemical analysis.

Circulating factor associated with increased glomerular permeability to albumin in recurrent focal segmental glomerulosclerosis.

Background: Heavy proteinuria and progressive renal injury recur after transplantation in up to 40 percent of patients with renal failure caused by idiopathic focal segmental glomerulosclerosis. A circulating factor may be responsible for this recurrence.

Methods: To determine whether patients with focal segmental glomerulosclerosis have a circulating factor capable of causing glomerular injury, we tested serum samples from 100 patients with the disorder in an in vitro assay of glomerular permeability to albumin.

Identification of the alpha 3 chain of type IV collagen as the common autoantigen in antibasement membrane disease and Goodpasture syndrome.

Antiglomerular basement membrane (GBM) antibodies can cause glomerulonephritis or pulmonary hemorrhage by themselves or Goodpasture syndrome when they occur together. It is unknown if variations in antibody reactivity contribute to the different patterns of organ involvement seen in this disease. This study examines the reactivity of the alpha 1-alpha 6 NC1 domains of Type IV collagen, the putative autoantigen, in sera from patients with anti-GBM antibodies after various clinical presentations of lung hemorrhage and renal injury.

Structure and composition of type IV collagen of bovine aorta.

To determine the chain composition of type IV collagen of bovine thoracic aorta, we analyzed collagenase-solubilized carboxyl-terminal noncollagenous (NC1)-domains by high-pressure liquid chromatography, two-dimensional electrophoresis, immunoblotting and enzyme-linked immunoassay. In addition to the classical alpha 1- and alpha 2-chains, we found small amounts of the recently discovered alpha 3-, alpha 4- and alpha 5-chains. The alpha 3- and alpha 4-chains were, collectively, 7-13% of the total, and the alpha 5-chain was present in a low amount.

Specificity of Goodpasture autoantibodies for the recombinant noncollagenous domains of human type IV collagen.

Type IV collagen has recently emerged as a family composed of five known chains (alpha 1-alpha 5), each of which contains a carboxyl-terminal noncollagenous domain (NC1) of approximately 230 amino acids. The NC1 domain of the alpha 3(IV) chain is the probable target for autoantibodies in patients with Goodpasture syndrome (GP), as evidenced from studies employing bovine type IV collagen. In the present experiments, the specificity of GP antibodies for the five NC1 domains of human type IV collagen was determined by using recombinant NC1 domains as the antigen.

Molecular characteristics of the Goodpasture autoantigen.

Goodpasture syndrome is an autoimmune disease causing rapidly progressive glomerulonephritis and pulmonary hemorrhage. The clinical manifestations are caused by autoantibodies that bind to a constituent, termed the Goodpasture autoantigen, of alveolar and glomerular basement membranes. Searches for the identity of this constituent have recently culminated in the discovery of two new chains (alpha 3 and alpha 4) of type IV collagen and the identification of the alpha 3 chain as the Goodpasture autoantigen.

The pathogenesis of Alport syndrome involves type IV collagen molecules containing the alpha 3(IV) chain: evidence from anti-GBM nephritis after renal transplantation.

Mutations in the COL4A5 collagen gene have been implicated as the primary defect in Alport syndrome, a heritable disorder characterized by sensorineural deafness and glomerulonephritis that progresses to end-stage renal failure. In the present study, the molecular nature of the defect in Alport glomerular basement membrane (GBM) was explored using anti-GBM alloantibodies (tissue-bound and circulating) produced in three Alport patients subsequent to renal transplantation. The alloantibodies bound to the alpha 3(IV)NC1 domain of type IV collagen and not to any other basement membrane component.

Purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli.

In this report we describe the purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli. Nucleotide sequence encoding porcine prorelaxin was inserted into an E. coli expression vector, pOTS, and the recombinant plasmid was transformed into the E.

Type IV collagen of Engelbreth-Holm-Swarm tumor matrix: identification of constituent chains.

The noncollagenous hexamer (NC1) domain of type IV collagen from Engelbreth-Holm-Swarm (EHS) sarcoma matrix was subjected to electrophoretic, amino-terminal amino acid sequence, and immunochemical analysis to determine which of the five known kinds of alpha(IV) chains are present. Electrophoretic analysis, whether by one-dimensional or two-dimensional electrophoresis, showed that nonlathyritic and lathyritic hexamer gave nearly identical patterns. Amino-terminal amino acid sequence analysis of hexamer subunits, transblotted from two-dimensional gels, revealed that the hexamer subunits were derived exclusively from the alpha 1 and alpha 2 chains.

Goodpasture syndrome. Localization of the epitope for the autoantibodies to the carboxyl-terminal region of the alpha 3(IV) chain of basement membrane collagen.

The autoantibodies of patients with Goodpasture syndrome are primarily targeted to the noncollagenous (NC1) domain of the alpha 3(IV) chain of basement membrane collagen (Saus, J., Wieslander, J., Langeveld, J.

Extracellular matrix (mesoglea) of Hydra vulgaris. I. Isolation and characterization.

Hydrozoans such as Hydra vulgaris, as with all classes of Cnidaria, are characterized by having their body wall organized as an epithelial bilayer with an intervening acellular layer termed the mesoglea. The present study was undertaken to determine what extracellular matrix (ECM) components are associated with Hydra mesoglea. Using polyclonal antibodies generated from vertebrate ECM molecules, initial light and electron microscopic immunocytochemical studies indicated the presence of type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin immunoreactive components in Hydra mesoglea.

Glomerular basement membrane. Identification of dimeric subunits of the noncollagenous domain (hexamer) of collagen IV and the Goodpasture antigen.

The noncollagenous (NC1) domain hexamer of glomerular basement membrane (GBM) collagen is composed of a multiplicity of monomeric and dimeric subunits, and specific subunits are the targets for anti-GBM autoantibodies of patients with Goodpasture (GP) syndrome. The identity of GBM monomers has been established and the alpha 3(IV)NC1 monomer identified as the one that binds GP antibodies (Gunwar, S., Saus, J.

Alveolar basement membrane: molecular properties of the noncollagenous domain (hexamer) of collagen IV and its reactivity with Goodpasture autoantibodies.

The noncollagenous domain hexamer of collagen IV from bovine alveolar basement membrane was excised with bacterial collagenase, purified under nondenaturing conditions, and characterized. The hexamer is comprised of four distinct subunits [alpha 1(IV)NC1, alpha 2(IV)NC1, alpha 3(IV)NC1, and alpha 4(IV)NC1]. Each subunit exists in both monomeric and dimeric (disulfide-crosslinked) form, and both monomers and dimers have charge isoforms.

Properties of the collagenous domain of the alpha 3(IV) chain, the Goodpasture antigen, of lens basement membrane collagen. Selective cleavage of alpha (IV) chains with retention of their triple helical structure and noncollagenous domain.

A third chain, alpha 3(IV), of basement membrane collagen was recently discovered and was identified as the primary target for the autoantibodies of patients with Goodpasture syndrome (Saus, J., Wieslander, J., Langeveld, J.

Glomerular basement membrane. Identification of a fourth chain, alpha 4, of type IV collagen.

The noncollagenous domain hexamer of collagen IV from bovine glomerular basement membrane was further investigated to determine the types of collagen chain from which subunits M2*b and M3 are derived. M2*b was shown to be a shorter form, containing 9 fewer residues, of M2*a which was previously established as the noncollagenous domain of a third chain, alpha 3, of collagen IV (Saus, J., Wieslander, J.

Unusual dissociative behavior of the noncollagenous domain (hexamer) of basement membrane collagen during electrophoresis and chromatofocusing.

The noncollagenous domain of basement membrane collagen exists as a hexamer upon excision with bacterial collagenase. Two hexamer subtypes, differing in subunit composition, have been identified and several additional subtypes are possible because at least two, and possibly more, triple-helical molecules, differing in chain composition, exist in authentic basement membranes (Saus J, Wieslander J, Langeveld JPM, Quinones S, and Hudson BG. (1988) Identification of the Goodpasture antigen as the alpha 3(IV) chain of collagen IV.