Biomedical subjects

R J Butkowski

Publications and source records attributed to R J Butkowski.

At least 37 records · Page 2Linked to original sources

PubMed1992

Partial protein sequence of the globular domain of alpha 4(IV) collagen chain: sites of sequence variability and homology with alpha 2(IV).

The globular domain (NC) of alpha 4(IV) collagen chain was partially sequenced and compared with the NC domain of other collagen IV chains. The alpha 4(IV) NC domain was found to be most closely related to alpha 2(IV) NC domain but distinct from the NC domain of alpha 1(IV), alpha 2(IV), alpha 3(IV) and alpha 5(IV) collagen chains. Partial sequence, representing nearly one half of alpha 4(IV) NC domain, shows 56%, 69%, 51% and 54% identity with the corresponding NC domains of alpha 1(IV), alpha 2(IV), alpha 3(IV) and alpha 5(IV) collagen chains, respectively. A short, highly polar, region of variable sequence is found near the carboxy terminus of alpha 4(IV) NC domain. This sequence corresponds to a non-conserved region among NC domains, suggesting functional specialization at this site. It exhibits high surface probability with predicted structural differences among NC domains. These results confirm uniqueness of alpha 4(IV) NC domain and indicate its structural relatedness to other NC domains of collagen IV.

Amino Acid Sequence↗

PubMed1991

X-chromosome inactivation in the liver of female heterozygous OTC-deficient sparse-furash mice.

X-chromosome inactivation patterns were investigated in livers of nine spfash female heterozygous ornithine transcarbamylase (OTC)-deficient mice. Quantitative morphometric analysis of cellular mosaicism was performed on sections of frozen liver reacted with purified anti-OTC antibody and prepared for immunofluorescent microscopy. Analysis of enzymatic OTC activity was performed on sections of these livers using a radiochromatographic technique. Several areas of cellular mosaicism were seen in each of the histological sections that were studied. The distribution of the volume fraction of the liver tissue cells having cells with normal OTC content among the nine mice ranged from 20 to 70% and it correlated (r = 0.8, P = 0.005) with the enzymatic activities of the respective livers. The extreme variegation of mosaic patches in the liver suggests the high probability that a single needle biopsy will be diagnostic in females heterozygous for an OTC mutation. This study also suggests that at the time of X inactivation, the number of primordial liver embryonic cells is small and the observed variegation of liver mosaicism probably results from complex migration patterns of liver cells during fetal development. This study shows that the spfash mouse is a suitable animal model for quantitative studies of X-chromosome inactivation in liver using immunohistochemical staining of OTC protein.

Animals↗

PubMed1991

Renal entactin (nidogen): isolation, characterization and tissue distribution.

Entactin/nidogen (E/N) was isolated from bovine renal tubular basement membrane. Apparent molecular weight, amino acid composition, and molecular configuration by electron microscopy rotary shadowing were similar to that of nidogen from EHS mouse tumor. The identity of bovine E/N was confirmed using a thrombin derived peptide, the sequence of which corresponded to a region within mouse and human E/N. Monoclonal and polyclonal anti-E/N antibodies were used to determine the distribution of E/N in human kidney by immunofluorescent and immunoelectron microscopy. E/N was present in all renal basement membranes and was distributed through the full width of the glomerular basement membrane (GBM) with accentuation along its epithelial aspects. E/N distribution was similar to that of novel collagen chain alpha 3(IV) NC domain in the GBM. In the mesangium, E/N was distributed mainly in the peripheral mesangial region that is bounded by the GBM, while classical collagen chain alpha 1(IV) NC as present diffusely throughout the mesangium. In the developing nephron, E/N was present in basement membranes of the ureteric bud, primitive vesicle and S-form. In all instances, E/N co-localized with laminin B2 chain. Prominent E/N detection within the mesangium was observed in diseases where mesangial expansion was present. This process was also seen in early diabetic nephropathy, but disappeared with disease progression. However, all thickened diabetic renal basement membranes showed an increase in E/N which was also present in Kimmelstiel-Wilson lesions. E/N was observed in the GBM "spikes" of membranous glomerulonephritis and in epithelial crescents associated with various disorders. The association between E/N, laminin and type IV collagen chains observed in the normal kidney were maintained in disorders with altered E/N distribution. We could not detect any changes in the distribution of E/N in other acquired and hereditary kidney diseases. These observations reflect the involvement of E/N in the structure and disease alteration of renal basement membranes and mesangial matrix.

Amino Acid Sequence↗

PubMed1991

Distribution of tubulointerstitial nephritis antigen and evidence for multiple forms.

A monoclonal antibody (A8) to a basement membrane component (TIN antigen), which is associated with autoimmune tubulointerstitial nephritis, was developed and utilized to characterize tissue distribution and properties of TIN antigen by immunofluorescence microscopy and immunoblotting. Results were confirmed with polyclonal goat anti-rabbit and human autoantibodies. TIN antigen was found in basement membranes of kidney cortex, small intestines, skin, and cornea, but was not detected in the renal medulla. Within the kidney cortex proximal tubular basement membrane (TBM) showed the strongest staining. TIN antigen was also detected in Bowman's capsule, distal TBM, peritubular capillaries, and focally in the interstitium, but not in glomerular basement membrane or mesangial matrix. Immunoblotting of SDS-extracted human, rabbit, mouse, and Brown Norway rat TBM with A8 revealed predominantly a 58 kD TIN antigen; however, other reactive components were detected in minor quantities. Bovine TBM contained components of 52 kD, 45 kD and 35 kD in varying concentrations. Immunoblotting of isolated rabbit TIN antigen revealed the major 58 kD component that was characterized previously, and minor components of 300 kD, 175 kD, 160 kD and 50 kD. TIN antigen was not detected in Lewis rat TBM by immunofluorescence or immunoblotting. These studies suggest the following: 1) TIN antigen may be synthesized as a high molecular weight glycoprotein that is processed to smaller forms; 2) it may be covalently associated with other basement membrane components; 3) the antibody reactive epitope may be present on multiple TBM components; and 4) high molecular weight forms may represent aggregates of TIN antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Characterization of a tubular basement membrane component reactive with autoantibodies associated with tubulointerstitial nephritis.

A kidney tubular basement membrane (TBM) component that is bound by antibodies from individuals with anti-TBM antibody-associated tubulointerstitial nephritis (TIN) was purified and characterized (TIN antigen). TIN antigen was prepared from rabbit TBM by extraction with guanidine and purified by ion-exchange, gel filtration, and reversed-phase chromatography. Based upon yields of protein and antibody reactivity, TIN antigen accounts for about 9% of the mass of TBM and thus is a major component of this basement membrane. A predominant 58-kDa form comprises about 90% of purified TIN antigen, and a 50-kDa form accounts for the remainder. The two forms share the amino-terminal sequence Ser-Ile-Phe-Gln-Gly-Gln-Tyr-X-Arg-Ser-Phe-Gly- and give similar tryptic peptide maps, indicating that they are structurally related. Their amino acid compositions overall are similar to laminin and entactin/nidogen. The absence of hydroxyproline and hydroxylysine and the low levels of glycine in TIN antigen indicate that it is noncollagenous. No similarities were found between other known proteins and sequences of tryptic peptides and the amino terminus of TIN antigen, suggesting that it is distinct from other characterized basement membrane components. A goat polyclonal antibody toward rabbit TIN antigen showed the same kidney distribution as human antibodies and was completely inhibited in enzyme-linked immunosorbent assay by purified TIN antigen. These data further support the idea that TIN antigen is the primary target for anti-TBM antibodies associated with TIN. This research presents methods to prepare TIN antigen for biochemical studies and investigations of its role in anti-TBM autoimmune TIN.

Amino Acid Sequence↗

PubMed1990

Alport syndrome, basement membranes and collagen.

Alport syndrome, an inherited disorder of the kidney, eye and ear, has fascinated nephrologists, pathologists, and geneticists for nearly a century. With the recent application of molecular biochemical and genetic techniques, this mysterious disease has begun to yield some of its secrets. Alport syndrome can now be viewed as a generalized disorder of basement membranes that appears to result from mutations in an X-chromosome-encoded basement membrane collagen chain. This chain, along with two other novel collagen chains, is absent from Alport basement membranes, in contrast to the classical chains of collagen IV. Phenotypic heterogeneity in Alport syndrome probably arises from allelic mutations at a single genetic locus. The phenomenon of post-transplant anti-glomerular basement membrane nephritis may be a manifestation of specific mutations at the Alport locus that prevent synthesis of the gene's protein product and the establishment of immunological tolerance.

Basement Membrane↗

PubMed1990

A method for preparation of renal tubular basement membrane.

A method is presented for the preparation of tubular basement membrane from renal cortex. The procedure utilizes a Polytron tissue disrupter, differential sieving, and for some species, sucrose density centrifugation. The procedure is especially useful for obtaining highly purified rabbit and bovine tubular basement membranes. Human tubular and glomerular basement membranes can be prepared with some cross contamination. The method offers the capability of rapidly generating large quantities of the basement membranes that retain the Goodpasture antigen and a TBM antigen associated with anti-TBM nephritis.

Animals↗

PubMed1990

Characterization of type IV collagen NC1 monomers and Goodpasture antigen in human renal basement membranes.

Monomeric subunits of the globular domain of type IV collagen from human renal basement membrane were isolated and characterized. The monomers, M24, M26, M28+, and M28 , which have been identified previously in human glomerular basement membrane, were characterized by amino acid analysis, amino-terminal sequencing, and electrophoretic mobility. The results indicate that M24 and M26 are derived from alpha 1(IV) and alpha 2(IV) collagen chains, respectively. Amino-terminal sequencing revealed that M28+, and M28 correspond to the globular domain of novel collagen chains. M28 has been characterized as the principal target antigen in Goodpasture's syndrome and antiglomerular basement membrane (GBM) nephritis, and both M28 species are absent from the GBM in Alport familial nephritis. The cationic charge of M28 appears to be a consequence of a relatively high concentration of basic amino acids when compared with other monomers. Previous studies of bovine GBM have demonstrated chains with amino-terminal sequence homology to M28+ and M28 .

Amino Acid Sequence↗

PubMed1990

Immunochemical analysis of carbamyl phosphate synthetase I and ornithine transcarbamylase deficient livers: elevated N-acetylglutamate level in a liver lacking carbamyl phosphate synthetase protein.

Liver tissue from 3 patients with carbamyl phosphate synthetase I deficiency and 11 patients with ornithine transcarbamylase deficiency was analyzed by Western blots for the presence of carbamyl phosphate synthetase I and ornithine transcarbamylase cross-reactive material with anti-rat enzyme antibodies. One patient with carbamyl phosphate synthetase I deficiency had no cross-reactive material with the antibody to this enzyme, one had reduced amounts, and one had similar to normal amounts. Only one of the patients with ornithine transcarbamylase deficiency had apparently normal amounts of enzyme protein in his liver, another patient had markedly reduced amounts, and the other nine patients had traces or absence of reactive ornithine transcarbamylase protein. N-acetylglutamate content in the carbamyl phosphate synthetase I deficient liver with absent enzyme protein, was higher (98.4 nmol/g) than that measured in 5 normal controls (41.6 +/- 19.3 mean +/- SD, n = 5) and much higher than the N-acetylglutamate level (2.2 nmol/g) in another carbamyl phosphate synthetase I deficient liver with normal amounts of enzyme protein. Hepatic N-acetylglutamate content in 3 patients with complete ornithine transcarbamylase deficiency was below the normal mean but within 2 standard deviations from the mean (8.5-12.8 nmol/g) whereas it was higher (30.7 nmol/g) in another partial ornithine transcarbamylase deficient patient. Most patients with ornithine transcarbamylase deficiency have markedly reduced or undetectable enzyme protein in their liver, therefore. Western blot analysis could help to establish this diagnosis in most patients. Absent carbamyl phosphate synthetase I enzyme protein may have caused elevation of the hepatic content of its cofactor, N-acetylglutamate, by an as yet unknown mechanism.

Adult↗

PubMed1990

Posttransplant anti-glomerular basement membrane nephritis in related males with Alport syndrome.

This report describes the development of anti-glomerular basement membrane (GBM) glomerulonephritis after kidney transplantation in related males with Alport syndrome. Antibodies in sera from one of these patients stained normal GBM, Bowman's capsule, tubular basement membranes, and epidermal basement membranes but did not stain tissues from an unrelated Alport male. The target antigen was found to be a 26 kd peptide of the noncollagenous domain of basement membrane collagen. This study provides further evidence of the importance of abnormalities of basement membrane collagen in the pathogenesis of the Alport nephropathy. We speculate that certain mutations at the Alport locus, such as large intragenic deletions or frame-shift mutations, may be associated with failure to develop immune tolerance to epitopes on this 26kd peptide. In the setting of permissive immune response and regulation, transplantation of a normal kidney may result in the generation of anti-GBM antibodies.

Basement Membrane↗

PubMed1989

Basement membrane collagen in the kidney: regional localization of novel chains related to collagen IV.

Variability in the collagen chain composition of renal basement membranes was demonstrated by immunofluorescent microscopy using polyclonal and monoclonal antibodies and correlating with imaging of the glomerular basement membrane by phase microscopy. Antibodies toward the globular domains of alpha 1(IV) and alpha 2(IV) collagen chains, triple helical and 7S domains of collagen IV bind within the glomerulus to mesangial matrix, along the subendothelial region of the glomerular capillary wall, and to all tubular and vascular basement membranes. The portion of glomerular basement membrane corresponding to the phase dense image is not reactive with these antibodies. A different binding pattern is seen with antibodies against two novel globular regions of basement membrane collagen chains which bind to the phase dense aspect of glomerular basement membrane and to Bowman's capsule. Human tubular basement membrane is not reactive, except along portions of the distal tubule, whereas bovine tubular basement membrane is diffusely reactive; mesangial matrix and extraglomerular vascular basement membranes are not reactive. Although a possible explanation for the regional distribution of basement membrane collagen antigens in the glomerulus may relate to antigen exposure, a more likely reason is that collagen chains are regionally expressed. The staining patterns suggest that the novel collagen chains have a selective tissue distribution compared with alpha 1(IV) and alpha 2(IV) chains and that the glomerular cells of origin of these collagen IV chains may differ.

Animals↗

PubMed1988

Structural heterogeneity of the noncollagenous domain of basement membrane collagen.

The noncollagenous domain of collagen from three different basement membranes of bovine origin (glomerular, lens capsule, and placental) was excised with bacterial collagenase, purified under nondenaturing conditions, and characterized. In each case the domain existed as a hexamer comprised of four distinct subunits (alpha 1 (IV) NC1, alpha 2 (IV) NC1, M2*, and M3). Each subunit exists in both monomeric and dimeric (disulfide-cross-linked) forms. Certain dimers also exist which contain nonreducible cross-links. The hexamers from the three membranes differ with respect to stoichiometry of subunits and subunit isoforms and to the degree of cross-linking of monomers into dimers. The minor subunits, M2* and M3, vary in quantity over a 20-fold range relative to the major ones among the three hexamers. The results indicate that: 1) at least two populations of triple-helical collagen molecules, differing in chain composition, exist in each membrane and that their relative proportions are tissue-specific; and 2) the chemical nature of the noncollagenous domain of these populations is tissue-specific with regard to subunit isoforms and relative proportion of reducible and nonreducible cross-links in dimers. A novel structural feature of the noncollagenous domain of basement membrane collagen was also evinced from these studies. Namely, that each of the four monomeric subunits exists in charge isoforms.

Amino Acids↗

PubMed1987

Localization of the Goodpasture epitope to a novel chain of basement membrane collagen.

The chain origins of subunits M1, M2*, and M3 previously described (Butkowski, R. L., Wieslander, J., Wisdom, B.J., Barr, J.F., Noelken, M.E., and Hudson, B.G. (1985) J. Biol. Chem. 260, 3739-3747) of the globular domain of basement membrane collagen were identified, by amino-terminal amino acid sequence analysis, with respect to their relationship to the chains of collagen IV. M1 comprises two polypeptides which correspond to the noncollagenous segments (NC1) of the alpha 1 ad alpha 2 chains of collagen IV. M2*, containing the Goodpasture epitope, and M3 are distinct from these two constituents and from each other but have Gly-X-Y triplets and hydroxyproline at their amino terminus, reflecting the fact that each has a collagen chain origin. These results indicate the presence of two new collagen chains in basement membrane. These new chains appear to be integral components of collagen IV molecules. Alternatively, they could represent new molecular species of basement membrane collagen containing a globular domain, comprising M2* and M3, with physicochemical properties very similar to those of collagen IV.

Amino Acid Sequence↗

PubMed1987

Identification of a target antigen in human anti-tubular basement membrane nephritis.

Sera from two patients with primary anti-tubular-basement-membrane-mediated tubulointerstitial nephritis, one a renal allograft recipient and the other with spontaneous anti-tubular-basement-membrane disease, were analyzed for the specificity of their autoantibodies. Both sera had circulating antibodies that reacted by ELISA with extracts of tubular basement membrane from several species, but failed to react significantly with extracts of glomerular basement membrane. Reactive antigen was solubilized with 6 M guanidine-HCl, 6 M urea, with reduction and alkylation, and with sodium dodecylsulfate. Digestion of the basement membrane with collagenase released relatively small quantities of antigen from the membrane, and trypsin and pepsin destroyed its antigenicity. The antigenic activity was characterized with respect to its size distribution by gel filtration and by immuno-overlay analysis of protein blots. Collectively, the results indicate that the major reactivity of both sera is directed towards a Mr 58,000 component that is unique to the tubular basement membrane. Minor reactivities toward high molecular weight components common to both glomerular and tubular basement membranes were detected by immuno-overlay analysis. This study identifies an antigen that is involved in human anti-tubular-basement-membrane-mediated tubulointerstitial nephritis, and demonstrates an advantage of the use of denaturing extraction over proteolytic methods to prepare the antigen.

Adult↗

PubMed1987

Alport familial nephritis. Absence of 28 kilodalton non-collagenous monomers of type IV collagen in glomerular basement membrane.

Alport-type familial nephritis (FN), a genetic disorder, results in progressive renal insufficiency and sensorineural hearing loss. Immunochemical and biochemical analyses of the non-collagenous (NC1) domain of type IV collagen isolated from the glomerular basement membranes (GBM) of three males with this disease demonstrate absence of the normally occurring 28-kilodalton (kD) NC1 monomers, but persistence of the 26- and 24-kD monomeric subunits derived from alpha 1 and 2 (both type IV) collagen chains, respectively.

Basement Membrane↗

PubMed1985

Properties of the globular domain of type IV collagen and its relationship to the Goodpasture antigen.

The globular domain of type IV collagen from bovine glomerular basement membrane was solubilized by collagenase digestion. Components of this domain include several monomer-size and structurally related dimer-size polypeptides. The monomer-size polypeptides were resolved into three fractions (M1, M2, and M3) with slightly different mobilities upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (nonreduced Mr = 24,500-28,300). Chemical and immunochemical studies indicate that each is a distinct component. M2 is reactive with antibodies from patients with Goodpasture syndrome. The molecular weight by sedimentation equilibrium was 32,000 for M2 and 28,000 for M1. The dimers were characterized as two classes, D1 and D2. D1 consists of two sets of nonreactive components (D1a-d and D1a,c) whereas D2 contains one set of four components (D2a-d), each of which is reactive with Goodpasture sera. Chemical and immunochemical studies indicate that a monomer-dimer relationship exists between M1 and D1 and between M2 and D2. The origin of M3 remains undetermined. Rabbit antibodies to type IV collagen alpha chains react with M1 and M2, and antibodies to M1 and M2 react with type IV collagen alpha chains, which provides additional evidence for the localization of the Goodpasture antigen to one of the chains of type IV collagen.

Amino Acids↗

PubMed1985

Tubular basement membrane changes in 2-amino-4,5-diphenylthiazole-induced polycystic disease.

Polycystic renal disease was induced in rats by feeding 2-amino-4,5-diphenylthiazole. Tubular (TBM) and glomerular basement membranes (GBM) were purified and analyzed for possible structural changes that may be a factor in the development of the tubular dilations and cysts. Changes in the relative quantities of TBM polypeptides were detected by sodium dodecylsulfate polyacrylamide gel electrophoresis. An overall increase in the concentration of high molecular weight components and a decrease in concentration of those of low molecular weight components were observed. Changes which were particularly notable included a twofold increase in a component of Mr = 380,000 and a decrease in one of Mr = 55,000 as analyzed without reduction of disulfide bonds. With reduction of disulfide bonds, the Mr = 380,000 component dissociates, whereas the Mr = 55,000 polypeptide does not, and polypeptides of Mr = 245,000 and 145,000 are observed to does not, and polypeptides of Mr = 245,000 and 145,000 are observed to increase about twofold in concentration (approximate molecular weights were determined using globular protein standards). These changes take place most rapidly from 4 to 8 weeks of drug administration and remain relatively constant between 8 and 16 weeks. If feeding of the drug is discontinued, the distribution of TBM polypeptides returns to normal. These results indicate that tubular basement membrane from animals with 2-amino-4,5-diphenylthiazole-induced polycystic renal disease is abnormal, and this should be considered as a possible contributing factor in the formation of cysts.

Amino Acids↗

PubMed1984

Goodpasture antigen of the glomerular basement membrane: localization to noncollagenous regions of type IV collagen.

The glomerular basement membrane antigen in Goodpasture syndrome is a collagenase-resistant molecule with a monomer molecular weight of about 26,000. Type IV collagen isolated from glomerular basement membrane contains collagenase-resistant sequences within its structure. Polyacrylamide gel electrophoresis, enzyme-linked immunosorbent assay, and chemical analysis were used to demonstrate that the collagenase-resistant sequences of type IV collagen contain Goodpasture antigen.

Anti-Glomerular Basement Membrane Disease↗