PubMed Health⌕ Search

Biomedical subjects

R J Butkowski

Publications and source records attributed to R J Butkowski.

At least 19 recordsLinked to original sources
PubMed1992

Role of antibodies to tubulointerstitial nephritis antigen in human anti-tubular basement membrane nephritis associated with membranous nephropathy.

We report three patients, from two unrelated families, with anti-tubular basement membrane (TBM) antibody nephritis associated with membranous nephropathy. This rare disorder is characterized by nephrotic syndrome, tubular dysfunction, and progression to renal failure. Direct immunofluorescent studies in these patients revealed linear IgG deposition along the proximal TBM, while circulating antibodies reacting with proximal TBM but not with glomerular basement membrane were identified by indirect immunofluorescence. Sera from all three patients reacted by enzyme-linked immunosorbent assay and Western immunoblotting with purified 58-kd tubulointerstitial nephritis (TIN) antigen isolated from TBM. Additional reactivity with a 175-kd component, which may be a higher-molecular-weight form of TIN antigen, was observed by immunoblotting. Since recurrent Fanconi syndrome was seen after transplantation in one patient, anti-TBM antibodies were removed by plasmapheresis prior to kidney transplantation in the other two patients. Neither patient has clinical evidence of recurrent anti-TBM nephritis in the allograft despite the posttransplantation reappearance of anti-TBM antibodies in the serum of one patient. Serologic and molecular HLA class I and class II polymorphism analysis has identified the presence of both HLA-B7 and -DRw8 antigens in two unrelated affected individuals (0.3% expected frequency in the white population). We conclude that sera from patients with anti-TBM nephritis associated with membranous nephropathy react with 58-kd TIN antigen previously implicated in the pathogenesis of primary anti-TBM nephritis. This rare autoimmune disorder may be HLA associated with B7 and/or DRw8, providing susceptibility to the disease. Further investigation is needed to understand the pathogenesis of recurrent anti-TBM nephritis in the renal allograft.

Antibodies, Anti-Idiotypic↗
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↗
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↗
PubMed1979

Rabbit tubular basement membrane. Isolation and analysis of polypeptides.

Renal tubules from rabbit kidneys were isolated from thin shavings of the kidney surface. Basement membrane was then prepared following sonication of the isolated tubules. To insure preservation of the integrity of the basement membrane polypeptides, the protease inhibitors, diisopropyl fluorophosphate, ethylenediaminetetraacetic acid, N-ethylmaleimide, and epsilon-amino-caproic acid were used at all stages of the preparations. The optimal conditions of sonication and centrifugation were established and the chemical composition of basement membrane prepared under these conditions was examined in detail. Glycine, hydroxyproline, and hydroxylysine were found in concentrations of 206, 65, and 18 residues per thousand, respectively, in basement membrane from young kidneys. About 38% of the basement membrane was found to be soluble in sodium dodecyl sulfate upon incubation at 90 degrees C, and to possess relatively low amounts of the amino acids characteristic of collagen. Electrophoretic analysis of this fraction revealed that the major subunits ranged in approximate molecular weight from 18,500 to greater than 10(6). When analyzed with disulfide bonds reduced, a molecular weight range from 31,000 to 275,000 was observed for this fraction. The sodium dodecyl sulfate-insoluble fraction could be dissolved upon reduction and alkylation and its composition was enriched in the amino acids characteristic of collagen. Polypeptides from this fraction were analyzed by electrophoresis in agarose and in agarose-acrylamide gels. The approximate molecular weight of the smallest component was 164,000. Additional polypeptides were observed whose molecular weights occurred in multimers of this component, up to 1.1 x 10(6), possibly indicating covalent cross-linked multimers of a basic collagen-like polypeptide(s).

Amino Acids↗
PubMed1977

Thrombin: structural features related to specificity.

A comparison of the primary structure of human thrombin with the structures of chymotrypsin, trypsin, elastase and factor Xabeta reveals several structural features which may be involved in the specificity of thrombin toward macromolecular substrates. Among the major structural differences noted in such a comparison are the insertions of five extended peptide regions in the primary structure of alpha-thrombin when compared to chymotrypsin. These insertions, which we refer to as "loops", have been designated A, B, C, D, and E. The A, B and C "loops" in human thrombin appear to be large enough to interact at or near the active active site if an alpha-thrombin-chymotrypsin three-dimensional structural homology is assumed. In beta-thrombin, the configuration of the A and B "loops" may be perturbed by proteolysis, and the ability of beta-thrombin to clot fibrinogen is thus reduced. Perturbation of the configuration of the C "loops" by proteolysis in the formation of gamma-thrombin may further reduce the ability of thrombin to bind fibrinogen.

Amino Acid Sequence↗
PubMed1976

Effect of polylysine on the activation of prothrombin. Polylysine substitutes for calcium ions and factor V in the factor Xa catalyzed activation of prothrombin.

Polylysine has been demonstrated to dramatically accelerate the rate of the factor Xa catalyzed activation of both prothrombin and prethrombin 1. Under the present experimental conditions (pH 8.0, 23 C), no detectable activation of prothrombin or prethrombin 1 occurs with either factor Xa or polylysine alone. The activation of prethrombin 2, the direct precursor of alpha-thrombin, by factor Xa is not stimulated by polylysine. The activation of either prothrombin or prethrombin 1 by factor Xa in the presence of polylysine is partially inhibited by the presence of 5 mM CaCl2. Electrophoretic analysis in sodium dodecyl sulfate showed that the products that were formed in the above activation system comigrated with the reaction products derived from prothrombin activated by factor Xa in the presence of calcium ions and phospholipid. It is suggested that polylysine stimulates the factor Xa-catalyzes activation of prothrombin by replacing the combination of calcium ions and factor V.

Animals↗
PubMed1975

Human prothrombin activation.

Human prothrombin has been purified from American Red Cross Factor IX concentrates. Studies of the activation of the human prothrombin with the use of sodium dodecyl sulfate electrophoretic analysis of activation products indicated that human prothrombin activation is similar to bovine prothrombin activation. Molecular weight analysis of human prothrombin and intermediated by sodium dodecyl sulfate co-electrophoresis with bovine prothrombin and its intermediates resulted in molecular weights of 70,000 for prothrombin, 51,000 for intermediate 1, 41,000 for intermediate 2, 23,000 for intermediate 3, and 13,000 for intermediate 4. Amino acid compositions of human prothrombin and intermediates are similar to those for bovine prothrombin and intermediates. NH2-terminal sequence studies of human prothrombin, intermediates, and alpha-thrombin A and B chains placed the intermediates in the parent human prothrombin molecule as described for the bovine system. Intermediate 3 is the NH2-terminal of prothrombin, and intermediate 1 is the COOH-terminal segment of the zymogen. Intermediate 4 is the NH2-terminal of intermediate 1. Intermediate 2', the immediate precursor of alpha-thrombin, is the COOH-terminal segment of intermediate 1. In general, a high degree of homology in the primary structure of prothrombin and intermediates was observed between the human and bovine system. The NH2-terminal sequences of human intermediate 2' and alpha-thrombin A chain are identical. However, human intermediate 2' isolated in a manner identical with that used for the isolation of bovine intermediate 2 is homologous with bovine intermediate 2, beginning with residue 14.

Amino Acid Sequence↗