Biomedical subjects

J J Enghild

Publications and source records attributed to J J Enghild.

At least 19 recordsLinked to original sources

PubMed2001

Altered expression of extracellular superoxide dismutase in mouse lung after bleomycin treatment.

The antioxidant enzyme extracellular superoxide dismutase (EC-SOD) is highly expressed in the extracellular matrix of lung tissue and is believed to protect the lung from oxidative damage that results in diseases such as pulmonary fibrosis. This study tests the hypothesis that proteolytic removal of the heparin-binding domain of EC-SOD results in clearance of the enzyme from the extracellular matrix of pulmonary tissues and leads to a loss of antioxidant protection. Using a polyclonal antibody to mouse EC-SOD, the immunodistribution of EC-SOD in normal and bleomycin-injured lungs was examined. EC-SOD labeling was strong in the matrix of vessels, airways, and alveolar surfaces and septa in control lungs. At 2 d post-treatment, a slight increase in EC-SOD staining was evident. In contrast, lungs examined 4 or 7 d post-treatment, showed an apparent loss of EC-SOD from the matrix and surface of alveolar septa. Notably, at 7 d post-treatment, the truncated form of EC-SOD was found in the bronchoalveolar lavage fluid of bleomycin-treated mice, suggesting that EC-SOD is being removed from the extracellular matrix through proteolysis. However, loss of EC-SOD through proteolysis did not correlate with a decrease in overall pulmonary EC-SOD activity. The negligible effect on EC-SOD activity may reflect the large influx of intensely staining inflammatory cells at day 7. These results indicate that injuries leading to pulmonary fibrosis have a significant effect on EC-SOD distribution due to proteolytic removal of the heparin-binding domain and may be important in enhancing pulmonary injuries by altering the oxidant/antioxidant balance in alveolar interstitial spaces.

Animals↗

PubMed2001

Covalent complexes of antigen and alpha(2)-macroglobulin: evidence for dramatically-increased immunogenicity.

A safe, effective, more potent adjuvant than currently available would be beneficial in developing new therapeutics and diagnostic reagents. We report here a technique for the rapid, efficient incorporation of non-proteolytic antigens into alpha(2)-macroglobulin (alpha(2)M; tradename, SynerVax), allowing us to covalently couple much larger subunit antigens to alpha(2)M than previously possible. Our goal was to determine if incorporation of HB, the monomeric form of Hepatitis B virus (HBV) surface antigen (HBsAg), into alpha(2)M would result in increased immune reactivity. Earlier attempts to immunize animals using HB did not generate significant levels of antibodies. Using HB complexes prepared with alpha(2)M we now report dramatically-increased immunogenicity of HB in BALB/c mice. Combining these soluble complexes with a depot-generating agent (alum), titers>1:1,000,000 are obtained with a single injection. This novel adjuvant technology should provide a valuable tool for the development of either prophylactic and therapeutic vaccines, or monoclonal antibodies against hitherto poorly-immunogenic subunit antigens.

Animals↗

PubMed2000

Purification and characterization of extracellular superoxide dismutase in mouse lung.

Extracellular superoxide dismutase (EC-SOD) is the major isozyme of SOD in arteries, but is also abundant in lungs. In particular, mouse lungs contain large amounts of EC-SOD compared to lungs in other mammals. This suggests that EC-SOD may have an amplified function in the mouse lung. This study describes the purification and characterization of mouse EC-SOD as well as its localization in mouse lung. Mouse EC-SOD exists primarily as a homotetramer composed of a pair of dimers linked through disulfide bonds present in the heparin-binding domains of each subunit. In addition, mouse EC-SOD can exist in active multimeric forms. We developed and utilized a polyclonal antibody to mouse EC-SOD to immunolocalize EC-SOD in mouse lung. EC-SOD labeling is strongest in the matrix of vessels, airways, and alveolar septa. This localization suggests that EC-SOD may have important functions in pulmonary biology, perhaps in the modulation of nitric oxide-dependent responses.

Amino Acid Sequence↗

PubMed2000

Secretion of extracellular superoxide dismutase in neonatal lungs.

Extracellular superoxide dismutase (EC-SOD), the only known enzymatic scavenger of extracellular superoxide, may modulate reactions of nitric oxide (NO) in the lungs by preventing reactions between superoxide and NO. The regulation of EC-SOD has not been examined in developing lungs. We hypothesize that EC-SOD plays a pivotal role in the response to increased oxygen tension and NO in the neonatal lung. This study characterizes rabbit EC-SOD and investigates the developmental regulation of EC-SOD activity, protein expression, and localization. Purified rabbit EC-SOD was found to have several unique biochemical attributes distinct from EC-SOD in other species. Rabbit lung EC-SOD contains predominantly uncleaved subunits that do not form disulfide-linked dimers. The lack of intersubunit disulfide bonds may contribute to the decreased heparin affinity and lower EC-SOD content in rabbit lung. EC-SOD activity in rabbit lungs is low before birth and increases soon after gestation. In addition, the enzyme is localized intracellularly in preterm and term rabbit lungs. Secretion of active EC-SOD into the extracellular compartment increases with age. The changes in EC-SOD localization and activity have implications for the neonatal pulmonary response to oxidative stress and the biological activity of NO at birth.

Aging↗

PubMed1999

Organization of the inter-alpha-inhibitor heavy chains on the chondroitin sulfate originating from Ser(10) of bikunin: posttranslational modification of IalphaI-derived bikunin.

Inter-alpha-inhibitor-derived bikunin was purified and the molecular mass was determined to be approximately 8.7 kDa higher than the prediction based on the protein sequence, suggesting extensive posttranslational modifications. These modifications were identified and characterized by a combination of protein and carbohydrate analytical techniques. Three modifications were identified: (i) glycosylation of Ser(10), (ii) glycosylation of Asn(45), and (iii) a heterogeneous truncation of the C-terminus. The Asn(45) associated glycan was shown to be a homogenous "complex type" biantennary structure. The chondroitin-4-sulfate (CS) chain attached to Ser(10) was analyzed by both matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and acrylamide gel electrophoresis after partial chondroitin ABC lyase digestion. The analyses showed that the CS chains were composed of 15 +/- 3 [GlcUA-GalNAc] disaccharide units. On average, every forth disaccharide was sulfated, and these sulfated disaccharides appeared to be more common near the reducing end. Anion exchange chromatography at pH 3. 4 of intact bikunin resulted in the isolation of four isotypes shown to differ only in the amount of sulfation. Heavy chain 1 (HC1) and heavy chain 2 (HC2) are attached to the CS by a novel cross-link [Enghild, J. J., Salvesen, G., Hefta, S. A., Thogersen, I. B., Rutherfurd, S., and Pizzo, S. V. (1991) J. Biol. Chem. 266, 747-751], and the order in which the two heavy chains are positioned on the CS was examined. The results indicate that HC1 is in close proximity to HC2 and both are near the less sulfated nonreducing end of the CS. Taken together, the data show the following organization of the IalphaI molecule: [GlcUA-GalNAc](a)-HC1-[GlcUA-GalNAc](b)-HC2-[GlcUA-GalNAc](c)-Gal -Gal-Xyl-Ser(10)-bikunin, (a + b + c = 12-18 disaccharides).

Alpha-Globulins↗

PubMed1999

Autocrine regulation of growth stimulation in human epithelial ovarian carcinoma by serine-proteinase-catalysed release of the urinary-type-plasminogen-activator N-terminal fragment.

Ovarian carcinomas secrete single-chain urinary-type plasminogen activator (scuPA) and expression of uPA is up-regulated relative to normal ovarian epithelium, leading to an enhanced proteolytic capacity which may facilitate invasion. Furthermore, the uPA receptor (uPAR) is present on ovarian carcinoma cells and is occupied in tumour tissues. In the present study, incubation of scuPA with serum-free conditioned medium from ovarian carcinoma cells resulted in release of a 14 kDa polypeptide. N-terminal sequence analysis identified this fragment as the uPA N-terminal fragment (NTF), which contains a growth-factor and a kringle domain. NTF generation was abolished by serine-proteinase inhibitors, but not inhibitors of matrix metalloproteinases, and was not enhanced by the addition of plasminogen or plasmin. To determine whether ovarian carcinoma-cell growth is altered by uPA, the effect of exogenous scuPA or NTF on proliferation was analysed. Both NTF and scuPA induced a dose-dependent increase in proliferation, with maximal stimulation obtained at 10-20 nM. Furthermore, blocking the interaction of endogenous uPA with uPAR using anti-NTF antibodies significantly inhibited proliferation. Together these data indicate that, in addition to enhancing the invasive activity of ovarian carcinoma cells via increased pericellular proteolysis, uPA also acts as a mitogen for ovarian carcinoma cells, suggesting a biochemical mechanism whereby uPA may contribute to ovarian carcinoma progression by modulating both cell invasion and proliferation.

Cell Division↗

PubMed1999

The heparin-binding domain of extracellular superoxide dismutase is proteolytically processed intracellularly during biosynthesis.

Extracellular superoxide dismutase (EC-SOD) is the only known extracellular enzyme designed to scavenge the superoxide anion. The purified enzyme exists in two forms when visualized by reduced SDS-polyacrylamide gel electrophoresis: (i) intact EC-SOD (Trp1-Ala222) containing the C-terminal heparin-binding domain and (ii) cleaved EC-SOD (Trp1-Glu209) without the C-terminal heparin-binding domain. The proteolytic event(s) leading to proteolysis at Glu209-Arg210 and removal of the heparin-binding domain are not known, but may represent an important regulatory mechanism. Removal of the heparin-binding domain affects both the affinity of EC-SOD for and its distribution to the extracellular matrix, in which it is secreted. During the purification of human EC-SOD, the intact/cleaved ratio remains constant, suggesting that proteolytic removal of the heparin-binding domain does not occur during purification (Oury, T. D., Crapo, J. D., Valnickova, Z., and Enghild, J. J. (1996) Biochem. J. 317, 51-57). This was supported by the finding that fresh mouse tissue contains both intact and cleaved EC-SOD. To study other possible mechanisms leading to the formation of cleaved EC-SOD, we examined biosynthesis in cultured rat L2 epithelial-like cells using a pulse-chase protocol. The results of these studies suggest that the heparin-binding domain is removed intracellularly just prior to secretion. In addition, the intact/cleaved EC-SOD ratio appears to be tissue-dependent, implying that the intracellular processing event is regulated in a tissue-specific manner. The existence of this intracellular processing pathway may thus represent a novel regulatory pathway for affecting the distribution and effect of EC-SOD.

Animals↗

PubMed1999

Angiostatin inhibits endothelial and melanoma cellular invasion by blocking matrix-enhanced plasminogen activation.

Angiostatin, a kringle-containing fragment of plasminogen, is a potent inhibitor of angiogenesis. The mechanism(s) responsible for the anti-angiogenic properties of angiostatin are unknown. We now report that human angiostatin blocks plasmin(ogen)-enhanced in vitro invasion of tissue plasminogen activator (t-PA)-producing endothelial and melanoma cells. Kinetic analyses demonstrated that angiostatin functions as a non-competitive inhibitor of extracellular-matrix (ECM)-enhanced, t-PA-catalysed plasminogen activation, with a Ki of 0.9+/-0.03 microM. This mechanism suggests that t-PA has a binding site for the inhibitor angiostatin, as well as for its substrate plasminogen that, when occupied, prevents ternary complex formation between t-PA, plasminogen and matrix protein. Direct binding experiments confirmed that angiostatin bound to t-PA with an apparent Kd [Kd(app)] of 6.7+/-0.7 nM, but did not bind with high affinity to ECM proteins. Together, these data suggest that angiostatin in the cellular micro-environment can inhibit matrix-enhanced plasminogen activation, resulting in reduced invasive activity, and suggest a biochemical mechanism whereby angiostatin-mediated regulation of plasmin formation could influence cellular migration and invasion.

Angiostatins↗

PubMed1999

Angiostatin binds ATP synthase on the surface of human endothelial cells.

Angiostatin, a proteolytic fragment of plasminogen, is a potent antagonist of angiogenesis and an inhibitor of endothelial cell migration and proliferation. To determine whether the mechanism by which angiostatin inhibits endothelial cell migration and/or proliferation involves binding to cell surface plasminogen receptors, we isolated the binding proteins for plasminogen and angiostatin from human umbilical vein endothelial cells. Binding studies demonstrated that plasminogen and angiostatin bound in a concentration-dependent, saturable manner. Plasminogen binding was unaffected by a 100-fold molar excess of angiostatin, indicating the presence of a distinct angiostatin binding site. This finding was confirmed by ligand blot analysis of isolated human umbilical vein endothelial cell plasma membrane fractions, which demonstrated that plasminogen bound to a 44-kDa protein, whereas angiostatin bound to a 55-kDa species. Amino-terminal sequencing coupled with peptide mass fingerprinting and immunologic analyses identified the plasminogen binding protein as annexin II and the angiostatin binding protein as the alpha/beta-subunits of ATP synthase. The presence of this protein on the cell surface was confirmed by flow cytometry and immunofluorescence analysis. Angiostatin also bound to the recombinant alpha-subunit of human ATP synthase, and this binding was not inhibited by a 2,500-fold molar excess of plasminogen. Angiostatin's antiproliferative effect on endothelial cells was inhibited by as much as 90% in the presence of anti-alpha-subunit ATP synthase antibody. Binding of angiostatin to the alpha/beta-subunits of ATP synthase on the cell surface may mediate its antiangiogenic effects and the down-regulation of endothelial cell proliferation and migration.

Adenosine Triphosphatases↗

PubMed1999

Alpha1-microglobulin chromophores are located to three lysine residues semiburied in the lipocalin pocket and associated with a novel lipophilic compound.

Alpha1-microglobulin (alpha1m) is an electrophoretically heterogeneous plasma protein. It belongs to the lipocalin superfamily, a group of proteins with a three-dimensional (3D) structure that forms an internal hydrophobic ligand-binding pocket. Alpha1m carries a covalently linked unidentified chromophore that gives the protein a characteristic brown color and extremely heterogeneous optical properties. Twenty-one different colored tryptic peptides corresponding to residues 88-94, 118-121, and 122-134 of human alpha1m were purified. In these peptides, the side chains of Lys92, Lys118, and Lys130 carried size heterogeneous, covalently attached, unidentified chromophores with molecular masses between 122 and 282 atomic mass units (amu). In addition, a previously unknown uncolored lipophilic 282 amu compound was found strongly, but noncovalently associated with the colored peptides. Uncolored tryptic peptides containing the same Lys residues were also purified. These peptides did not carry any additional mass (i.e., chromophore) suggesting that only a fraction of the Lys92, Lys118, and Lys130 are modified. The results can explain the size, charge, and optical heterogeneity of alpha1m. A 3D model of alpha1m, based on the structure of rat epididymal retinoic acid-binding protein (ERABP), suggests that Lys92, Lys118, and Lys130 are semiburied near the entrance of the lipocalin pocket. This was supported by the fluorescence spectra of alpha1m under native and denatured conditions, which indicated that the chromophores are buried, or semiburied, in the interior of the protein. In human plasma, approximately 50% of alpha1m is complex bound to IgA. Only the free alpha1m carried colored groups, whereas alpha1m linked to IgA was uncolored.

Animals↗

PubMed1999

Histologic distribution and biochemical properties of alpha 1-microglobulin in human placenta.

PROBLEM: The embryo is protected from immunologic rejection by the mother, possibly accomplished by immunosuppressive molecules located in the placenta. We investigated the distribution and biochemical properties in placenta of the immunosuppressive plasma protein alpha 1-microglobulin. METHOD OF STUDY: Placental alpha 1-microglobulin was investigated by immunohistochemistry and, after extraction, by electrophoresis, immunoblotting and radioimmunoassay. RESULTS: alpha 1-Microglobulin staining was observed in the intervillous fibrin and in syncytiotrophoblasts, especially at sites with syncytial injury. Strongly stained single cells in the intervillous spaces and variably stained intravillous histiocytes were noted. Solubilization of the placenta-matrix fraction and placenta membrane fraction released predominantly the free form of alpha 1-microglobulin, but, additionally, an apparently truncated form from the placenta-membrane fraction. The soluble fraction of placenta contained two novel alpha 1-microglobulin complexes. CONCLUSIONS: The biochemical analysis indicates the presence in placenta of alpha 1-microglobulin forms not found in blood. The histochemical analysis supports the possibility that alpha 1-microglobulin may function as a local immunoregulator in the placenta.

Electrophoresis, Polyacrylamide Gel↗

PubMed1998

Human procarboxypeptidase U, or thrombin-activable fibrinolysis inhibitor, is a substrate for transglutaminases. Evidence for transglutaminase-catalyzed cross-linking to fibrin.

Procarboxypeptidase U (EC 3.4.17.20) (pro-CpU), also known as plasma procarboxypeptidase B and thrombin-activable fibrinolysis inhibitor, is a human plasma protein that has been implicated in the regulation of fibrinolysis. In this study, we show that pro-CpU serves as a substrate for transglutaminases. Both factor XIIIa and tissue transglutaminase catalyzed the polymerization of pro-CpU and the cross-linking to fibrin as well as the incorporation of 5-dimethylaminonaphthalene-1-sulfonyl cadaverine (dansylcadaverine), [14C]putrescine, and dansyl-PGGQQIV. These findings show that pro-CpU contains both amine acceptor (Gln) and amine donor (Lys) residues. The amine acceptor residues were identified as Gln2, Gln5, and Gln292, suggesting that both the activation peptide and the mature enzyme participate in the cross-linking reaction. These observations imply that transglutaminases may mediate covalent binding of pro-CpU to other proteins and cell surfaces in vivo. In particular, factor XIIIa may cross-link pro-CpU to fibrin during the latter part of the coagulation cascade, thereby helping protect the newly formed fibrin clot from premature plasmin degradation. Moreover, the cross-linking may facilitate the activation of pro-CpU, stabilize the enzymatic activity, and protect the active enzyme from further degradation.

Binding Sites↗

PubMed1998

Expression of a copper-containing amine oxidase by human ciliary body.

PURPOSE: To examine the molecular structure and ultrastructural distribution of a novel amine oxidase in human ciliary body. METHODS: Human ciliary bodies were solubilized with a nonionic detergent. The solubilized material was subjected to affinity chromatography with 2B4.14.1, a monoclonal antibody which recognizes a family of ciliary body glycoproteins. Proteins eluted from the affinity column were further separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Peptides produced from a 2B4.14. 1-reactive protein with an approximate molecular weight of 100 kDa were analyzed by Edman degradation. The protein thus identified was further examined by Western blotting and immunoelectron microscopy with anti-peptide antisera. RESULTS: Peptide sequences from the 100 kDa ciliary body protein were identical to the predicted protein sequence of an amine oxidase identified recently in a human placental cDNA library. The identity of the ciliary body protein was confirmed by Western blotting with rabbit antiserum generated against the predicted carboxy-terminal peptide of human placenta amine oxidase. Western blotting under nonreducing conditions and following glycosidase digestion indicated that the native enzyme is a disulfide-linked homodimer with multiple N-linked oligosaccharide side chains. By immunoelectron microscopy, the ciliary body amine oxidase was localized to the plasma membranes of inner epithelial cells. CONCLUSIONS: Human placenta amine oxidase is present on the plasma membranes of ciliary body inner epithelial cells. This finding provides a potential explanation for amine oxidase enzyme activity detected in previous studies of anterior segment tissues. Though the functional role of human placenta amine oxidase in the eye is unclear, it may contribute to the production of H2O2 in aqueous humor.

Aged↗

PubMed1998

Comparative properties of two cysteine proteinases (gingipains R), the products of two related but individual genes of Porphyromonas gingivalis.

Proteolytic enzymes produced by Porphyromonas gingivalis are important virulence factors of this periodontopathogen. Two of these enzymes, referred to as arginine-specific cysteine proteinases (gingipains R), are the product of two related genes. Here, we describe the purification of an enzyme translated from the rgpB/rgp-2 gene (gingipain R2, RGP-2) and secreted as a single chain protein of 422 residues. The enzyme occurs in several isoforms differing in pI, molecular mass, mobility in gelatin zymography gels, and affinity to arginine-Sepharose. In comparison to the 95-kDa gingipain R1, a complex of catalytic and hemagglutinin/adhesin domains, RGP-2 showed five times lower proteolytic activity, although its activity on various P1-arginine p-nitroanilide substrates was generally higher. Gingipains R amidolytic activity, but not general proteolytic activity, was stimulated by glycyl-glycine. However, in cases of limited proteolysis, such as the inactivation of alpha-1-antichymotrypsin, glycyl-glycine potentiated inhibitor cleavage. In contrast, alpha-1-proteinase inhibitor was not inactivated by gingipains R and only underwent proteolytic degradation during boiling in reducing SDS-polyacrylamide gel electrophoresis treatment buffer. Similarly, native type I collagen was completely resistant to cleavage by gingipains but readily degraded after denaturation. Together, these data explain much of the controversy regarding gingipains structure and substrate specificity and indicate that these enzymes function as P. gingivalis virulence factors by proteolysis of selected target proteins rather than random degradation of host connective tissue components.

Adhesins, Bacterial↗

PubMed1998

Preparation and electron paramagnetic resonance characterization of spin labeled monoderivatives of horse cytochrome c.

Horse cytochrome c was reacted with the spin label (succinimidyl-2,2, 5,5-tetra-methyl-3-pyrroline-1-oxyl-carboxylate) using optimized conditions and the reaction products were separated by a combination of cation-exchange chromatography and HPLC. The purified cytochrome c derivatives were digested with TPCK treated trypsin and the resulting peptides were separated by reverse phase HPLC. The modified Lys residues were subsequently characterized by Edman degradation and mass spectrometry. These analyses showed that five distinct cytochrome c derivatives had been produced which were modified at the specific Lys residues including Lys8, Lys25, Lys72, Lys86 or Lys87, respectively. The electron paramagnetic resonance (EPR) spectra for each cytochrome c derivative revealed that for the spin label attached to Lys8 and Lys87 only one component contributes to the spectrum whereas for Lys25, Lys72 and Lys86 the spectrum consists of two components. The highest mobility with the rotational correlation time, tauB, of 0.38 ns was observed for Lys87. The longest tauB of 1.84 ns was obtained for Lys72. An attempt to correlate the spin label mobility with the local protein structure is presented. These mono derivatized cytochrome c molecules provide a unique tool for EPR studying the interaction between cytochrome c and the lipid bilayer, as well as cytochrome c oxidase and reductase.

Animals↗

PubMed1998

Posttranslational modifications of human inter-alpha-inhibitor: identification of glycans and disulfide bridges in heavy chains 1 and 2.

Inter-alpha-inhibitor (IalphaI) is a serine proteinase inhibitor found in high concentrations in human plasma. The protein is composed of a light inhibitory chain called bikunin and two heavy chains of unknown function. The three polypeptide chains are covalently assembled via a carbohydrate cross-link [Enghild, J. J., Salvesen, G., Hefta, S. A., Thogersen, I. B., Rutherfurd, S., & Pizzo, S. V. (1991) J. Biol. Chem. 266, 747-751]. The aim of this study was to complete the primary structure by characterizing additional covalent posttranslational modifications of the heavy chains. Analysis revealed three N-linked oligosaccharides located on Asn251 and Asn554 of heavy chain 1 and on Asn64 of heavy chain 2: all these were complex biantennary structures composed of (Asn)-GlcNAc2-Man-(Man-GlcNAc-Gal-SA)2. In addition, the IalphaI heavy chains carried several O-linked glycans located on Thr619 of heavy chain 1 and a cluster of four O-linked oligosaccharides on Thr612, Ser619, Thr621, and Thr637 of heavy chain 2. The oligosaccharides were short (Ser/Thr)-GalNAc-Gal-SA trisaccharides. The IalphaI heavy chains contain nine Cys residues, of which eight are involved in disulfide bridges. The unpaired Cys residue residing on heavy chain 1, Cys26, appears to be modified by dihexosylation. The other Cys residues exclusively form intrachain disulfide bridges. In heavy chain 1 the two disulfide bonds are formed between Cys210 and Cys213 and between Cys234 and Cys506, and in heavy chain 2, between Cys207 and Cys210 and between Cys596 and Cys597. Interestingly, three of these four disulfides are formed between Cys residues that are either adjacent or only two amino acid residues apart.

Alpha-Globulins↗

PubMed1998

Alpha1-microglobulin is found both in blood and in most tissues.

In this study we demonstrate that, in addition to blood, alpha1-microglobulin (alpha1m) is present in most tissues, including liver, heart, eye, kidney, lung, pancreas, and skeletal muscle. Western blotting of perfused and homogenized rat tissue supernatants revealed alpha1m in its free, monomeric form and in high molecular weight forms, corresponding to the complexes fibronectin-alpha1m and alpha1-inhibitor-3-alpha1m, which have previously been identified in plasma. The liver also contained a series of alpha1m isoforms with apparent molecular masses between 40 and 50 kD. These bands did not react with anti-inter-alpha-inhibitor antibodies, indicating that they do not represent the alpha1m-bikunin precursor protein. Similarly, the heart contained a 45-kD alpha1m band and the kidney a 50-kD alpha1m band. None of these alpha1m isoforms was present in plasma. Immunohistochemical analysis of human tissue demonstrated granular intracellular labeling of alpha1m in hepatocytes and in the proximal epithelial cells of the kidney. In addition, alpha1m immunoreactivity was detected in the interstitial connective tissue of heart and lung and in the adventitia of blood vessels as well as on cell surfaces of cardiocytes. alpha1m mRNA was found in the liver and pancreas by polymerase chain reaction, suggesting that the protein found in other tissues is transported via the bloodstream from the production sites in liver and pancreas. The results of this study indicate that in addition to its role in plasma, alpha1m may have important functions in the interstitium of several tissues. (J Histochem Cytochem 46:887-893, 1998)

Animals↗

PubMed1998

Accumulation of beta ig-h3 gene product in corneas with granular dystrophy.

We isolated and identified the major protein present in corneas with granular dystrophy (GCD). We compared Coomassie-blue-stained protein bands obtained on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) from the extracts of corneas with GCD, corneas with other disorders, and normal human corneal tissue. After SDS-PAGE and transfer to a polyvinylidene difluoride membrane, bands of interest were analyzed by amino acid sequencing and by Western blotting. Corneas with GCD were also examined immunohistochemically. On SDS-PAGE a 63-kd band just below albumin was present in extracts of all corneas. The albumin/63-kd ratio was normally approximately 3:1, suggesting that the protein is a dominant constituent of the cornea. This band was much more plentiful than normal in corneas with GCD. Amino-terminal sequence analysis of the protein revealed a Gly-Pro-Ala-Lys-Ser-Pro-Tyr-Gln-Leu-Val-Leu-Gln-His-Ser-Arg sequence indistinguishable from an amino-terminal protein sequence deduced from a cDNA clone designated beta ig-h3, and it as well as the abnormal accumulations in GCD cross-reacted with beta ig-h3 antiserum. The presence of excessive beta ig-h3 in human corneas with GCD together with reported mutations in the beta ig-h3 gene in GCD suggests that the mutated gene product is a fundamental constituent of the characteristic corneal accumulations in GCD.

Aged↗