Regulation of rat renal kallikrein expression by estrogen and progesterone.
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Animals↗
Biomedical subjects
J Chao
Publications and source records attributed to J Chao.
Storage of poliovirus vaccine.
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Drug Storage↗
Tissue kallikrein-binding protein is a serpin. I. Purification, characterization, and distribution in normotensive and spontaneously hypertensive rats.
Kallikrein-binding protein was purified to apparent homogeneity from rat serum by Affi-Gel Blue, DEAE-Sepharose CL-6B, Sephacryl S-200 chromatography, and preparative gel electrophoresis or high performance liquid chromatography. The purified protein migrates as a single band of 60 kDa in a sodium dodecyl sulfate-polyacrylamide gel under reducing conditions. It is an acidic protein with isoelectric points ranging from 4.2 to 4.6. The amino terminus of the binding protein is an Asp residue as determined by sequence analysis. It forms a 92-kDa sodium dodecyl sulfatestable complex with kallikrein with a t1/2 of 18 min. Western blot and radioimmunoassay showed a distribution of the kallikrein-binding protein in serum, urine, and various tissues with a 5-10-fold lower amount in spontaneously hypertensive rats (SHR) than in Wistar-Kyoto rats (WKY). A full length cDNA clone encoding the kallikrein-binding protein was isolated from a rat liver cDNA library by immunoscreening and the translated amino acid sequence matches the amino-terminal 29-amino acid sequence of the binding protein. The cDNA sequence shares 68.8% identity with human alpha 1-antichymotrypsin and is identical to that of a rat hepatic protein. Dot blot analysis shows that kallikrein-binding protein is expressed at high levels in the liver and at low levels in the lung, salivary gland, and kidney. Its mRNA level in the liver decreases by 2-fold after acute phase inflammation and is higher in male than in female rats. Genomic Southern blot analyses reveal restriction fragment length polymorphisms between SHR and WKY rats in the binding protein locus. The results indicate that rat kallikrein-binding protein belongs to the serpin superfamily and its level is significantly reduced in the spontaneously hypertensive rats.
Amino Acid Sequence↗
Urokinase- and tissue-type plasminogen activators are suppressed by cortisol in the involuting prostate of castrated rats.
The effects of cortisol on the inhibition of cell-death processes and suppression of plasminogen-activator (PA) activity during involution of the rat ventral prostate gland were investigated to determine the principal type of PA activated by castration and inhibited by this hormone and whether the mechanism responsible for decreased PA activity involved reductions in enzyme synthesis or increased activity of a PA inhibitor. By using the technique of fibrin-agarose zymography, three bands of PA activity were detected at 4 and 7 days after castration: a major band with a molecular mass of approx. 30 kDa and two minor bands of 48 kDa and 64 kDa. Both the 30 kDa and 48 kDa activities were inhibited with anti-[urokinase-type PA (u-PA)] IgG. The 64 kDa activity was inhibited by anti-[tissue-type PA (t-PA)] IgG. In addition to retarding prostatic involution, daily administration of cortisol to the castrated animals suppressed all three bands of PA activity. A comparison of the pattern of total PA activity and of e.l.i.s.a. estimates of u-PA concentration during the castration-induced rise and after cortisol inhibition indicated a near perfect correlation between the two parameters. Northern-blot analysis using prostatic polyadenylated RNA revealed that the level of u-PA mRNA was highest at 4 and 7 days after castration and that cortisol treatment repressed u-PA mRNA to a level similar to that in non-castrated controls. Neither Northern hybridizations nor reverse zymography detected RNA transcripts or activity corresponding to the PA inhibitor PAI-1 in any of the prostate samples. Western-blot analysis revealed that, although the amount of arginine esterase A, another prostatic proteinase, also increased after castration, the rise in concentration of this protein was not blocked by glucocorticoid administration. Together our findings indicate the following: (1) the predominant form of PA activity induced in the prostate after castration and inhibited by cortisol is a 30 kDa form of u-PA. Although less prominent, t-PA and a 48 kDa form of u-PA follow a similar pattern of induction and inhibition; (2) changes in u-PA activity in response to castration and cortisol treatment are due to alterations in the level of u-PA mRNA and protein rather than in the activity of PAI-1; (3) not all castration-induced proteinases in the prostate are inhibited by cortisol.
Animals↗
Identification, purification, and localization of tissue kallikrein in rat heart.
A tissue kallikrein has been isolated from rat heart extracts by DEAE-Sepharose and aprotinin-affinity column chromatography. The purified cardiac enzyme has both N-tosyl-L-arginine methyl ester esterolytic and kinin-releasing activities, and displays parallelism with standard curves in a kallikrein radioimmunoassay, indicating it to have immunological identity with tissue kallikrein. The enzyme is inhibited by aprotinin, antipain, leupeptin and by high concentrations of soybean trypsin inhibitor, but stimulated by lima-bean or ovomucoid trypsin inhibitor and low concentrations of soybean trypsin inhibitor. By using a specific monoclonal antibody to tissue kallikrein in Western blot as well as active-site labelling with [14C]di-isopropyl fluorophosphate, the cardiac enzyme was identified as a protein of 38 kDa, a molecular mass identical with that of tissue kallikrein. Immunocytochemistry at the electron-microscopic level localized this enzyme to the sarcoplasmic reticulum and granules of rat atrial myocytes. Two cardiac kallikrein precursors, (38 and 40 kDa) were identified from the translation in vitro of heart mRNA by immunoprecipitation and electrophoresis of [35S]methionine-labelled cell-free translation products. Kallikrein mRNA in the rat heart was also demonstrated by dot-blot analysis using a tissue kallikrein cDNA probe. These results indicate that the tissue kallikrein gene is expressed in the rat heart and that the purified enzyme is indistinguishable from tissue kallikrein with respect to enzymic and immunological characteristics.
Animals↗
Differential interactions of human kallikrein-binding protein and alpha 1-antitrypsin with human tissue kallikrein.
The characteristics of a new kallikrein-binding protein in human serum and its activities were studied. Both the kallikrein-binding protein and alpha 1-antitrypsin form 92 kDa SDS-stable and heat-stable complexes with human tissue kallikrein. In non-SDS/PAGE, the mobility of these complexes differ. Complex-formation between kallikrein and the binding protein is inhibited by heparin, whereas that between kallikrein and alpha 1-antitrypsin is heparin-resistant. In normal or alpha 1-antitrypsin-deficient-serum, the amount of 92 kDa SDS-stable complex formed upon addition of kallikrein is not related to serum alpha 1-antitrypsin levels. The rate of complex-formation between kallikrein and the binding protein is 12 times higher than that between kallikrein and alpha 1-antitrypsin. Purified alpha 1-antitrypsin, which exhibits normal elastase binding, has a kallikrein-binding activity less than 5% of that of serum. Binding of tissue kallikrein in serum is not inhibited by increasing elastase concentrations, and elastase binding in serum is not inhibited by excess tissue kallikrein. A specific monoclonal antibody to human alpha 1-antitrypsin does not bind to either 92 kDa endogenous or exogenous kallikrein complexes isolated from human serum. The studies demonstrate a new tissue kallikrein-binding protein, distinct from alpha 1-antitrypsin, is present in human serum.
Binding, Competitive↗
Purification and characterization of a kallikrein-like T-kininogenase.
A T-kininogenase has been purified to homogeneity from rat submandibular gland extracts by DEAE-Sepharose chromatography and preparative gel electrophoresis. The purified protein has an apparent Mr of 28,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and splits into heavy and light chains with Mr of 22,000 and 6,000 in the presence of dithiothreitol. It is an acidic glycoprotein with pI of 4.65-4.75. The carbohydrate moiety is located on the light chain and binds concanavalin A and wheat germ agglutinin. The active site serine residue of the heavy chain is labeled with [14C]diisopropylfluorophosphate and visualized by fluorography. NH2-terminal amino acid sequences of the light and heavy chains reveal 74-84% identity to rat tissue kallikrein, tonin, and other kallikrein-related enzymes. The enzyme cleaves T-kininogen to release T-kinin which was separated by high performance liquid chromatography on a reverse phase C18 column and identified by a kinin radioimmunoassay. Its T-kininogenase but not N-tosyl-L-arginine methyl ester esterase activity can be enhanced 10-fold in the presence of dithiothreitol. The esterolytic activity of the enzyme is inhibited by soybean trypsin inhibitor, aprotinin, leupeptin, and antipain; whereas lima bean and ovomucoid trypsin inhibitors stimulate its activity. The enzyme is localized at the granular convoluted tubule and striated duct cells in rat submandibular glands by immunohistochemistry. The results indicate that T-kininogenase belongs to the group of structurally similar yet distinct kallikrein-like serine proteases.
Amino Acid Sequence↗
Molecular cloning and primary structure of rat alpha 1-antitrypsin.
A cDNA clone encoding rat alpha 1-antitrypsin has been isolated from a lambda gt-11 rat liver cDNA library using an antigen-overlay immunoscreening method. The nucleotide sequence of this cDNA clone is 1306 base pairs in length and has a coding region of 1224 base pairs which can be translated into an alpha 1-antitrypsin precursor protein consisting of 408 amino acid residues. The cDNA sequence contains a termination codon, TAA, at position 1162 and a polyadenylation signal sequence, AATAAT, at position 1212. The calculated molecular weight of the translated mature protein is 43,700 with 387 amino acid residues; this differs from purified rat alpha 1-antitrypsin's apparent molecular weight of 54,000 because of glycosylation. Five potential glycosylation sites were identified on the basis of the cDNA sequence. The translated mature protein sequence from the cDNA clone matches completely with the N-terminal 33 amino acids of purified rat alpha 1-antitrypsin, which has an N-terminal Glu. The cDNA encoding rat alpha 1-antitrypsin shares 70% and 80% sequence identity with its human and mouse counterparts, respectively. The reactive center sequence of rat alpha 1-antitrypsin is highly conserved with respect to human alpha 1-antitrypsin, both having Met-Ser at the P1 and P1' residues. Genomic Southern blot analysis yielded a simple banding pattern, suggesting that the rat alpha 1-antitrypsin gene is single-copy. Northern blot analysis using the cDNA probe showed that rat alpha 1-antitrypsin is expressed at high levels in the liver and at low levels in the submandibular gland and the lung.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Prevalence of lifestyle risk factors in a family practice.
The prevalence of eight adverse lifestyle risk factors which are linked to increased morbidity and mortality was determined. Smoking, excess alcohol use, obesity, lack of exercise, lack of seatbelt use, use of hazardous transportation vehicles, and inadequate rest were studied. A random sample of 147 patients from a private family practice were selected in a cross-sectional survey. Data were collected by mailed questionnaire. The most prevalent adverse habits were lack of seatbelt use (71%) and sedentary lifestyle (44%). Patients with multiple adverse health habits had poorer self-reported physical as well as emotional well-being. Using a factor analysis of the correlations among eight habits, no significant clustering of habits emerged as indicators of high risk. Thus, no abbreviated list of adverse lifestyle risk factors for clinical use was found that could replace a complete review of risk factors.
Adult↗
Kallikrein multigene families and the regulation of their expression.
Tissue kallikreins are a group of closely related serine proteinases that are represented by multigene families in mice and rats. The existence of similar, large, kallikrein-like gene families in other mammalian species is currently a matter of dispute. We have surveyed a number of vertebrate species using genomic DNA Southern blotting and screened a human genomic library with a monkey kallikrein cDNA probe. The hybridization patterns of the genomic Southern blots and the characterization of 19 independent human clones using restriction analysis and Southern blotting indicate that other mammalian species may have multiple kallikrein-like genes as well. The regulatory mechanisms that govern the expression, activity, and bioavailability of tissue kallikreins are likewise complex. At the level of transcription, hormones, dietary factors, and tissue-specific factors are known to affect the expression of tissue kallikrein genes. At the posttranslational level, kallikrein activity and bioavailability are regulated by enzymatic activation, circulating autoantibodies, and binding proteins. We have demonstrated the presence of kallikrein-binding proteins in humans and rats, and, furthermore, we have shown reduced levels of this binding protein in a hypertensive rat model.
Animals↗
Sex dimorphism and inflammatory regulation of T-kininogen and T-kininogenase.
Studies were carried out in order to better understand hormonal and inflammatory regulation of the T-kininogen and T-kininogenase system. T-kininogen from rat serum and T-kininogenase from rat submandibular gland were purified to homogeneity, and specific antisera to the purified proteins were generated. Simple, sensitive and specific radioimmunoassays were developed for measuring both T-kininogen and T-kininogenase. The assays incorporated a modified poly(ethylene glycol) technique for separating free from antibody-bound forms. Optimal combinations of poly(ethylene glycol) and gamma-globulin were found, yielding low background and high specific binding. The assays can detect a minimum of 160 pg of T-kininogen and 80 pg of T-kininogenase per tube. Serial dilutions of sera from normal and turpentine-treated rats showed complete parallelism with the T-kininogen standard curve. T-kininogen levels in rat serum and rat tissues increased more than 10-fold following turpentine treatment, while T-kininogenase levels in the submandibular gland and other tissues remained unchanged. Through use of a kinin-directed kininogen monoclonal antibody, Western blots of two-dimensional gels of serum following acute inflammation showed increased levels of several kininogens which vary in both molecular weight and isoelectric point. Analysis of serum kininogen levels shows sexual dimorphism, with female rats having 3.9-fold higher levels than males. Contrarily, T-kininogenase levels in the submandibular gland of male rats are 2.4-fold higher than those in females. The studies also showed that the T-kininogen and T-kininogenase system is regulated by sex hormones. T-kininogen is an acute-phase protein whose rapid increase and mobilization following inflammation may provide a primary defense against proteolytic damage during trauma.
Animals↗
Characterization of genes encoding rat tonin and a kallikrein-like serine protease.
Tissue kallikreins are a group of serine proteases which may function as peptide hormone processing enzymes. Two rat kallikrein genomic clones (RSKG-5 and RSKG-50) were sequenced and characterized. The rat tonin gene and a kallikrein-like gene were found in clones RSKG-5 and RSKG-50, respectively. The tonin gene is 4146 base pairs in length, with both the variant CCAAA and TTTAAA boxes in the 5'-end region and an AATAAA polyadenylation signal at the 3' end of the gene. It has five exons which are separated by four introns. Sequence analysis of 3.7-kb 5' upstream and 7.5-kb 3' downstream of the tonin gene failed to reveal a second kallikrein gene. Sequence comparisons of the RSKG-5 exons with tonin cDNA revealed that only one base in the 3'-noncoding region was different from that in the previously reported rat tonin cDNA. Characteristic TC- and TG-repeated sequences were also found in the first and second introns of the tonin gene. The tonin gene encodes a preprotonin of 259 amino acids (aa). The active enzyme consists of 235 aa and is preceded by a deduced signal peptide of 17 aa and a profragment of 7 aa. Northern blot analysis indicates that RSKG-5 is expressed in a sex-dependent manner in rat submandibular gland, with a higher level expressed in males. The RSKG-50 gene was truncated at an EcoRI site in the second intron, excluding its 5' end. Compared to the coding sequence of pancreatic kallikrein, 12 nucleotides have been deleted in exon 3 of the RSKG-50 gene. The nucleotide sequences of the third, fourth, and fifth exons of the RSKG-50 gene encode a polypeptide of 188 aa residues. The translated peptide is 80% homologous to rat pancreatic kallikrein and 75% homologous to rat tonin in the corresponding regions. Key residues in the RSKG-50 gene product indicate a serine protease with kallikrein-like cleavage specificity at basic amino acids.
Amino Acid Sequence↗
Structural analysis of a rat renal kallikrein gene.
A renal kallikrein gene has been isolated, sequenced and characterized from a rat genomic library using a kallikrein cDNA probe. The kallikrein gene is 4160 bases in length and consists of 5 exons and 4 introns. The deduced sequence of the gene encodes an 18 amino acid (a.a.) signal peptide, a 6 a.a. propeptide and a 237 a.a. active enzyme with an N-terminal valine. A sequence comparison of this and tissue kallikrein (pancreatic kallikrein) indicates the key a.a. residues for serine protease activity (HIS-ASP-SER) and cleavage specificity at basic a.a. Northern blot analysis using a specific oligonucleotide probe reveals that this gene is expressed specifically in the kidney but not in the pancreas. This gene is also expressed non-specifically in the submandibular gland which expresses all kallikrein family genes. The expression of this kidney-specific kallikrein gene is regulated by steroid hormones.
Adrenalectomy↗
Human kallistatin, a new tissue kallikrein-binding protein: purification and characterization.
A new and specific tissue kallikrein-binding protein was identified in mammalian serum and in secreted transformed-cell culture media (Chao et al., Biochem. J. 239: 325-331, 1986). We have designated this kallikrein-binding protein as "kallistatin". Human kallistatin has been purified from serum, using chromatographic steps including DEAE-Sephadex, hydroxylapatite, Cibacron blue-Sepharose, Sephacryl S200, and preparative polyacrylamide gel electrophoresis. The purified kallistatin consists of a single polypeptide chain with an apparent molecular weight of approximately 54 kDa and isoelectric point of approximately 5.0. Kallistatin was eluted as a single peak on reverse-phase HPLC. The purified kallistatin and 125I-labelled human tissue kallikrein form a approximately a 92 kDa SDS- and heat-stable complex. The complex formation is pH dependent and is inhibited by 0.1% (W/V) of deoxycholate or SDS but not by 0.5% (W/V) of Triton X-100, digitonin, Lubrol or CHAPS. A approximately 54 kDa protein was identified in partially purified kallistatin by polyclonal anti-kallistatin antibodies in Western blot analysis and by its binding to 125I-labelled-human tissue kallikrein in ligand blotting. The role of kallistatin in regulating tissue kallikrein activity and metabolism may now be evaluated.
Blotting, Western↗
Sex dimorphism and estrogen regulation of kininogens in rat serum, adrenal gland and kidney.
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Adrenal Glands↗
Renal kallikrein mRNA localization by in situ hybridization.
Tissue kallikrein gene expression in rat kidney was examined by in situ hybridization histochemistry. A rat tissue kallikrein cDNA probe, 534 bases in length and complementary to the 3' end of kallikrein mRNA was first used in Northern blot analysis to demonstrate the existence of tissue kallikrein mRNA in rat kidney. Then, kallikrein mRNA's localization in rat kidney sections was studied in situ hybridization histochemistry using the same probe. Positive signals were concentrated in the renal cortex at the vascular pole of the glomeruli and to a lesser degree, the distal tubular cells. Prehybridization with the unlabeled probe can abolish the positive signal; the same result can also be achieved by pretreatment of the tissue section with ribonuclease. By using the same technique, tissue kallikrein mRNA was also localized in granular convoluted tubule and striated duct cells of rat submandibular gland. The results suggest a new site of renal kallikrein synthesis at the vascular pole of the glomerulus. These findings, coupled with the previous studies that tissue kallikrein can participate in activation and releasing of renin, raise a potential physiological role of kallikrein in renin release or prorenin processing at juxtaglomerular cells.
Animals↗
Restriction fragment length polymorphisms mapped in spontaneously hypertensive rats using kallikrein probes.
We have explored the role of kallikrein-kinin system in essential hypertension using spontaneously hypertensive rats (SHR) as an animal model. A rat tissue kallikrein complementary (c) DNA (RSK 1105) was used as a probe in Southern blot hybridization to detect restriction fragment length polymorphisms (RFLPs) in SHR. Using 23 different restriction endonucleases, we have identified five RFLPs involving alterations in restriction fragment lengths for the restriction enzymes Bgl II, Dra I, Nde I, Sph I, and Bcl I. Three of the enzymes, Nde I, Sph I, and Bgl II, generate multiple polymorphic fragments. We have further mapped these RFLPs with two additional probes, both from the rat renal kallikrein gene RSKG 7. The 5' probe, consisting of sequences approximately 2000 base pair (bp) 5' of the first exon, recognizes RFLPs in DNA digested with Bcl I and Sph I. The 3' probe, approximately 4400 bp away from the fifth exon, recognizes polymorphic fragments in DNA digested with Bcl I, Dra I and Nde I. These findings indicate possible differences in tissue kallikrein genes or their regulatory regions in SHR that could contribute to the pathogenesis of hypertension in this animal model.
Animals↗
Human urinary kallikrein. Complete amino acid sequence and sites of glycosylation.
Human glandular kallikrein was purified from urine and subjected to detailed structural characterization. The protein was carboxymethylated with iodoacetic acid and digested with TPCK-trypsin, Staphylococcal aureus V-8 protease and endo LysC peptidase. The resulting peptide fragments were separated by reverse-phase HPLC using C-4 columns and acetonitrile-trifluoroacetic acid gradient elution. The complete amino acid sequence of the carboxymethylated derivative was elucidated by sequence analysis and alignment of peptides derived from different proteolytic cleavages. A procedure using in situ CNBr cleavage of a large endo LysC peptidase-derived peptide followed by direct sequencing was carried out to provide overlap for two glycosylation sites at residues 78 and 84. Three Asn-linked glycosylation sites were confirmed by the direct sequence analysis of the isolated glycopeptides. However, the third glycosylation at Asn-144 occurs only in 60% of kallikrein molecules. Reverse-phase HPLC effectively separates two species of HUK which correspond to molecules glycosylated and non-glycosylated at Asn-144, respectively. The human urinary kallikrein contains 238 amino acid residues with Ile and Ser as N- and C-terminal amino acids, respectively. The primary structure is completely identical to that deduced from a human genomic DNA sequence (F.K. Lin et al., manuscript in preparation) and is different in one amino acid (Lys-162 vs. Glu-162) from that deduced from pancreatic or kidney cDNA sequence.
Amino Acid Sequence↗