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L Chao

Publications and source records attributed to L Chao.

At least 145 records · Page 8Linked to original sources
PubMed1994

Human tissue kallikrein induces hypotension in transgenic mice.

We investigated the role of the kallikrein-kinin system in blood pressure control by developing transgenic mice overexpressing human tissue kallikrein. Two lines of transgenic mice carrying the human tissue kallikrein gene under the control of the mouse metallothionein metal-responsive promoter were established. Human tissue kallikrein was identified in pancreas, salivary gland, kidney, liver, and spleen of the transgenic mice by a specific radioimmunoassay for human tissue kallikrein. The immunoreactive human tissue kallikrein reached high levels in the circulation. The linear displacement curves for the transgenic product were parallel with the human tissue kallikrein standard curve, indicating their immunologic identity. The expression of human tissue kallikrein transcript in the transgenic mice was further confirmed by Northern blot analysis and by reverse transcription-polymerase chain reaction followed by Southern blot. Both lines of transgenic mice had significantly lowered blood pressure (86.4 +/- 13.5 mm Hg [mean +/- SD], n = 8 and 78.9 +/- 12.4 mm Hg, n = 8) compared with control mice (100.9 +/- 5.0 mm Hg, n = 8). Induction with zinc did not lower the blood pressure further despite elevated expression of the transgene. Administration of aprotinin, a potent tissue kallikrein inhibitor, restored the blood pressure of the transgenic mice but had no significant effect on control littermates. Our findings raise the possibility of tissue kallikrein being a powerful modulator of blood pressure and provide a new animal model for the study of blood pressure regulation.

Animals↗
PubMed1994

Molecular cloning and characterization of a novel kallikrein transcript in colon and its distribution in human tissues.

1. We have cloned and characterized a new species of kallikrein cDNA from a human colon cDNA library. The new kallikrein cDNA clone contains a part of intron 2 of the tissue kallikrein gene which is spliced to the remaining exon sequences. It does not contain exons 1 and 2. 2. An in-frame open reading frame is present in the new kallikrein cDNA allowing translation of a 216-amino acid product. The intron-containing kallikrein transcript was detected in salivary glands, pancreas, kidney, colon, prostate gland, testis, spleen, and lung by reverse-transcription/polymerase chain reaction followed by Southern blot analysis using an intron-containing kallikrein-specific oligonucleotide probe. 3. The results indicate that the new species of kallikrein may be processed by alternative splicing or arises from a different transcription initiation site.

Amino Acid Sequence↗
PubMed1994

Histopathology of lymphatic tissues in transgenic mice expressing human tissue kallikrein gene.

BACKGROUND: Tissue kallikrein is a member of a family of closely related serine proteinases whose genetics are currently under intense investigation, but whose functions are still poorly understood. Functions of human tissue kallikrein, other than production of inflammatory kinins, are not readily amenable to investigation. A current mechanism for examining the function of a gene product is to introduce the gene with tissue-specific and/or inducible promoters into a suitable host that can then be experimentally manipulated. EXPERIMENTAL DESIGN: Five transgenic mouse lines were established by backcross matings of transgenic founder mice containing the human tissue kallikrein gene. The inserted genomic material included the full-length kallikrein coding sequence as well as 800 bp in the upstream promoter region and 300 bp in the 3' noncoding region (PHK). Two of the five strains contained, in addition to the complete coding region and 3' flanking sequence, an upstream, zinc-inducible metallothionein promoter (MRE-PHK). Tissues from animals containing human kallikrein gene constructs (as determined by Southern blot of tail DNA) were examined histologically, and compared with control tissues from siblings negative for the gene. RESULTS: Transgenic mice exhibited tissue pathology in several lymphatic organs. Cytoarchitecture was disrupted in both thymus and spleen. The distinction between cortex and medulla in the thymus was usually blurred, and cytolysis was common. Spleens exhibited decreased T cell-dependent zones (periarteriolar sheath), with active hematopoietic foci throughout the red pulp. In lymph nodes, cortical nodules were rare. The deep cortex (paracortical area) was usually normal in heterozygotes, but often depleted in homozygous animals. Comparable results were obtained in all five transgenic strains. CONCLUSIONS: Expression of human tissue kallikrein appears to exert a profound effect on the cytoarchitecture of lymphatic tissues and a general decrease in lymphocytes, particularly in T cell-dependent areas. These findings presumably reflect altered function of lymphatic tissues in transgenic mouse strains carrying the human kallikrein gene.

Animals↗
PubMed1993

Kallistatin: a novel human serine proteinase inhibitor. Molecular cloning, tissue distribution, and expression in Escherichia coli.

We have recently purified a novel human serine proteinase inhibitor (serpin), designated as kallistatin, which binds to tissue kallikrein and inhibits kallikrein's kininogenase and amidolytic activities. In the present studies, we have cloned a full-length cDNA encoding kallistatin from human liver RNA by the polymerase chain reaction. The cDNA is 1284 base pairs in length and encodes 427 amino acid residues, including a 26-residue signal peptide and a 401-residue mature peptide. The translated amino acid sequence of kallistatin matches with the protein sequence and shares 44-46% sequence identity with human alpha 1-antichymotrypsin, protein C inhibitor, corticosteroid-binding globulin, alpha 1-antitrypsin, thyroxin-binding globulin, and rat kallikrein-binding protein. Kallistatin is a new member of the serpin superfamily with a unique reactive site P1-P1' of Phe-Ser. Four potential glycosylation sites are found in the translated amino acid sequence of kallistatin. In a Southern blot analysis following reverse transcription and polymerase chain reaction, kallistatin was found to be expressed in human liver, stomach, pancreas, kidney, aorta, testes, prostate, artery, atrium, ventricle, lung, renal proximal tubular cell, and a colonic carcinoma cell line T84. A genomic Southern blot using the full-length kallistatin cDNA probe revealed simple banding patterns suggesting the gene encoding kallistatin is single-copied. The kallistatin cDNA encoding the mature peptide was expressed in Escherichia coli. The recombinant kallistatin forms an SDS-stable complex with 125I-human tissue kallikrein and has a molecular mass of 40 kDa. The cloning of human kallistatin cDNA established the identity of the novel kallikrein inhibitor and its expression in a functional form in E. coli provides means for studying its structure-function relationship through protein engineering.

Amino Acid Sequence↗
PubMed1993

Molecular cloning and sequence analysis of the monkey and human tissue kallikrein genes.

Cynomolgus monkey renal kallikrein cDNA and genomic human tissue kallikrein gene were cloned. The monkey gene encodes a 257 amino acid (aa) preprokallikrein and exhibits 95% and 92% homology to the human at nucleotide (nt) and aa level, respectively. The monkey gene encodes a 233-aa mature kallikrein versus a 238-aa in human. The human kallikrein gene and urinary kallikrein both contain a Lys-162 instead of the reported Glu-162. Human, monkey and rat renal/pancreatic kallikrein genes evolve with a N-glycosylation containing domain (aa 81-87) which is absent in porcine and is non-glycosylable in mice. Only human kallikrein evolves with an additional Thr-108 and with a N-glycosylation site at aa-141.

Amino Acid Sequence↗
PubMed1993

Expression and characterization of rat kallikrein-binding protein in Escherichia coli.

Rat kallikrein-binding protein is a novel serine-proteinase inhibitor that forms a covalent complex with tissue kallikrein. We have purified rat kallikrein-binding protein and cloned the cDNA and the gene encoding rat kallikrein-binding protein [Chao, Chai, Chen, Xiong, Chao, Woodley-Miller, Wang, Lu and Chao (1990) J. Biol. Chem. 265, 16394-16401; Chai, Ma, Murray, Chao and Chao (1991) J. Biol. Chem. 266, 16029-16036]. In the present study, we have expressed rat kallikrein-binding protein in Escherichia coli with a T7-polymerase/promoter expression system. A high level of expression was detected by an e.l.i.s.a. with an average of 24.2 mg of recombinant rat kallikrein-binding protein per 1 of culture. The recombinant protein appeared as a major protein in a crude extract of Escherichia coli on SDS/PAGE. It showed a molecular mass of 43 kDa and was recognized by polyclonal antibody to the native rat kallikrein-binding protein in Western-blot analysis. The recombinant rat kallikrein-binding protein has been purified to apparent homogeneity by DEAE-Sepharose CL-6B, hydroxyapatite Bio-Gel HPHT and Mono P 5/5 column chromatography. The purified recombinant rat kallikrein-binding protein showed immunological identity with the native rat kallikrein-binding protein purified from rat serum, in a specific e.l.i.s.a. To confirm the fidelity of the expression, the N-terminal ten amino acids of the recombinant rat kallikrein-binding protein were sequenced and were shown to match perfectly with those of the native rat kallikrein-binding protein. The purified recombinant rat kallikrein-binding protein formed SDS- and heat-stable complexes with rat tissue kallikrein (rK1) and T-kininogenase (rK10) in vitro, but not with other enzymes in the rat kallikrein gene family, such as tonin (rK2) and S3 protein (rK9), which indicates enzyme-specific binding. The properties of the recombinant rat kallikrein-binding protein including its size, charge, complex formation with target enzymes and immunological characteristics were compared with those of the native protein. This expression system provides a simple way to obtain a large amount of the biologically active recombinant protein, to study structure-function relationships of the rat kallikrein-binding protein and its interaction with its target enzymes.

Animals↗
PubMed1993

Molecular characterization of glucose-6-phosphate dehydrogenase (G6PD) deficiency in patients of Chinese descent and identification of new base substitutions in the human G6PD gene.

The underlying DNA changes associated with glucose-6-phosphate dehydrogenase (G6PD)-deficient Asians have not been extensively investigated. To fill this gap, we sequenced the G6PD gene of 43 G6PD-deficient Chinese whose G6PD was well characterized biochemically. DNA samples were obtained from peripheral blood of these individuals for sequencing using a direct polymerase chain reaction (PCR) sequencing procedure. From these 43 samples, we have identified five different types of nucleotide substitutions in the G6PD gene: at cDNA 1388 from G to A (Arg to His); at cDNA 1376 from G to T (Arg to Leu); at cDNA 1024 from C to T (Leu to Phe); at cDNA 392 from G to T (Gly to Val); at cDNA 95 from A to G (His to Arg). These five nucleotide substitutions account for over 83% of our 43 G6PD-deficient samples and these substitutions have not been reported in non-Asians. The substitutions found at cDNA 392 and cDNA 1024 are new findings. The substitutions at cDNA 1376 and 1388 account for over 50% of the 43 samples examined indicating a high prevalence of these two alleles among G6PD-deficient Chinese. Our findings add support to the notion that diverse point mutations may account largely for much of the phenotypic heterogeneity of G6PD deficiency.

Asian↗
PubMed1993

The advance of Muller's ratchet in a haploid asexual population: approximate solutions based on diffusion theory.

Asexual populations experiencing random genetic drift can accumulate an increasing number of deleterious mutations, a process called Muller's ratchet. We present here diffusion approximations for the rate at which Muller's ratchet advances in asexual haploid populations. The most important parameter of this process is n0 = N e-U/s, where N is population size, U the genomic mutation rate and s the selection coefficient. In a very large population, n0 is the equilibrium size of the mutation-free class. We examined the case n0 > 1 and developed one approximation for intermediate values of N and s and one for large values of N and s. For intermediate values, the expected time at which the ratchet advances increases linearly with n0. For large values, the time increases in a more or less exponential fashion with n0. In addition to n0, s is also an important determinant of the speed of the ratchet. If N and s are intermediate and n0 is fixed, we find that increasing s accelerates the ratchet. In contrast, for a given n0, but large N and s, increasing s slows the ratchet. Except when s is small, results based on our approximations fit well those from computer simulations.

Computer Simulation↗
PubMed1993

Regulation of kininogen gene expression and localization in the lung after monocrotaline-induced pulmonary hypertension in rats.

Pyrrolizidine monocrotaline (MCT) from plant seed produces pulmonary endothelial cell injury, pulmonary hypertension, and inflammation in rats, providing a useful animal model for studying progressive pulmonary vascular disease. Kininogen is the precursor of proinflammatory kinins and may also exert anti-inflammatory actions by inhibiting cysteine proteinases. Given the potential roles of kininogen in vascular injury and inflammation, we have investigated the regulation of kininogen gene expression in the MCT-induced pulmonary hypertensive rat model. Sprague-Dawley rats, in groups of six, were given a single subcutaneous injection of monocrotatine (60 mg/kg body wt) and sacrificed 10 and 20 days later. Northern blot hybridization using a kininogen cDNA probe showed kininogen gene expression in the liver, lung, and kidney. MCT treatment induced a time-dependent increase in kininogen mRNA levels, whereas it reduced rat alpha 1-antitrypsin and kallikrein-binding protein mRNA levels in the liver. Similarly, kininogen mRNA levels were low in the normal lung and were increased 7.5- and 13.7-fold, respectively, after MCT injection for 10 and 20 days. Immunoreactive kininogen levels in perfused liver and lung extracts of rats receiving MCT injection increased up to 20-fold, as measured by a T-kininogen radioimmunoassay. Western blot analyses showed that a 68-kilodalton immunoreactive kininogen increased in the serum and lung extracts of MCT-treated rats compared to those in the control rats. In control rats, immunostaining for kininogen in the lung was most marked in venous endothelial cells and alveolar macrophages. After MCT treatment, staining for kininogen increased dramatically throughout the lung tissues, often covering the epithelial surfaces of alveoli and bronchi. The present studies have shown that the toxin MCT altered the synthesis and distribution of pulmonary kininogen and suggest that the kininogen/kinin system may be associated with the pulmonary vascular injury, remodeling, and inflammation seen in this animal model.

Animals↗
PubMed1992

Kallistatin: a novel human tissue kallikrein inhibitor. Purification, characterization, and reactive center sequence.

A novel human tissue kallikrein inhibitor designated as kallistatin has been purified from plasma to apparent homogeneity by polyethylene glycol fractionation and successive chromatography on heparin-Agarose, DEAE-Sepharose, hydroxylapatite, and phenyl-Superose columns. A purification factor of 4350 was achieved with a yield of approximately 1.35 mg per liter of plasma. The purified inhibitor migrates as a single band with an apparent molecular mass of 58 kDa when analyzed on SDS-polyacrylamide gel electrophoresis under reducing conditions. It is an acidic protein with pI values ranging from 4.6 to 5.2. No immunological cross-reactivity was found by Western blot analyses between kallistatin and other serpins. Kallistatin inhibits human tissue kallikrein's activity toward kininogen and tripeptide substrates. The second-order reaction rate constant (ka) was determined to be 2.6 x 10(4) M-1 s-1 using Pro-Phe-Arg-MCA. The inhibition is accompanied by formation of an equimolar, heat- and SDS-stable complex between tissue kallikrein and kallistatin, and by generation of a small carboxyl-terminal fragment from the inhibitor due to cleavage at the reactive site by tissue kallikrein. Heparin blocks kallistatin's complex formation with tissue kallikrein and abolishes its inhibitory effect on tissue kallikrein's activity. The amino-terminal residue of kallistatin is blocked. Sequence analysis of the carboxyl-terminal fragment generated from kallistatin reveals the reactive center sequence from P1' to P15', which shares sequence similarity with, but is different from known serpins including protein C inhibitor, alpha 1-antitrypsin, and alpha 1-antichymotrypsin. The results show that kallistatin is a new member of the serpin superfamily that inhibits human tissue kallikrein.

Amino Acid Sequence↗
PubMed1992

Molecular cloning and characterization of rKlk10, a cDNA encoding T-kininogenase from rat submandibular gland and kidney.

We have cloned and determined the nucleotide sequence of a novel kallikrein-like mRNA, designated rKlk10*, from rat submandibular gland and kidney with the aid of the polymerase chain reaction (PCR). This cDNA contains 737 base pairs comprising the sequence encoding a mature protein of 235 amino acid residues, partial zymogen peptide, and 3' noncoding sequence. Sequence comparisons showed that rKlk10 mRNA shares 87 and 88% sequence identity with rat tissue kallikrein at nucleic acid and amino acid levels, respectively. It encodes a 26,428-Da acidic protein whose derived amino acid sequence matches completely with the partial amino acid sequence of a kallikrein-like enzyme designated as T-kininogenase, K10 protein, or antigen-gamma purified from rat submandibular gland [Xiong et al. (1990) J. Biol. Chem. 265, 2822-2827; Gutman et al. (1991) Eur. J. Biochem. 784, 1-5; Berg et al. (1991) Biochem. J. 280, 19-25]. The protein encoded by rKlk10 retains the key amino acid residues determining kallikrein cleavage specificity. Northern blot analysis with an rKlk10-specific oligonucleotide probe showed that its mRNA level in the submandibular gland is decreased dramatically by administration of the beta agonist isoproterenol. Tissue-specific expression of rKlk10 was analyzed by Northern blotting and Southern blotting of PCR-amplified cDNA, which showed that rKlk10 is expressed at high levels in the submandibular gland and low levels in the kidney but not in seven other tissues including prostate, liver, heart, adrenal gland, testes, pituitary, and pancreas. rKlk10 cDNAs cloned from the kidney and submandibular gland show sequence identity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Biochemical characterization and substrate specificity of rat prostate kallikrein (S3): comparison with tissue kallikrein, tonin and T-kininogenase.

A tissue kallikrein-like enzyme encoded by S3 mRNA was purified to homogeneity from rat prostate gland. The apparent molecular mass of the prostate enzyme is 32 kDa as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The intact 32 kDa enzyme is split into two bands of lower molecular mass, 18 and 14 kDa, under reducing conditions on SDS-PAGE. NH2-terminal amino acid sequence analyses of the intact enzyme and heavy and light chains revealed the identity to the translated sequence of a prostate kallikrein cDNA (S3). Isoelectric focusing indicated that the prostate enzyme is a basic protein with pI of 7.30-7.45. Specific activities of the prostate kallikrein toward angiotensin I, angiotensinogen and rat low M(r) kininogen as well as tripeptide chromogenic substrates were compared with those of tissue kallikrein, tonin and T-kininogenase. The kinin-releasing activity is inhibited by leupeptin, antipain, benzamidine and soybean trypsin inhibitor. A sensitive and specific radioimmunoassay for the rat prostate kallikrein shows that the immunoreactive kallikrein levels in prostate and submandibular gland were 23.78 +/- 2.62 micrograms/mg protein (n = 5) and 12.29 +/- 2.25 micrograms/mg protein (n = 5), respectively. The results indicate that the prostate kallikrein S3 is expressed at high levels in both prostate and submandibular glands.

Amino Acid Sequence↗
PubMed1992

Differential regulation of kininogen gene expression by estrogen and progesterone in vivo.

Kininogens which have multifunctional domains, serve as the precursors of potent vasoactive kinin peptides and also function as cysteine proteinase inhibitors. Given its potential role in blood pressure homeostasis and inflammation, we have examined the regulation of rat kininogen gene expression by sex hormones in vivo. Our studies indicate a differential regulation of kininogen gene expression in rat liver by estrogen and progesterone. Northern and dot blot analysis using a rat low molecular weight kininogen cDNA probe show that kininogen mRNA levels in the liver of female rats are 4-fold higher than those in male rats. Ovariectomy results in a reduction of kininogen transcripts in the liver, while estradiol replacement of the ovariectomized rats increases kininogen mRNA levels. Similarly, Northern blot analysis using a kallikrein cDNA probe shows that estradiol treatment induces an increase of kallikrein gene expression in the kidney of the same animals. In contrast, progesterone treatment of the ovariectomized rats results in an increase in renal kallikrein mRNA levels while it reduces kininogen gene expression as compared to vehicle-treated ovariectomized animals. Immunoreactive kininogen levels in the serum, analyzed by a direct radioimmunoassay and Western blot, are increased by estradiol but slightly decreased by progesterone treatment. Western blot of serum proteins on a two-dimensional polyacrylamide gel reveals that in estradiol-treated ovariectomized rats, the levels of several 68,000 Da kininogens varying in charge are markedly higher than those in ovariectomized rats. The results indicate that estrogen is one of the determinants in regulating low molecular weight kininogen gene expression in vivo. The impact of estrogen-regulated kininogen expression on cardiovascular function awaits further investigation.

Animals↗
PubMed1992

Expression of kallikrein-binding protein and alpha 1-antitrypsin genes in response to sex hormones, growth, inflammation and hypertension.

We have recently purified rat kallikrein-binding protein (RKBP) and alpha 1-antitrypsin (alpha 1-AT) to homogeneity and isolated, sequenced cDNAs encoding these potential regulators of tissue kallikreins. Characterization of the cDNA and the gene has established the identity of the kallikrein-binding protein as a new member of the serpin (serine proteinase inhibitor) superfamily. Using the cDNA probes in Northern blot hybridization, we found a differential regulation of RKBP and alpha 1-AT gene expression in the liver. Ovariectomy results in a 67% reduction of RKBP mRNA levels but a 30% increase of alpha 1-AT mRNA levels. Estradiol or progesterone treatment of the ovariectomized rats increases RKBP transcripts by 2.5- and 6.5-fold, respectively, but reduces alpha 1-AT mRNA level by 30% and 45%, respectively. In contrast to kininogen expression, both RKBP and alpha 1-AT mRNA levels in the liver are at the lowest at birth and rapidly increase during growth and development. Rats injected with endotoxin from 4 to 24 h show a time-dependent decrease of RKBP mRNA levels while the same treatment induces alpha 1-AT gene expression. RKBP mRNA levels in the normotensive Wistar Kyoto (WKY) rats are higher than those in the spontaneously hypertensive rats (SHR) while there are no differences of alpha 1-AT mRNA levels between SHR and WKY.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute-Phase Reaction↗
PubMed1992

Evolution of the kallikrein gene family.

All kallikrein-like genes that have been studied to date are composed of 5 exons and the tertiary structures of the encoded enzymes are remarkably similar. In the mouse and rat, these genes are highly conserved, tightly linked and tandemly arranged. In other species, such as the human, the family is less well defined and seems to be much smaller than that of the mouse and rat. Although extensively studied, the exact physiologic significance is not known for many kallikrein gene family members, however, they are thought to play important roles in processing biologically important peptide precursors. Given the potential importance of these mammalian enzymes as a group of highly selective peptide processing enzymes, it would be helpful to know more about the ways in which this family varies from species to species, especially with respect to the size of the family in each species. The evolutionary mechanisms which have shaped this family of genes are largely unknown, however, enough data has been generated to begin understanding the pathway by which this gene family has evolved.

Animals↗
PubMed1992

Localization of kallikrein gene family proteases in rat tissues.

Monoclonal antibodies specific for three kallikrein gene family enzymes (tissue kallikrein, esterase A and tonin) have been used to determine the tissue and cellular distributions of these proteases as well as their association with other relevant molecules (kininogen, kallikrein-binding protein, and Na,K-ATPase alpha-subunit). Secretion of these enzymes from salivary glands was also analyzed. The results of these localization studies provide important clues to the functions of different members of this closely related family of serine proteases.

Animals↗