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Biomedical subjects

L Chao

Publications and source records attributed to L Chao.

At least 127 records · Page 7Linked to original sources
PubMed1995

Regulatory elements in the promoter region of the renal kallikrein gene in normotensive vs hypertensive rats.

The renal kallikrein-kinin system has been implicated in the pathogenesis of hypertension. The expression level of the renal kallikrein gene in the kidney is significantly lower in spontaneously hypertensive rats (SHR) as compared with that of normotensive (SD and WKY) rats. Deletion analysis showed that the fragment -356/-188 of the promoter contains a transcriptional silencer(s) and the GC rich region located between -77 and -187 is the minimal essential element for directing the expression of the CAT reporter gene in mouse L cells. In the kidney of normotensive vs hypertensive rats, the nuclear protein factors NF1/CTF and SP1 bind differently to the renal kallikrein promoter, but similarly in the salivary gland. The differential transcriptional regulation of the rat renal kallikrein gene in the kidney may be responsible for the genetic difference between normotensive and hypertensive rats.

Animals↗
PubMed1995

A novel insertional mutation in the TC21 gene activates its transforming activity in a human leiomyosarcoma cell line.

TC21 is the fourth member of the ras gene family to exhibit oncogenic activation in human tumor cells. To assess the prevalence of activated TC21 oncogenes in human tumors, we have developed sensitive single-strand conformational polymorphism (SSCP) conditions and immunological reagents for the detection of both single base alterations and/or overt overexpression in a wide spectrum of human tumor cell lines and surgical samples. In an initial examination of 33 human tumor specimens, we observed a novel nine basepair three amino acids insertion at TC21 codon 24 in one human uterine leiomyosarcoma cell line, SK-UT-1. This mutant allele when transfected into NIH3T3 cells, displayed high transforming activity comparable to that of the Leu72 oncogenic mutant identified by expression cDNA cloning from a human ovarian carcinoma cell line. Comparing the level of GTP-binding by the mutant and normal TC21 products revealed that this novel lesion increases the GTP-bound form of the TC21 molecule. These findings imply that the mechanism by which mutations activate the oncogenic properties of this ras-related molecule is analogous to that of previously known ras family members.

3T3 Cells↗
PubMed1995

Molecular cloning, tissue-specific expression, and cellular localization of human prostasin mRNA.

We have purified a novel human serine proteinase, designated as prostasin, from seminal fluid (Yu et al., 1994). In the present study, we have cloned and characterized the full-length cDNA encoding prostasin and identified its tissue-specific expression and cellular localization. A cDNA fragment was obtained by polymerase chain reaction using degenerate oligonucleotide primers derived from the NH2-terminal and internal amino acid sequences. A full-length cDNA sequence encoding prostasin was obtained by amplification of the 5'- and 3'-ends of the cDNA. It contains a 1,032-base coding region, a 572-base 3'-noncoding region and a 138-base 5'-noncoding sequence. Prostasin cDNA encodes a protein of 343 amino acids, which consists of a 32-amino acid signal peptide and a 311-amino acid proprostasin. Proprostasin is then cleaved between Arg12 and Ile13 to generate a 12-amino acid light chain and a 299-amino acid heavy chain, which are associated through a disulfide bond. The deduced amino acid sequence of the heavy chain has 34-42% identity to human acrosin, plasma kallikrein, and hepsin. A potential N-glycosylation site at Asn127 and the catalytic triad of His53, Asp102, and Ser206 have been identified. The deduced prostasin has a unique 19-amino acid hydrophobic portion at the COOH terminus, which makes it suitable to anchor in the cell membrane. Carboxyl-terminal sequencing of purified prostasin indicates that the hydrophobic portion is removed and that there is a cleavage between Arg290 and Pro291 during secretion. Southern blot analysis, following a reverse transcription polymerase chain reaction, indicates that prostasin mRNA is expressed in prostate, liver, salivary gland, kidney, lung, pancreas, colon, bronchus, renal proximal tubular cells, and prostate carcinoma LNCaP cells. Cellular localization of prostasin mRNA was identified within epithelial cells of the human prostate gland by in situ hybridization histochemistry.

Amino Acid Sequence↗
PubMed1995

Molecular cloning, purification and in situ localization of human colon kallikrein.

We have cloned and characterized a full-length cDNA encoding tissue kallikrein from a human colon carcinoma cell line (T84). The nucleic acid sequence of the colon kallikrein cDNA is identical to that of renal/pancreatic or tissue kallikrein cDNA. Reverse-transcription PCR followed by Southern-blot analysis using specific oligonucleotide probes showed expression of tissue kallikrein in human colon, pancreas and kidney. Tissue kallikrein mRNA was localized in glandular epithelial cells (goblet cells) in colon by in situ hybridization histochemistry. Human colon kallikrein was purified to apparent homogeneity by DEAE-Sepharose Cl-6B, aprotinin-affinity, and HQ/M perfusion chromatography. The purified colon kallikrein migrated as a broad, 40-45 kDa band in SDS/PAGE and was recognized by antibodies to human tissue kallikrein. The linear displacement curves for the colon kallikrein in an RIA were parallel with the human tissue kallikrein standard curve, indicating their immunological identity. The N-terminal sequence of the purified colon kallikrein matches completely with that of purified urinary or tissue kallikrein. These results indicate that human colon kallikrein is transcribed from the tissue kallikrein gene.

Amino Acid Sequence↗
PubMed1995

Intramuscular delivery of rat kallikrein-binding protein gene reverses hypotension in transgenic mice expressing human tissue kallikrein.

The tissue kallikrein-kinin system has been postulated to play a role in blood pressure regulation. The activity of tissue kallikrein is controlled by a number of factors in vivo. Rat kallikrein-binding protein (RKBP) is a serine proteinase inhibitor which binds to and inhibits tissue kallikrein's activity in vitro. We have recently developed several hypotensive transgenic mouse lines which express human tissue kallikrein. In order to investigate the role of RKBP in blood pressure regulation, we delivered the RKBP to these transgenic mice by intramuscular injection. Expression of the RKBP was detected in skeletal muscle by reverse transcription-polymerase chain reaction and Southern blot analysis at 10, 20, 30, and 40 days post-injection. Immunoreactive RKBP levels in the muscle and serum of these mice were quantified by a RKBP-specific enzyme-linked immunosorbent assay and Western blot analysis. The levels of RKBP mRNA and immunoreactive protein were detectable at 10 days post-injection and increased significantly at 20 and 30 days. During this period, RKBP delivery significantly increased systemic blood pressure in the kallikrein transgenic mice to a level comparable to that of normotensive control mice. The RKBP and vector DNA delivery had no effect on the blood pressure of normotensive control mice. No serum antibodies to RKBP or its DNA were detected in the mice 40 days post injection. These results suggest that the increase of systemic blood pressure by RKBP delivery in these hypotensive transgenic mice may be mediated by inhibiting tissue kallikrein activity.

Animals↗
PubMed1995

Purification and characterization of tissue kallikrein-like proteinases from the black sea bass (Centropristis striata) and the southern frog (Rana berlandieri).

Serine proteinases were isolated from the pyloric caeca of the black sea bass (Centropristis striata) and the pancreas of the Southern frog (Rana berlandieri) and were purified to apparent homogeneity by aprotinin affinity column chromatography, reverse phase high performance liquid chromatography and gel filtration FPLC liquid chromatography to produce products with molecular masses of approximately 27,000 Da and isoelectric points from 4.2 to 5.0. Both enzymes were kallikrein-like and were bound by diisopropylfluorophosphate; had pH optima from 9 to 10; showed high specificity for the hydrolysis of arginine peptide bonds and low to moderate affinity for lysine bonds at the P1 substrate recognition sites; were inhibited by aprotinin, benzamidine, leupeptin, and soybean trypsin inhibitor; generated kinin from kininogen and were highly stable at room temperature. Differences between the enzymes were observed relative to their hydrophobicities, substrate specificities, stabilities at acidic pHs in the presence and absence of calcium, and the amounts of kinin generated from kininogen. Many of the fish trypsins, previously identified as anionic trypsins, may actually be more kallikrein-like.

Amino Acid Sequence↗
PubMed1995

Cellular localization of tissue kallikrein and kallistatin mRNAs in human kidney.

The renal kallikrein-kinin system has been implicated in the regulation of blood pressure and sodium/water excretion. The activity of renal kallikrein is controlled by a number of factors in vivo. Kallistatin is a newly identified serine proteinase inhibitor (serpin) which binds to tissue kallikrein and inhibits its enzymatic activity in vitro. To understand the role of kallistatin in modulating tissue kallikrein's function in vivo, we examined the anatomical relationship between human tissue kallikrein and kallistatin in the kidney by in situ hybridization histochemistry. Tissue kallikrein and kallistatin gene transcripts were identified using digoxigenin-labeled riboprobes at the cellular level. Antisense and sense riboprobes corresponding to the 3' region of the human kallikrein and kallistatin mRNAs were synthesized by in vitro transcription and used for hybridization. Using an antisense kallikrein riboprobe, sites of kallikrein synthesis were localized in the distal tubules, collecting ducts and Henle's loops of the kidney. To a lesser degree, juxtaglomerular cells were also stained. Kallistatin mRNA was found at the same sites where kallikrein mRNA was localized. The most intense signals of both kallikrein and kallistatin were seen in the distal tubules and collecting ducts. Hybridization was specific for the target mRNA since sense kallikrein or kallistatin riboprobe did not bind to the sections. Immunoreactive human renal kallikrein and kallistatin levels were measured in the kidney and urine by immunoassays using specific antibodies. Co-localization of kallikrein and kallistatin mRNA in the kidney suggests a potential role of kallistatin in regulating tissue kallikrein's function.

Carrier Proteins↗
PubMed1995

Muscle delivery of human kallikrein gene reduces blood pressure in hypertensive rats.

We recently found that transgenic mice expressing human tissue kallikrein develop sustained hypotension. The result suggests that a continuous supply of human tissue kallikrein could have a prolonged effect on blood pressure reduction. In the present study, we investigated the potential of using human tissue kallikrein for gene therapy by injecting a kallikrein gene construct into the skeletal muscle of spontaneously hypertensive rats. Expression of the human tissue kallikrein messenger RNA in spontaneously hypertensive rats was identified by reverse transcription-polymerase chain reaction with Southern blot. Human tissue kallikrein was detected in the injected animals by an enzyme-linked immunosorbent assay. Injection of the human kallikrein gene into spontaneously hypertensive rats caused a significant reduction of systemic blood pressure, ranging from 15 to 26 mm Hg, compared with the control group. The differences were significant 1 week after the injection and continued for more than 2 months. Blood pressure reduction could be reversed after the administration of the bradykinin antagonist Hoe 140. The results indicate that somatic delivery of the human tissue kallikrein gene induces a sustained reduction of systemic blood pressure in spontaneously hypertensive rats. The present study raises the possibility of applying kallikrein gene therapy to the treatment of human hypertensive diseases.

Animals↗
PubMed1995

Human atrial natriuretic peptide gene delivery reduces blood pressure in hypertensive rats.

Chronic infusion of atrial natriuretic peptide (ANP) has been shown to cause natriuresis, diuresis, and hypotension in rats and humans. We explored the effect of a continuous supply of ANP by somatic ANP delivery on genetically hypertensive rats. A DNA construct containing the human ANP gene fused to the Rous sarcoma virus 3'-long terminal repeat (RSV-LTR) was injected intravenously into spontaneously hypertensive rats (SHR) through the tail vein. Expression of human ANP in SHR was identified in the heart, lung, and kidney by radioimmunoassay and reverse transcription-polymerase chain reaction followed by Southern blot analysis. A single injection of naked ANP plasmid DNA (12.3 kb) caused a significant reduction of systemic blood pressure in young SHR (4 weeks old), and the effect continued for 7 weeks. The differences were significant at 1 to 2 weeks (n = 6, P < .05) and 3 to 6 weeks after injection (n = 6, P < .01) A maximal blood pressure reduction of 21 mm Hg in young SHR was observed 5 weeks after injection with ANP DNA (159.4 +/- 3.02 mm Hg, mean +/- SEM, n = 6) compared with SHR injected with vector DNA alone (180.2 +/- 3.02 mm Hg, mean +/- SEM; n = 6; P < .01). Somatic gene delivery of human ANP DNA had no effect on the blood pressure of adult SHR (12 weeks old). After ANP gene delivery, there were significant increases in urinary volume and urinary potassium output (n = 6, P < .05) but not in body weight, heart rate, water intake, urinary sodium output, urinary creatine, and urinary protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗
PubMed1995

Direct gene delivery of human tissue kallikrein reduces blood pressure in spontaneously hypertensive rats.

Hypertension is a multigene and multifactorial disorder affecting approximately 25% of the population. To demonstrate potential therapeutic effects of human tissue kallikrein in hypertension, spontaneously hypertensive rats were subjected to somatic gene therapy. Two human tissue kallikrein DNA constructs, one under the promoter control of the metallothionein metal response element and the other under the control of the Rous sarcoma virus 3'-LTR, were generated. We delivered naked DNA constructs into spontaneously hypertensive rats via intravenous injection. The expression of human tissue kallikrein in rats was identified in the heart, lung, and kidney by reverse transcription polymerase chain reaction followed by Southern blot analysis and an ELISA specific for human tissue kallikrein. A single injection of both human kallikrein plasmid DNA constructs caused a sustained reduction of blood pressure which began 1 wk after injection and continued for 6 wk. A maximal effect of blood pressure reduction of 46 mmHg in rats was observed 2-3 wk after injection with kallikrein DNA as compared to rats with vector DNA (n = 6, P < 0.05). The hypotensive effect caused by somatic gene delivery of human tissue kallikrein in hypertensive rats is reversed by subcutaneous injection of aprotinin, a potent tissue kallikrein inhibitor. No antibodies to either human tissue kallikrein or kallikrein DNA were detected in rat sera after injection of the human kallikrein gene. These results show that direct gene delivery of human tissue kallikrein causes a sustained reduction in systolic blood pressure in genetically hypertensive rats and indicate that the feasibility of kallikrein gene therapy for treating human hypertension should be studied.

Animals↗
PubMed1995

Cognitive complexity and scanning by Chinese and American students in an American university.

75 Chinese and 75 American students' perceptual styles of cognitive complexity and scanning (focus versus nonfocus) were analyzed. The American students, 22 Chinese women, and 18 Chinese social science and humanities students scored as more complex than the 53 Chinese men and 57 Chinese natural science and engineering students. It was also observed that the Chinese men and American women scored as more focused than the 22 Chinese women and 43 American men.

Adult↗
PubMed1995

Biochemistry, regulation and potential function of kallistatin.

Components of the tissue kallikrein-kinin system include tissue kallikrein, kallistatin (kallikrein-binding protein), kininogen, kinin, bradykinin B1 and B2 receptors, and kininases. Tissue kallikrein is a serine proteinase which is capable of cleaving kininogen substrate to release the vasoactive kinin peptide. The binding of kinin to its specific receptor at target organs can produce a wide spectrum of biological effects. Kinin generation is primarily determined by the activity and availability of kallikrein since the level of kininogen is not a rate-limiting factor. Kallikrein levels are controlled by its rate of synthesis, activation, inactivation and clearance. The synthesis of tissue kallikrein is regulated transcriptionally, and its activity is regulated through post-translational processing and inactivation by inhibitors. Kallistatin is a newly discovered serine proteinase inhibitor (serpin) which forms a specific and covalently-linked complex with tissue kallikrein. Kallistatin may regulate tissue kallikrein's activity, bioavailability and clearance rate at the post-translational level. The major site of kallistatin synthesis is the liver with lower expression levels in the pancreas and kidney. Unlike many other serpins which are only present in the plasma, kallistatin is found in various tissues, cells and bodily fluids. The fact that both tissue kallikrein and kallistatin are widely distributed in tissues suggests kallistatin's role as a potential regulator of kallikrein outside the circulation. Protein purification and molecular cloning techniques have been used to study the structure, regulation and function of the components of the kallikrein-kinin system and for exploring their roles in ion transport, inflammation and blood pressure regulation. Considerable progress has been made in recent years to achieve these goals. This article provides an overview of the biochemical properties and potential physiological and pathophysiological roles of kallistatin.

Animals↗
PubMed1994

Cloning, sequence analysis and expression of the gene encoding the mouse bradykinin B2 receptor.

The bradykinin B2 receptor (B2R) mediates most of the biological effects of kinins. In the present study, we have cloned and sequenced the gene (B2R) from a mouse embryonic stem (ES) cell genomic library. Mouse B2R is 7 kb in length containing three exons of 0.3, 0.23 and 3.7 kb separated by two introns of 2.5 and 0.41 kb. The first and second exons are noncoding while the third exon contains the full-length coding region and a long 3' noncoding region. An ATG translation start codon, TGA stop codon and a polyadenylation signal (AATAAA) were identified in the third exon. B2R encodes a protein of 41,470 Da and 366 amino acids (aa) forming seven transmembrane (TM) domains. At the aa level, B2R shares 91 and 82% sequence identity with the rat and human B2R, respectively. In the 5' flanking region, a consensus TATA box, a putative cAMP-response element, a putative phorbol ester-response element, a putative AP-1-binding site and a putative IL-6-response element were identified. Southern blot analysis following reverse transcription and PCR showed that B2R is expressed in most mouse tissues, except the liver and spleen, which is consistent with the wide distribution of B2R activity as deduced from pharmacological studies.

Amino Acid Sequence↗
PubMed1994

Structure and chromosomal localization of the gene (BDKRB2) encoding human bradykinin B2 receptor.

The bradykinin B2 receptor (BDKRB2) has high affinity for the intact kinins, which mediate a wide spectrum of biological effects, including pain, inflammation, vasodilation, and smooth muscle contraction and relaxation. In the present study, we have cloned and sequenced the gene encoding human bradykinin B2 receptor from a human genomic library. The B2 receptor gene contains three exons separated by two introns. The first and second exons are noncoding, while the third exon contains the full-length coding region, which encodes a protein of 364 amino acids forming 7 transmembrane domains. The human B2 gene shares high sequence identity with rat and mouse B2 receptor genes and significant similarity with the gene encoding the angiotensin II type I receptor in the nucleotide sequence and exon-intron arrangement. In the 5' flanking region, a consensus TATA box and several putative transcription factor-binding sites have been identified. Genomic Southern blot analysis showed that the B2 receptor is encoded by a single-copy gene that was localized to chromosome 14q32 by in situ hybridization. In a Southern blot analysis following reverse transcription and polymerase chain reaction, the human B2 receptor was found to be expressed in most human tissues.

Amino Acid Sequence↗
PubMed1994

Molecular cloning, sequence analysis, and chromosomal localization of the human protease inhibitor 4 (kallistatin) gene (PI4).

The gene encoding human protease inhibitor 4 (kallistatin; gene symbol PI4), a novel serine proteinase inhibitor (serpin), has been isolated and completely sequenced. The kallistatin gene is 9618 bp in length and contains five exons and four introns. The structure and organization of the kallistatin gene are similar to those of the genes encoding alpha 1-antichymotrypsin, protein C inhibitor, and alpha 1-antitrypsin. The kallistatin gene is also similar to the genes encoding rat and mouse kallikrein-binding proteins. The first exon of the kallistatin gene is a noncoding 89-bp fragment, as determined by primer extension. The fifth exon, which contains 308 bp of noncoding sequence, encodes the reactive center of kallistatin. In the 5'-flanking region of the kallistatin gene, 1125 bp have been sequenced and a consensus promoter segment with potential transcription regulatory sites, including CAAT and TATA boxes, an AP-2 binding site, a GC-rich region, a cAMP response element, and an AP-1 binding site, has been identified within this region. The kallistatin gene was localized by in situ hybridization to human chromosome 14q31-q32.1, close to the serpin genes encoding alpha 1-antichymotrypsin, protein C inhibitor, alpha 1-antitrypsin, and corticosteroid-binding globulin. In a genomic DNA Southern blot, kallistatin-related genes were identified in monkey, mouse, rat, bovine, dog, cat, and a ground mole. The patterns of hybridization revealed clues of human serpin evolution.

Amino Acid Sequence↗
PubMed1994

Molecular cloning and sequence analysis of rat bradykinin B2 receptor gene.

The bradykinin B2 receptor mediates the effect of kinin. In order to understand the structure and regulation of its expression, we have cloned and sequenced the gene encoding the rat bradykinin B2 receptor and its 5' flanking region from a rat genomic library. The B2 receptor gene spans 7.3 kb in length and contains three exons which are separated by two introns. It encodes a peptide of 366 amino acids. The transcription initiation site was mapped by the primer extension assay. A variant TATA box sequence, an IL-6 response element and a cAMP response element were identified in the 5' flanking region of the rat bradykinin B2 receptor gene.

Amino Acid Sequence↗
PubMed1994

Prostasin is a novel human serine proteinase from seminal fluid. Purification, tissue distribution, and localization in prostate gland.

A novel serine proteinase, designated as prostasin, has been purified from human seminal fluid to apparent homogeneity by DEAE-Sepharose CL-6B and aprotinin-affinity chromatography. The purified protein migrates as two close bands with an apparent molecular mass of 40 kDa on SDS-polyacrylamide gel electrophoresis under reducing conditions. It can be labeled with [14C]diisopropyl fluorophosphate and has a pI ranging from 4.5 to 4.8. Sequence analysis reveals that the two protein bands have an identical NH2-terminal amino acid sequence which is different from any known protein sequence in the SwissPro or GenBank data base. The NH2-terminal 20-amino acid sequence shares 50-55% identity with human alpha-tryptase, elastase 2A and 2B, chymotrypsin, acrosin, and the catalytic chains of hepsin, plasma kallikrein, and coagulation factor XI. Prostasin has trypsin-like activity with a pH optimum of 9.0, hydrolyzing peptidyl fluorogenic substrates: D-Pro-Phe-Arg-MCA, D-Phe-Phe-Arg-MCA, D-Val-Leu-Arg-MCA, and Z-Gly-Pro-Arg-AFC. It is inhibited by aprotinin, antipain, leupeptin, and benzamidine. The tissue distribution of prostasin was determined by a newly developed radioimmunoassay. Linear displacement curves for immunoreactive prostasin in body fluids and tissues were parallel with the standard curve of purified prostasin, indicating their immunological identity. Immunoreactive prostatin levels were 8.61 +/- 0.42 microgram/ml in the seminal fluid and 0.201 +/- 0.029 microgram/ml in urine. Prostasin is present at high levels in the prostate gland (143.7 +/- 15.9 ng/mg protein), moderate levels (2-6 ng/mg protein) in colon, lung, kidney, pancreas, salivary gland, liver, and bronchi, but it is not detected in the brain, muscle, testis, ventricle, atrium, and aorta. Immunohistochemical localization reveals that prostasin is present in epithelial cells and ducts of the prostate gland. These studies indicate that prostasin purified from seminal fluid is a novel serine proteinase and originates from the prostate gland.

Amino Acid Sequence↗