Biomedical subjects

L Chao

Publications and source records attributed to L Chao.

At least 109 records · Page 6Linked to original sources

PubMed1996

Purification and characterization of salivary kallikrein from an insectivore (Scalopus aquaticus): substrate specificities, immunoreactivity, and kinetic analyses.

We report the successful one-step separation of tissue kallikrein from the salivary glands of an insectivore, the Eastern Atlantic mole (Scalopus aquaticus) by perfusion chromatography. Purified mole salivary kallikrein was characterized as a 30-kDa serine proteinase with a pI of 5.3 and a pH optimum of 9.0. It was readily recognized by human tissue kallikrein antibody in immunoblot analyses. It preferentially hydrolyzes fluorogenic peptidyl substrates with arginyl residues, rather than lysyl residues at the P1 substrate recognition site, indicating that it is like other mammalian kallikreins. Mole kallikrein efficiently releases kinin from low molecular weight human, dog, and bovine kininogen substrates with specific activities similar to that of human tissue kallikrein. Steady state kinetics performed with the synthetic tripeptidyl substrates, Phe-Phe-Arg-, Pro-Phe-Arg, and Val-Leu-Arg-7-amino-4-methylcoumarin, gave K(m) values for mole kallikrein of 3.3, 46.1, and 2.8 microM, respectively, and specificity constants, kcat/K(m), of 3818, 165, and 8714 s-1 pM-1, respectively. Mole kallikrein, when compared with human and rat tissue kallikreins, more closely resembles human kallikrein based on immunoreactivity and kininogenase activity. Mole kallikrein appears to be a member of a single gene or small multigene family. S. aquaticus is recommended for studying the evolution of mammalian proteins and may offer advantages over rodent models for biomedical research.

Amino Acid Sequence↗

PubMed1996

Asymmetries generated by transcription-coupled repair in enterobacterial genes.

Although certain replication errors occur at different frequencies on each of the complementary strands of DNA, it remains unclear whether this bias is prevalent enough during chromosome replication to affect sequence evolution. Here, nucleotide substitutions in enteric bacteria were examined, and no difference in mutation rates was detected between the leading and lagging strands, but in comparing the coding and noncoding strands, and excess of C-->T changes was observed on the coding strand. This asymmetry is best explained by transcription-coupled repair on the noncoding strand. Although the vast majority of mutations are thought to arise from spontaneous errors during replication, this result implicates DNA damage as a substantial source of mutations in the wild.

Chromosome Mapping↗

PubMed1996

Structure and chromosomal localization of the human prostasin (PRSS8) gene.

Prostasin, denoted as PRSS8, is a newly identified human serine proteinase that shares high sequence identity with acrosin, plasma kallikrein, and hepsin (Yu et al., 1994, 1995). In the present study, a full-length PRSS8 gene has been isolated and characterized. A 7-kb PRSS8 gene fragment has been sequenced, including a 1.4-kb 5'-flanking region, the 4.4-kb PRSS8 gene, and a 1.2-kb 3'-flanking region. The gene consists of six exons and five introns based on comparison with its cDNA sequence. The sizes of these exons are 417, 18, 163, 272, 167, and 899 bp, while those of the introns are 243, 1763, 271, 85, and 92 bp. A number of potential regulatory elements have been revealed in the 5'-flanking region, including an AP2 site, two erythroid-specific promoter elements, and a sterol regulatory element. In addition, there are a variant GC box and a variant AP1 site in the promoter region. The transcription initiation site of the PRSS8 gene has been defined at the G residue and its adjacent A residue in a sequence CTCATGACT, which is similar to an initiator element CTCANTCT. Between the transcription initiation site and these putative regulatory elements, there is an AC-rich repetitive sequence that spans over 300 bp. Human PRSS8 is a single-copy gene and has been localized on chromosome 16p11.2 by in situ hybridization.

Amino Acid Sequence↗

PubMed1996

Genomic DNA sequence, expression, and chromosomal localization of the human B1 bradykinin receptor gene BDKRB1.

We have cloned and sequenced the human B1 bradykinin receptor gene (BDKRB1), which contains an uninterrupted coding exon. A putative promoter was identified by linking various lengths of the 5'-flanking region of the B1 receptor gene coding sequence to a CAT reporter and assaying for CAT activity. Deletion analysis showed that a 300-bp fragment in the promoter region is sufficient to direct the synthesis of the reporter and that an enhancer-like element is present between -1842 and -812. A genomic Southern blot using the B1 cDNA revealed that the receptor is encoded by a single-copy gene. The gene is located on chromosome 14q32.1-q32.2, in close proximity to the B2 receptor gene. Northern blot analysis identified a 1.7- to 1.8-kb mature mRNA transcript of the B1 receptor gene in the kidney and pancreas. A widespread tissue distribution of the B1 gene expression was identified by RT-PCR-Southern blot analysis using specific oligonucleotide probes.

Chloramphenicol O-Acetyltransferase↗

PubMed1996

Expression and cellular localization of the kallikrein-kinin system in human ocular tissues.

Tissue kallikrein is a serine proteinase which processes kininogens to release bioactive kinins. Kinins mediate a variety of biological processes through the interaction with kinin receptors. Kinins are involved in the regulation of blood pressure and local blood flow, vasodilation, smooth muscle contraction and relaxation, production of pain and inflammation, and stimulation of cell proliferation. The tissue kallikrein-kinin system has been implicated in a number of pathophysiological processes such as hypertension, allergy and diabetes mellitus. In the present study, we have identified the expression and localization of components of the kallikrein-kinin system in the human eye by reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analyses, and in situ hybridization histochemistry. RT-PCR and Southern blot analyses have detected mRNAs of the key components of the system including tissue kallikrein, low molecular weight kininogen, and bradykinin B1 and B2 receptors at high levels in human retina, choroid and ciliary body, and relatively low levels in the optic nerve. In situ hybridization has identified cellular localization of these four mRNAs in ocular tissues. They are expressed in retinal neuronal cells including the outer nuclear layer, inner nuclear layer and ganglion cell layer. These mRNAs were also identified in endothelial cells of ocular blood vessels, ciliary muscle and lens epithelial cells. The sense riboprobes showed negative staining, which indicates the specificity of the antisense riboprobes. These results suggest that the tissue kallikrein-kinin system is produced endogenously in human ocular tissues. Similar expression patterns of kallikrein, kininogen and kinin receptors indicate that the kallikrein-kinin system may function in an autocrine or paracrine fashion in the eye.

Base Sequence↗

PubMed1996

Tissue kallikreins in evolutionarily diverse vertebrates.

A search for tissue kallikreins in lower vertebrates resulted in the discovery of three novel kallikreins. Tissue kallikrein was isolated from the salivary gland of the Eastern Atlantic mole, Scalopus aquaticus, and the pancreas of the Southern frog, Rana berlandieri. A prokallikrein was identified in skeletal muscle of the black sea bass, Centropristis striata. These enzymes range in molecular mass from 27 to 36 kDa and are acidic proteins with pIs between 4.2 and 5.3. Bass prokallikrein was activated by trypsin cleavage. These novel kallikreins were compared with human and rat tissue kallikreins in regard to immunoreactivity, molecular weight, isoelectric point, extinction coefficient, susceptibility to serine proteinase inhibitors and their ability to cleave low molecular weight dog kininogen to release kinin peptides.

Animals↗

PubMed1996

High level expression of human tissue kallikrein in the circulation induces hypotension in transgenic mice.

In order to create an animal model expressing a high level of tissue kallikrein in the circulation, the human tissue kallikrein gene was placed under the control of a mouse albumin enhancer and promoter to target its expression to liver. Three lines of transgenic mice carrying the human tissue kallikrein gene were established. The major site of human tissue kallikrein synthesis was identified in the liver of transgenic mice, and a high level of human tissue kallikrein was secreted into the mouse circulation. The systolic blood pressures of these transgenic mice are about 15-20 mmHg lower than that of the control mice. Administration of aprotinin, a potent tissue kallikrein inhibitor, restored normal blood pressure in these animals. These studies show that a high level of foreign tissue kallikrein in the circulation plays a role in blood pressure regulation.

Animals↗

PubMed1996

Tissue kallikrein inhibitors in mammals.

We have discovered, purified and cloned a new kallikrein-binding protein (KBP or kallistatin) from humans and rodents. Kallistatins are members of the serine proteinase inhibitor (serpin) superfamily. They are acidic glycoproteins with molecular masses of 58-62 kDa and pI values of 4.6-5.2. Kallistatin forms a SDS-stable complex with tissue kallikrein and inhibits kallikrein's activities. Human kallistatin has a unique cleavage site with Phe-Phe-Ser at the P2-P1-P1' positions. The protein sequence of mature human kallistatin shares 44-46% identity with other serpins such as human alpha 1-antitrypsin, protein C inhibitor and rat kallikrein-binding protein. The kallistatin genes display the typical five exon-four intron serpin gene structure. The human kallistatin gene is localized on chromosome 14q31-32.1 and the RKBP gene is on chromosome 6. Kallistatin is evolutionarily diverse but functionally conserved in mammalian species. This overview summarizes the biochemistry, molecular biology and potential physiology and/or pathophysiology of this new tissue kallikrein inhibitor.

Animals↗

PubMed1996

Cellular localization of bradykinin B1 receptor mRNA in the human kidney.

Cellular localization of the B1 receptor mRNA in the human kidney was identified by in situ hybridization histochemistry using digoxigenin-labeled riboprobe. With the antisense riboprobe, the B1 receptor mRNA was found mostly in the parietal layer of Bowman's capsule and the thin segment of Henle's loop. The renal carcinoma cells were stained with the B1 receptor riboprobe. These results showed the cellular localization of human renal B1 receptor mRNA and revealed sites of bradykinin action in regulating renal function under normal and pathological conditions.

Base Sequence↗

PubMed1996

Kallistatin, a novel human tissue kallikrein inhibitor: levels in body fluids, blood cells, and tissues in health and disease.

Kallistatin, a human serine proteinase inhibitor, is a newly identified tissue kallikrein inhibitor. It binds strongly to tissue kallikrein but weakly to other serine proteinases such as chymotrypsin and elastase. The tissue distribution and changes in kallistatin levels in human diseases were characterized by using specific monoclonal and polyclonal antibodies against kallistatin. Kallistatin antigen levels in blood cells, fluids, and tissues measured with a specific enzyme-linked immunosorbent assay showed displacement curves that were parallel with those in purified kallistatin, indicating their immunologic identity. Expression of kallistatin mRNA in platelets, neutrophils, lymphocytes, monocytes, endothelial cells, hepatocytes, and colon and prostate carcinoma cells was identified by reverse transcription-polymerase chain reaction followed by Southern blot analysis. Plasma kallistatin concentration was 22.1 +/- 3.5 micrograms/ml in 30 normal subjects and 21.1 +/- 3.8 micrograms/ml in 5 patients with C1 inhibitor deficiency. A significantly reduced kallistatin level (7.2 +/- 2.5 micrograms/ml, p < 0.001) was seen in plasma samples from 9 patients with liver disease and 10 patients with sepsis (7.7 +/- 3.5 micrograms/ml, p < 0 .001). Further, kallistatin levels in 10 women taking oral contraceptives (19.8 +/- 3.8 micrograms/ml) and 21 pregnant women (14.9 +/- 3.3 microg/ml) were significantly lower than those seen in healthy individuals. These data suggest that kallistatin is found in plasma, is produced mostly in the liver, and can be consumed during sepsis. Its consumption in sepsis may indicate a protective role to prevent blood pressure lowering.

Antibodies, Monoclonal↗

PubMed1996

Cellular localization of low-molecular-weight kininogen and bradykinin B2 receptor mRNAs in human kidney.

Kininogen is the precursor of the kinin peptide, which binds to kinin receptors and mediates a broad spectrum of physiological effects. To understand the function of kinin in the kidney, we have identified the cellular localization of the human low-molecular-weight (LMW) kininogen and bradykinin B2 receptor mRNAs in the human kidney by in situ hybridization histochemistry. Kininogen mRNA was found in the juxtaglomerular cells, mesangial areas, epithelium of parietal and visceral (podocytes) layers of Bowman's capsule, proximal and distal tubules, thin and thick segments of Henle's loop, collecting ducts, and the endothelial cells of the blood vessels. B2 receptor mRNA was colocalized with kininogen mRNA in the kidney except the podocytes. The most intense signals were observed in the distal tubules and collecting ducts for both kininogen and B2 receptor mRNAs. No signals were observed in the interstitial cells and macula densa. Control sections did not stain with either the kininogen or B2 receptor sense riboprobe. A Northern blot showed that the expression of LMW kininogen is in the liver and the kidney. Reverse transcription-polymerase chain reaction Southern blot showed expression of B2 receptor mRNA in the endothelial cells, renal proximal tubular cells, and kidney. Our results show the sites of action of kinin in the human kidney and provide further insight into the physiological role of the kallikrein-kinin system on renal function.

Base Sequence↗

PubMed1996

Differential regulation of kallikrein, kininogen, and kallikrein-binding protein in arterial hypertensive rats.

This study was designed to determine whether the kallikrein-kinin system exerts a protective action in hypertension induced by chronic inhibition of nitric oxide synthase. N omega-nitro-L-arginine methyl ester (L-NAME, 40 mg/100 ml water) was given orally to Sprague-Dawley rats, while controls received regular tap water. Hepatic kininogen mRNA levels in the L-NAME-treated group were 2.9- and 2.5-fold higher at 3 and 4 wk, respectively, compared with control rats, whereas kallikrein-binding protein (KBP) mRNA levels were 82% and 45% of the values found in control rats at 3 and 4 wk, respectively. There was no significant change in hepatic alpha 1-antitrypsin mRNA levels under the same conditions. At 3 and 4 wk post L-NAME treatment, renal kallikrein mRNA levels were 2.5- and 3.4-fold higher than in controls, whereas renal beta-actin mRNA levels were similar between groups. Changes in the transcript levels of renal kallikrein, kininogen, and KBP were consistent with their protein levels. Immunoreactive total kininogen and low-Mr kininogen levels in sera and tissue kallikrein levels in kidney were significantly higher in the L-NAME-treated group, whereas KBP levels in the circulation were lower compared with controls. Systolic blood pressure was increased by 58 +/- 4 mmHg after 4 wk of L-NAME treatment. This effect was enhanced in rats given L-NAME in combination with HOE-140, a bradykinin B2-receptor antagonist, at the dose of 100 micrograms/day ip (79 +/- 5 vs. 58 +/- 4 mmHg, P < 0.05). This difference was confirmed by direct measurement of mean blood pressure (MBP). An intra-arterial bolus injection of 200 ng bradykinin significantly decreased MBP of L-NAME-treated rats, and this effect was blunted in the group treated with the bradykinin antagonist (-29 +/- 3 vs. -9 +/- 2 mmHg, P < 0.01). These results suggest that enhanced kallikrein and kininogen synthesis may have a protective role against the cardiovascular effects induced by chronic inhibition of nitric oxide synthesis.

Animals↗

PubMed1996

Expression and cellular localization of tissue kallikrein-kinin system in human adrenal gland.

The tissue kallikrein-kinin system has been implicated in regulating blood pressure and electrolyte homeostasis. To understand the function of this system, we identified the expression and cellular localization of its components including tissue kallikrein, kallistatin, kininogen, and bradykinin B1 and B2 receptors in human adrenal gland. Reverse transcription-polymerase chain reaction followed by Southern blot analysis showed that these five components of this system were all expressed in human adrenal gland. In situ hybridization histochemistry with respective digoxigenin-labeled antisense riboprobes revealed localization of kallikrein transcript throughout the adrenal cortex and medulla except the zona glomerulosa, whereas kallistatin mRNA was only localized in the zona fasciculata. Low-molecular-weight kininogen and B2 receptor mRNAs were colocalized in the zona glomerulosa and zona fasciculata and also in the zona reticularis and chromaffin cells but to a lesser degree. The B1 receptor mRNA was stained in the zona fasciculata and medulla. These results show the expression and differential colocalization of the components of the tissue kallikrein-kinin system and reveal the potential action sites of this system in the adrenal gland.

Adrenal Glands↗

PubMed1996

High-salt diet upregulates kininogen and downregulates tissue kallikrein expression in Dahl-SS and SHR rats.

Tissue kallikrein cleaves low-molecular-weight (low-M(r)) kininogen to produce the vasoactive kinin peptide. It has been suggested that hypertensive patients with low urinary kallikrein excretion may have a defect in sodium handling. In this study, we examined the effect of a high-salt diet on the expression of tissue kallikrein and kininogen genes in Dahl salt-sensitive rats (Dahl-SS), spontaneously hypertensive rats (SHR), and normotensive Sprague-Dawley rats (SD) by Northern and Western blot analysis and radioimmunoassay. Control and experimental groups received normal and high-salt diets containing 0.4% and 8% NaCl, respectively, for 6 wk. High-salt diet induced a significant time-dependent increase of blood pressure in both strains of hypertensive rats and a slight but significant increase of blood pressure in normotensive SD rats. Hepatic kininogen mRNA levels of both Dahl-SS and SHR on a high-salt diet increased 2.4-fold and 2.0-fold, respectively, while alpha 1-antitrypsin mRNA levels were not changed in rats receiving high-salt diet. Immunoreactive total kininogen and low-M(r) kininogen (58 kDa) levels in sera increased in response to high-salt diet in both strains of hypertensive rats. In SD rats, the low-M(r) kininogen level in sera was unaltered, whereas total kininogen increased in response to high-salt diet. Tissue kallikrein mRNAs in the kidney and salivary glands of Dahl-SS, SHR, and SD rats were reduced, whereas beta-actin mRNA was not altered by high-salt diet. Similarly, immunoreactive intrarenal kallikrein levels were reduced in these rats in response to high-salt diet. These studies show that increases in blood pressure after salt loading in Dahl-SS and SHR are accompanied by increases in low-M(r) kininogen. Tissue kallikrein gene expression in hypertensive Dahl-SS and SHR and normotensive SD rats is suppressed after salt loading. These findings show that reduced renal kallikrein expression and increased kininogen expression is regulated at the transcriptional level during salt loading.

Animals↗

PubMed1996

Functional analysis of human tissue kallikrein in transgenic mouse models.

Clinical studies show that an inverse correlation exists between blood pressure and urinary kallikrein levels. It has been postulated that the tissue kallikrein-kinin system contributes to the maintenance of normal blood pressure. To test this hypothesis, we have established transgenic mice that overexpress human tissue kallikrein under the promoter control of the mouse metallothionein gene and a liver-targeted albumin gene. These animals secrete human tissue kallikrein in plasma at levels 10- to 40-fold higher than that found in normal human serum, and they are chronically hypotensive. This hypotensive effect can be reversed by the injection of aprotinin, a potent tissue kallikrein inhibitor, or Hoe 140, a specific bradykinin receptor antagonist. Transgenic mice overexpressing human tissue kallikrein show a sustained reduction in blood pressure throughout their life spans, indicating the lack of sufficient compensatory mechanisms to reverse the hypotensive effect of kallikrein. Somatic gene delivery of rat kallikrein-binding protein by muscle injection increases the blood pressure of the hypotensive transgenic mice to levels comparable with those in normotensive control mice. These results indicate that a direct link exists between kallikrein gene expression and alterations in blood pressure. In addition, we have developed normotensive transgenic mice that harbor the human tissue kallikrein gene containing 801 bp of its native promoter. The tissue distribution pattern of human kallikrein in these transgenic mice is similar to that in human tissues, with the highest level in the pancreas and much lower levels in the kidney and salivary gland. These transgenic mice provide new animal models for investigating the tissue-specific regulation of tissue kallikrein and its role in altering blood pressure.

Animals↗

PubMed1996

Antisense inhibition of the brain kallikrein-kinin system.

We used antisense oligodeoxynucleotide (ODN) strategy, based on interference of information flow from gene to protein, to determine the role of kininogen and bradykinin B2 receptor genes in the pathogenesis of genetic hypertension in rats. Mean blood pressure of 9-week-old spontaneously hypertensive rats (SHR) increased 4 hours after acute intracerebroventricular injection of synthetic 18-mer antisense ODNs targeting the translation initiation codon of kininogen mRNA (from 164 +/- 5 to 181 +/- 4 mm Hg, P < .01) or bradykinin B2 receptor mRNA (from 161 +/- 5 to 185 +/- 8 mm Hg, P < .01) and then returned to basal levels within 24 hours. Prolonged vasopressor effects were observed after repeated injections of antisense ODN targeting kininogen mRNA. Antisense ODNs to kininogen and B2 receptor mRNAs increased blood pressure of normotensive Wistar-Kyoto rats only slightly compared with SHR (from 116 +/- 3 to 124 +/- 1 and from 116 +/- 2 to 126 +/- 4 mm Hg, respectively; P < .05). Cardiovascular responses were confirmed by the use of antisense ODNs targeted to bind to different non-overlapping regions of kininogen or B2 receptor mRNA. Microinjection of antisense ODN to B2 receptor mRNA into the nucleus tractus solitarii increased mean blood pressure in SHR and prevented the vasodepressor effect induced by intranuclear microinjection of bradykinin. No significant change in mean blood pressure was induced in either strain by intravenous injection of antisense ODNs or by central injection of sense or scrambled ODNs. A strong fluorescent signal was detected at the level of the hippocampus, thalamus, hypothalamus periventricularis, midbrain, and cerebrum 1 hour after central injection of fluorescein isothiocyanate-conjugated antisense ODNs. Kininogen levels were significantly lower in the brain of rats given intracerebroventricular antisense kininogen ODN compared with controls. Our results indicate that the brain kallikrein-kinin system plays a role in the central regulation of blood pressure and suggest that this system may exert a protective action against further elevations of blood pressure levels in SHR.

Animals↗

PubMed1996

Kallistatin in human ocular tissues: reduced levels in vitreous fluids from patients with diabetic retinopathy.

PURPOSE: Kallistatin is a serine proteinase inhibitor, which binds to tissue kallikrein and inhibits its proteolytic activity. This study is to determine the expression, cellular localization and the potential function of kallistatin in the eye. METHODS: Tissue kallikrein-kallistatin complex formation was performed to detect the kallikrein-binding activity in ocular tissues. Immunoreactive kallistatin was detected and quantified by an enzyme-linked immunosorbent assay using polyclonal antibody specific to human kallistatin. In situ hybridization histochemistry was employed to localize the kallistatin mRNA in human eyes using an antisense riboprobe of kallistatin. RESULTS: We have identified active kallistatin in the cornea, ciliary body, sclera, choroid, optic nerve, retina, vitreous and aqeous fluids. Kallistatin binds to tissue kallikrein and forms an SDS-stable complex. Immunoreactive kallistatin was identified in these tissues. Linear dose-dependent curves of the tissue extracts of the retina and choroid are parallel to that of purified human kallistatin, suggesting their immunological identity. The kallistatin mRNA was identified in the ciliary muscle, lens epithelial cells, all the layers of retina cells, optic nerve, choroid and vascular endothelial cells. These cells were not stained by the sense riboprobe under the same conditions, indicating the specificity of the hybridization. We also compared immunoreactive kallistatin levels in vitreous fluids from 18 patients with diabetic retinopathy and 17 non-diabetic subjects. The results show that diabetic subjects have significantly lower kallistatin levels (233.0 +/- 14.6 ng/mg protein) compared to non-diabetic subjects (334.1 +/- 26.9 ng/mg protein). CONCLUSIONS: Kallistatin is produced endogenously in the eye and the decrease in the vitreous kallistatin levels may be involved in diabetic retinopathy.

Adult↗

PubMed1996

High level of circulating human tissue kallikrein induces hypotension in a transgenic mouse model.

We established a unique transgenic mouse model in liver-targeted expression of human tissue kallikrein using a mouse albumin enhancer and promoter. Northern blot analysis and ELISA showed that human tissue kallikrein was predominantly expressed in the liver of transgenic mice and secreted into the circulation at a high level. The transcript was also detected in the kidney, pancreas, salivary gland and heart at a low level by reverse transcription-polymerase chain reaction followed by Southern blot analysis. Systolic blood pressures were measured by the tail-cuff method, all three independent transgenic mouse lines are hypotensive (84.6 +/- 1.0 mmHg, n = 17; 84.5 +/- 1.5 mmHg, n = 9; 83.1 +/- 0.8 mmHg, n = 13, P < 0.01) compared with the control mice (100.9 +/- 0.9 mmHg, n = 17). Administration of aprotinin, a potent tissue kallikrein inhibitor or Hoe 140, a bradykinin receptor antagonist, restored the blood pressure of transgenic mice but had no significant effect on control littermates. These studies show that over-production of tissue kallikrein in the circulation plays a role in blood pressure regulation.

Animals↗