Search PubMed⌕ Search

Biomedical subjects

J Chao

Publications and source records attributed to J Chao.

At least 181 records · Page 10Linked to original sources
PubMed1992

Comparative studies on P2 specificity of wild-type rat tissue kallikrein, Y99H:W215G mutant and tonin.

To probe residues responsible for P2 specificity, we have recently created a mutant enzyme from rat tissue kallikrein with Tyr99 to His and Trp215 to Gly exchange (Y99H:W215G) using site-specific mutagenesis. In the present study, additional characterization of substrate specificities of both wild-type tissue kallikrein, Y99H:W215G mutant and native tonin was performed using synthetic Ac-X-Arg-pNA substrates especially designed for testing P2 specificity. Kinetic analyses of Km and kcat demonstrate a clear correlation between dramatically reduced affinity for hydrophobic P2 side-chain and the loss of the Tyr99-Trp215 hydrophobic pair. Analyses of rat tonin reveal a correlation between increased reaction rate and P2 hydrophilicity although tonin displays similar pattern in P2 affinity as compared with tissue kallikrein, suggesting a less hydrophobic environment in the substrate-binding pocket of rat tonin. The results strongly support the hypothesis that Tyr99-Trp215 interaction is the major determinant for P2 specificity and that the presence of a hydrophobic side-chain in P2 position substantially facilitates substrate hydrolysis of tissue kallikrein-like enzymes.

Amino Acid Sequence↗
PubMed1992

Effects of diabetes and insulin on expression of kallikrein and renin genes in the kidney.

We previously showed that renal prokallikrein synthesis is reduced in streptozotocin (STZ)-diabetic rats. Plasma renin activity is also reduced in diabetic rats. To investigate the molecular mechanisms underlying these changes, we examined the effects of diabetes and insulin treatment on renal kallikrein and renal renin mRNA levels and the activities of these enzymes. Rats made diabetic by STZ were either treated with 1.5 to 1.75 U PZI insulin daily to maintain moderate hyperglycemia (plasma glucose 200 to 300 mg/dl, D + I) or left untreated to produce severe hyperglycemia (plasma glucose greater than 400 mg/dl, D). Control (C) rats were also studied. After three weeks, renal kallikrein mRNA was reduced 50% in D rats. A proportional reduction in immunoreactive kallikrein was also observed (37.8 +/- 2.5 vs. 55.8 +/- 6.8 ng/mg protein, D vs. C, P less than 0.001). Kallikrein mRNA and immunoreactive kallikrein levels in D + I rats were not different from C rats. Renin mRNA level was also markedly reduced in D rats, compared to C rats. This was associated with reduced plasma renin concentration (4.5 +/- 0.2 vs. 10.5 +/- 1.6 ng Ang I/ml/hr, D vs. C, P less than 0.01). However, renal renin concentration was unchanged (0.84 +/- 0.17 vs. 0.84 +/- 1.3 micrograms Ang I/mg protein/hr, D vs. C). In D + I rats, renin mRNA level and plasma renin concentration were not different from C levels. However, renal renin concentration was increased (1.49 +/- 0.27 micrograms Ang I/mg protein/hr) compared to C rats (P less than 0.05). beta-actin mRNA levels were unchanged in either diabetic rat group.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1992

Specificity determinants of rat tissue kallikrein probed by site-directed mutagenesis.

Site-specific mutagenesis was employed to study structure-function relationships at the substrate binding site of rat tissue kallikrein. Four kallikrein mutants, the Pro219 deletion (P219del), the 34-38 loop Tyr-Tyr-Phe-Gly to Ile-Asn mutation [YYFG(34-38)IN], the Trp215----Gly exchange (W215G) and the double mutant with Tyr99----His and Trp215----Gly exchange (Y99H:W215G) were created by site-directed mutagenesis to probe their function in substrate binding. The mutant proteins were expressed in Escherichia coli at high levels and analyzed by Western blot. These mutant enzymes were purified to apparent homogeneity. Each migrated as a single band on SDS-PAGE, with slightly lower molecular mass (36 kDa) than that of the native enzyme, (38 kDa) because of their lack of glycosylation. The recombinant kallikreins are immunologically identical to the native enzyme, displaying parallelism with the native enzyme in a direct radioimmunoassay for rat tissue kallikrein. Kinetic analyses of Km and kcat using fluorogenic peptide substrates support the hypothesis that the Tyr99-Trp215 interaction is a major determinant for hydrophobic P2 specificity. The results suggest an important role for the 34-38 loop in hydrophobic P3 affinity and further show that Pro219 is essential to substrate binding and efficient catalysis of tissue kallikrein.

Amino Acid Sequence↗
PubMed1992

Spatial and temporal expression of kallikrein and its mRNA during nephron maturation.

To determine the relationships between nephron maturation and the ontogeny of intrarenal kallikrein, the expression and distribution of kallikrein and its mRNA were examined in fetal, neonatal, and adult Sprague-Dawley rats. Kallikrein-like immunoreactivity was first expressed 1 day before birth in the upper limb of S-shaped bodies. Immediately after birth, kallikrein immunostaining was present along the apical membranes of inner cortical distal tubules, whereas subcapsular less mature comma-shaped vesicles did not express kallikrein. After completion of nephrogenesis, kallikrein was also seen in the distal and connecting tubules of the outer cortex. In situ hybridization localized the kallikrein message in the distal tubules of inner cortical nephrons in newborns and outer cortex in adults. The transition from newborn to adult life was associated with six- and fourfold increase in renal kallikrein content and mRNA accumulation, respectively. We conclude that immature distal tubules have the transcriptional and translational capacity to express the kallikrein gene and protein and that the renal kallikrein gene is developmentally regulated. The spatial and temporal changes in intrarenal kallikrein expression during ontogeny are consistent with the hypothesis that the kallikrein-kinin system plays a role in the maturation of renal functions.

Animals↗
PubMed1992

Localization, synthetic regulation, and biology of renal atriopeptin-like prohormone.

We recently demonstrated the synthesis and secretion of an atriopeptin (AP)-like prohormone in rat neonatal and adult cortical kidney cell cultures. However, these cultures contained proximal as well as distal tubular epithelial cells; thus characterization of the peptide synthetic cell was not possible. Also, by immunohistochemical techniques, we localized this AP-like prohormone to the distal cortical nephron in adult rat kidney. In this study, we examined further details of the kidney cortical cell type that expresses and secretes this AP-like peptide in adult renal cortical cell cultures, its regulation by adenylate cyclase via adenosine 3',5'-cyclic monophosphate (cAMP) generation, and its ability to stimulate guanylate cyclase. Tubular fragments were derived from cortical tissue of adult Sprague-Dawley rats and separated into four fractions on Percoll density gradient. Cell cultures generated from fraction 3 secreted 5- to 10-fold the amount of this renal peptide compared with fractions 2 and 4. Further cell culture characterization was performed by agonist-stimulated cAMP formation, kallikrein localization, and prostaglandin E2 formation. From these analyses, it was determined that tissue band 3 was enriched for distal cortical connecting tubules. To further evaluate whether mammalian distal nephron synthesizes an AP-like protein, we determined that two immortalized mouse cell lines, derived from either the distal convoluted tubule or cortical collecting tubule, synthesized a radiolabeled AP after being pulsed with [35S]-methionine.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Differential developmental expression of the rat kininogen genes.

The rat liver expresses two low molecular weight kininogens (T-KG and K-KG). Although they share 90% of the nucleotide sequence in their 5' flanking regions, T- and K-KG genes are differentially regulated. The T-KG gene is inducible, and its protein is a potent thiol-protease inhibitor. In contrast, K-KG gene is expressed constitutively and encodes the precursor of the vasoactive nonapeptide bradykinin. To further elucidate the differential regulation of T- and K-KG genes, we examined their developmental expression in the Sprague-Dawley rat. Northern blots of total liver RNA were probed with oligonucleotides complementary to T and K-KG mRNA under high-stringency conditions. A single T-KG mRNA (1.8 kb) and two K-KG mRNA species (1.6 and 2.3 kb) were consistently detected at all ages studied. Steady state T-KG mRNA levels increased 3.5-fold at birth and remained high during the 1st week of postnatal life only to decline thereafter. T-KG immunoreactivity in the liver and plasma determined by Western blot analysis paralleled T-KG mRNA expression. In marked contrast, K-KG mRNA expression was not altered during the transition from fetal to neonatal life, nor was it affected by postnatal maturation. The results demonstrate that the fetal rat liver synthesizes kininogens and that T- and K-KG genes are differentially regulated during development. Up-regulation of T-KG synthesis after birth may serve a protective function in the newborn via its antiprotease activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗
PubMed1992

Physician agreement with US Preventive Services Task Force recommendations.

BACKGROUND: No large-scale work has yet assessed the reactions of physicians to the report of the US Preventive Services Task Force (USPSTF), despite its potential for fostering a consensus among practitioners. This study undertook a survey of family physicians to assess their agreement with the recommendations of the Task Force. METHODS: A survey containing the verbatim summary recommendations of the USPSTF was mailed to all 1784 active members of the Ohio Academy of Family Physicians. RESULTS: No evidence of selection bias was found among the 898 responding physicians. The average physician agreed with 88% of the recommendations. For a number of recommendations, however, particularly those in which the Task Force differed with the American Cancer Society, there was a high level of disagreement. Physician disagreement with the recommendations was associated with older age, not having completed a residency, male sex, less prior exposure to the USPSTF guidelines, and greater perception of the impracticality of applying them. CONCLUSIONS: The high level of agreement with most USPSTF recommendations implies that they represent an emerging consensus about which preventive services should be delivered. Attempts at USPSTF guideline dissemination should focus on recommendations with high agreement. Additional research is needed to assess the reasons for disagreement.

Age Factors↗
PubMed1992

Continuing medical education software: a comparative review.

Personal microcomputers can be used by physicians for continuing medical education (CME). Advantages of computerized CME include local control over the topic, time, place, and pace of instruction. Computers can be interactive, providing selected information that depends on the desires and needs of the physician. Learners have different preferred styles of receiving information, and computer programs can be written to appeal to a particular style of learning. Three examples of commercial software programs available for CME are reviewed. Cyberlog should appeal to those who like to learn from reading textbooks but also desire simple computer graphics and case simulations to reinforce key concepts. Patient Simulator II is a detailed patient simulation for those who learn best by doing. A subscription to Discotest provides patient management problems, and multiple choice questions (no graphics) based on information in Scientific American Medicine. No one program is suitable for all family physicians.

Computer Simulation↗
PubMed1992

In vivo catabolism of human kallikrein-binding protein and its complex with tissue kallikrein.

We recently identified and purified a novel human kallikrein-binding protein (HKBP) from human plasma. The HKBP forms a 92 kd sodium dodecyl sulfate-stable and heat-stable complex with tissue kallikrein. This study was undertaken to characterize the plasma clearance and tissue distribution of exogenously administered HKBP and its complex with tissue kallikrein. Human tissue kallikrein was first incubated with purified HKBP, and the high-molecular-weight complex was separated from unbound proteins on a high-pressure liquid chromatography gel filtration column. Tissue kallikrein, kallikrein-binding protein, and their complex were labeled with iodine-125 and then injected intravenously into Sprague-Dawley rats. The disappearance rates of trichloracetic acid-precipitable radioactivity from the circulation were determined. The clearance profile of HKBP shows a nonlinear pattern with an apparent half-life of 65 minutes (n = 4). The plasma clearance of HKBP complexed with kallikrein shows a similar profile but a shorter half-life of 33 minutes (n = 3). HKBP and its complex with kallikrein were mainly taken up by the liver but to a lesser degree by the kidney, lung, and other tissues. Labeled human kallikrein has an apparent half-life of 8 minutes (n = 4), and its clearance consists of a fast and a slow component. The data indicate that kallikrein-HKBP complex is cleared from the circulation two times faster than that of the binding protein alone and that it persists in the circulation four times longer than kallikrein alone. The results support the notion that more than one pathway exists for the metabolism of tissue kallikrein and that HKBP plays a role in modulating tissue kallikrein's bioavailability.

Animals↗
PubMed1991

Molecular cloning and sequence analysis of the mouse kallikrein-binding protein gene.

A genomic clone (MKBP-10) encoding the mouse kallikrein-binding protein (MKBP) was isolated from a mouse genomic DNA library by screening with a rat kallikrein-binding protein (RKBP) cDNA probe. The total sequenced region of the MKBP gene spans 8615 base pairs. The exon and intron locations of the RKBP gene were identified by similarity with the RKBP gene. The MKBP gene encodes a prepeptide of 417 amino acid residues which exhibits 71% homology with RKBP. A TATA box sequence was located in the 5' flanking region of the MKBP gene by similarity with the consensus sequence TATAAAA.

Amino Acid Sequence↗
PubMed1991

Molecular cloning and analysis of the rat kallikrein-binding protein gene.

The gene encoding rat kallikrein-binding protein (RKBP), a serine protease inhibitor, has been isolated and analyzed with the aid of the polymerase chain reaction. The gene is approximately 10 kilobases in length with four introns of approximately 2.2, 1.8, 0.9, and 0.84 kilobases. This gene is composed of five exons and encodes a polypeptide of 416 amino acid residues. The reactive center region of RKBP is encoded by the fifth exon with the putative P1-P1' residues being Lys-Ser. The organization of the RKBP gene is homologous to those of human alpha 1-antitrypsin and alpha 1-antichymotrypsin in size and arrangement of exons and introns, suggesting that they belong to the same subgroup of serpins. In the 5'-flanking region of the RKBP gene, a variant TATA box sequence, ATAAATA, is found 20 base pairs upstream from the transcription initiation site. The 5'-flanking region of the RKBP gene was able to direct transcription of the reporter gene chloramphenicol acetyltransferase when transfected into a rat hepatoma cell line. An internal promoter-like region was found in the first intron of the RKBP gene, downstream from the transcription initiation site and upstream from the translation initiation codon, however, it was unable to direct expression of the chloramphenicol acetyltransferase reporter gene in our experiments. The expression of RKBP in rat liver was induced by sex hormone treatment as indicated by dot-blot analysis. A genomic Southern blot using an RKBP cDNA probe revealed multiple bands suggesting that the RKBP gene belongs to a family of highly conserved genes.

Amino Acid Sequence↗
PubMed1991

Purification and characterization of recombinant tissue kallikrein from Escherichia coli and yeast.

A full-length rat tissue kallikrein cDNA was constructed by oligonucleotide engineering through an extension of RSK 1105, a partial cDNA clone containing 534 bp of the 3' end of tissue kallikrein, followed by site-directed mutagenesis to remove the vector sequence from within the chimaeric coding sequence. The cDNA has been cloned both into the plasmid pET3b under the control of the T7 promoter/polymerase system, and into the shuttle vector PYE directed by the alpha-factor promoter. Expression in Escherichia coli was detected by direct radioimmunoassay, and recombinant kallikrein of 36 kDa was identified by Western-blot analysis using both polyclonal and monoclonal antibodies to rat tissue kallikrein, and by autoradiography of 14C-labelled L-amino acid-labelled-protein synthesis in the presence of rifampicin. Expression in yeast was also detected by direct radioimmunoassay, and recombinant kallikrein was identified by Western-blot analysis with a molecular mass of 39 kDa. The recombinant kallikrein from yeast, however, remained mostly inactive. Kallikrein was purified to apparent homogeneity from E. coli by DEAE-Sepharose CL-6B and aprotinin-affinity column chromatography and confirmed by the N-terminal ten-amino-acid sequence, which matched the deduced sequence from the cDNA. Both E. coli and yeast recombinant kallikreins have Tos-Arg-OMe-esterolytic and kininogenase activities similar to those of purified tissue kallikrein. Comparisons were made between recombinant kallikreins and rat tissue kallikrein with respect to size, charge, substrate specificity, susceptibility to inhibitors and immunological properties. Our results open the way for the study of kallikrein structure-function relationships through protein engineering.

Amino Acid Sequence↗
PubMed1991

Imaging cytometry by multiparameter fluorescence.

A system is described for performing multicolor fluorescence image cytometry of cell preparations. After the setting up stage, the operation is automatic: the microscope fields are found and focused; then images are acquired for each fluorophore, corrected and analyzed, without any operator interaction. Human peripheral blood lymphocytes on microscope slides were used as a test system. In these experiments, three fluorescent antibodies were used to identify lymphocyte sub-populations, and a DNA content probe was used to identify all nucleated cells. The cell subset percentages determined by image cytometry were comparable to percentages obtained when cells from the same preparation were analyzed by flow cytometry. Multicolor fluorescence imaging cytometry can potentially be extended to the analysis of cells in smears, fine needle biopsies, imprints, and tissue sections.

Algorithms↗
PubMed1991

Kininogen and kinin in experimental spinal cord injury.

Activation of the kallikrein-kinin system has been implicated in the pathogenesis of vasogenic brain edema and posttraumatic vascular injury. We determined the levels of kininogen and kinin in an experimental spinal cord injury model in the rat. Kininogen content in traumatized cord segments increased in a time-dependent manner. Western blot analysis showed that the kininogen in traumatized cord comigrates with 68K low-molecular-weight kininogen or T-kininogen. Trypsin treatment of the kininogen in traumatized cord released both bradykinin and T-kinin, which were separated by HPLC and quantified with a kinin radioimmunoassay. Endogenous kinin levels in the frozen spinal cord also increased up to 40-fold 2 h after injury as compared with controls. The results demonstrate an increased accumulation of kininogen and its conversion to vasoactive kinins in experimental spinal cord injury.

Animals↗
PubMed1991

Design and implementation of full-frame, bit-allocation image-compression hardware module. Work in progress.

A hardware module was designed and built to implement the full-frame, bit-allocation image-compression algorithm in a clinical setting. The algorithm transforms an entire image without prepartitioning into small subimages. This adaptation eliminates block artifacts at subimage borders that can mimic relevant pathologic conditions. The quality of 1,024- and 2,048-pixel images compressed at a rate up to 10:1 with a custom-designed processor board (which contains four digital signal processors that transform and quantize separate rows and columns of an image independently with a two-pass cosine transform) and a 16-Mbyte frame buffer was found to be diagnostically acceptable in preliminary receiver operating characteristic studies. The module can compress a 1,024-pixel image in 4 seconds in a general-purpose computer system; images can be compressed in 1 second with the addition of a custom-designed data transporter. Copies of the compression module are being installed in the authors' department and in collaborating hospitals for laboratory and clinical evaluation.

Computers↗
PubMed1991

Glandular kallikrein gene expression is selectively down-regulated by glucocorticoids in pancreatic AR42J cells.

In this study we investigated the effects of steroid hormones on glandular kallikrein gene expression in the rat pancreatic acinar cell line AR42J. Using a cloned complementary DNA probe and a polyclonal antibody we demonstrated expression of a true glandular kallikrein gene and protein in AR42J cells by Western and Northern blot analysis. Dexamethasone resulted in a time-dependent parallel decrease of kallikrein messenger RNA and protein with a maximum at 12 and 72 h (30 +/- 10 and 8 +/- 0.5% of control, respectively, P less than 0.05, n = 6). In contrast, dexamethasone stimulated gene expression of two other serine proteases, chymotrypsin and trypsin, approximately 3 to 4-fold. The decrease of kallikrein concentration was dose dependent with half-maximal effects at 5 x 10(-8) M and maximal effects at 10(-7) M dexamethasone (23 +/- 6% of control, n = 3). The glucocorticoid antagonist RU 38486 blocked the glucocorticoid-induced decrease in cellular kallikrein content in a dose-dependent manner. Complete inhibition was observed at equimolar doses of dexamethasone and the antagonist. The inhibitory effect of dexamethasone was completely reversible after hormone withdrawal for 24 h. Neither estrogen, progesterone, testosterone, or aldosterone had significant effects on kallikrein expression. These data suggest that down-regulation of pancreatic kallikrein gene expression occurs selectively in response to glucocorticoids at a pretranslational level, mediated most likely by the glucocorticoid receptor.

Animals↗
PubMed1991

Prolactin (PRL) processing by kallikrein: production of the 21-23.5K PRL-like molecules and inferences about PRL storage in mature secretory granules.

We have previously described a series of C-terminally-clipped forms of PRL, the 21-23.5K PRL-like molecules (PLMs). Because we noted estrogen (E2) induction of PLMs and E2 also induces a pituitary glandular kallikrein, we have investigated the possibility that processing of PRL by kallikrein is responsible for the production of the PLMs. Subcellular fractionation of pituitaries from control or E2-treated female rats showed total kallikrein to be concentrated 1- to 4-fold and 6- to 20-fold in the granules (vs. original homogenate) from control and E2-treated animals, respectively. Cleavage of purified PRL by kallikrein resulted in the formation of large quantities of a number of the PLMs. Incubation of secretory granules derived from control or E2-treated animals under the same conditions showed no cleavage of PRL in the absence of a limiting granule membrane and a small production of the PLMs in the presence of a granule membrane. Production in the latter instance was slightly greater in granules derived from E2-treated animals. Addition of purified kallikrein to secretory granules from control or E2-treated animals in the presence or absence of granule membranes, resulted in the additional production of large amounts of only the smallest PLM (PLM 9), indicating a lack of cleavage of the two most C-terminal sites for the enzyme. Increasing the concentration of beta-mercaptoethanol to 0.64 M, to ensure monomerization of the granule PRL, had no effect on endogenous kallikrein activity of the number of products resulting from the addition of exogenous kallikrein to granules. In summary: 1) purified kallikrein can cleave purified PRL to form products which run with the same isoelectric point and mol wt values as the PLMs; 2) kallikrein is present in PRL secretory granules; 3) some PLM production occurs in a membrane-bound secretory granule fraction, but none occurs after removal of the membrane; this intragranular production, like cleavage of purified PRL with purified kallikrein, is dependent on the presence of a detergent and a reducing agent; 4) cleavage of granular PRL by exogenous kallikrein is limited to a single site and the more C-terminal sites are protected; and 5) protection of the C-terminus is not removed by intermolecular and intramolecular disulfide bond reduction. We conclude: 1) that pituitary glandular kallikrein is a strong candidate for the enzyme responsible for the production of the PLMs, and 2) that there is some element of PRL storage, other than intermolecular disulfide bonds, which involves the C-terminus of the molecule.

Animals↗