Screening of complementary DNA library using radiolabeled antigen.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
J Chao
Publications and source records attributed to J Chao.
Upregulation of renin-angiotensin system and downregulation of kallikrein in obstructive nephropathy.
The purpose of this study was to delineate the effects of prolonged (1 and 5 wk) unilateral ureteral obstruction (UUO) on the intrarenal renin-angiotensin and kallikrein-kinin systems in the rat. Systolic blood pressure (SBP) and plasma angiotensin (ANG) II levels were significantly higher at 1 and 5 wk of obstruction than in sham-operated groups. Also, plasma renin activity and ANG I levels were elevated at 1 wk (P < 0.05), and plasma angiotensin-converting enzyme (ACE)-kininase II activity was elevated at 5 wk (P < 0.05). Blockade of ANG II receptors with losartan (Dup 753) prevented the rise in SBP after UUO and normalized SBP in chronically hypertensive UUO rats. Renin mRNA levels and ANG II content were elevated in the obstructed kidneys at 1 and 5 wk compared with sham-operated kidneys (P < 0.05). ACE-kininase II activity was elevated in both the obstructed and contralateral kidneys at 5 wk compared with sham-operated kidneys (P < 0.05). In marked contrast to renin, total immunoreactive kallikrein contents and tissue kallikrein mRNA levels in the obstructed kidneys were reduced to 25% of sham-operated kidneys both at 1 and 5 wk (P < 0.001). The results indicate that urinary obstruction activates renin and suppresses kallikrein gene expression. Activation of ACE-kininase II by UUO also serves to enhance intrarenal ANG II generation and kinin degradation. The results implicate ANG II overproduction and kinin deficiency in the pathogenesis of UUO-induced hypertension and intrarenal vasoconstriction.
Angiotensin II↗
Tissue kallikrein and tonin levels in submandibular glands of STZ-induced diabetic rats and the effects of insulin.
The levels of tissue kallikrein, tonin, and other kallikrein-like proteinases were determined in extracts of rat submandibular glands 3 mo after the induction of diabetes with STZ (65 mg/kg i.v.). Total kallikrein-like proteinase activity was assayed catalytically with the fluorogenic substrate DVLR-AFC. Tissue kallikrein was assayed by using the same substrate in the presence of SBTI. Activity of other kallikrein-like proteinases was defined as the difference between the total kallikrein-like activity and that of tissue kallikrein. Tonin was assayed by using the substrate ZVKKR-AFC in the presence of aprotinin. Results were compared with age-matched controls and with diabetic rats that had received daily insulin injections for the last week of the test period. The results showed that all activities were significantly reduced in diabetic glands compared with controls. Insulin treatment restored concentrations of tissue kallikrein activity, whereas the activities of tonin and other kallikrein-like proteinases were unchanged. RIA supported these findings. The results indicate that in rat submandibular glands, insulin affects the synthesis of kallikrein-like proteinases in different ways and, allowing for the slowness of the processes involved, insulin may exert a direct influence on the regulation of tissue kallikrein synthesis but only have indirect effects on the synthesis of tonin and the closely related kallikrein-like proteinases.
Amino Acid Sequence↗
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↗
Lead poisoning in children.
In October 1991, the Centers for Disease Control issued new recommendations lowering the acceptable blood lead level. This change was based on data indicating that irreversible adverse neurodevelopmental effects occur in children with chronic low-level exposure to lead. The primary source of lead poisoning is still lead-based paint, particularly in older urban housing. Other sources of lead poisoning are also common. Almost all U.S. children are considered at risk of lead poisoning and should be screened for lead poisoning. Parents should be provided with information about potential sources of lead during routine well-child visits. Multidisciplinary efforts should be made to reduce all lead exposure. Oral chelation therapy on an outpatient basis is now available to treat less severe cases of lead poisoning.
Ambulatory Care↗
A prospective review system of nonurgent consultation requests in a family medicine residency practice.
BACKGROUND: This study describes a system for prospectively reviewing non-urgent consultation or referral requests in a family practice residency program. METHODS: A committee composed of four rotating family physicians met weekly during a 22-month period to review all non-urgent proposed consultations. The committee either approved consultations or made other recommendations to the primary care physician for clinical management. Data were prospectively recorded to determine what types of referrals were most commonly made and approved, and if the number of consultations changed after the system was instituted. RESULTS: The committee reviewed 930 non-urgent consultation requests. The consultation request rate was 2.5 per 100 patients seen. The number of consultation requests declined significantly over the study period. (P = .0007) Seventy-two percent of consultation requests were approved; 28% were not and resulted in recommendations for alternate management. Non-approval was most likely for referrals to specialists in podiatry, rheumatology, endocrinology, neurology, gastroenterology, cardiology, and allergy. The approval rate for consultations was not significantly related to patient age, sex, or type of health insurance. Patients with multiple consultation requests were more likely to have a prepaid health insurance plan or governmental insurance than private insurance. CONCLUSIONS: Prospective review of consultations and referrals can result in a decrease in the rate of consultations and referrals made by family physicians. Alternative management can frequently be recommended. Potential benefits of this system may include significant medical care cost savings and education for physicians.
Adolescent↗
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↗
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↗
Inhibition of rat tissue kallikrein gene family members by rat kallikrein-binding protein and alpha 1-proteinase inhibitor.
The regulation of tissue kallikrein activity by plasma serine proteinase inhibitors (serpins) was investigated by measuring the association rate constants of six tissue-kallikrein family members isolated from the rat submandibular gland, with rat kallikrein-binding protein (rKBP) and alpha 1-proteinase inhibitor (alpha 1-PI). Both these serpins inhibited kallikreins rK2, rK7, rK8, rK9 and rK10 with association rate constants in the 10(3)-10(4) M-1.s-1 range, whereas only 'true' tissue kallikrein rK1 was not susceptible to alpha 1-PI. This results in slow inhibition of rK1 by plasma serpins, which could explain why this kallikrein is the only member of the gene family identified so far that induces a transient decrease in blood pressure when injected in minute amounts into the circulation.
Animals↗
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↗
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↗
Substrate specificities of tissue kallikrein and T-kininogenase: their possible role in kininogen processing.
The present studies demonstrate the importance of subsite interactions in determining the cleavage specificities of kallikrein gene family proteinases. The effect of substrate amino acid residues in positions P3-P'3 on the catalytic efficiency of tissue kallikreins (rat, pig, and horse) and T-kininogenase was studied using peptidyl-pNA and intramolecularly quenched fluorogenic peptides as substrates. Kinetic analyses show the different effects of D-amino acid residues at P3, Pro at P'2, and Arg at either P'1 or P'3 on the hydrolysis of substrates by tissue kallikreins from rat and from horse or pig. T-Kininogenase was shown to differ from tissue kallikrein in its interactions at subsites S2, S'1, and S'2. As a result of these differences, Abz-FRSR-EDDnp with Arg at P'2 is a good substrate for tissue kallikreins from horse, pig, and rat but not for T-kininogenase. Abz-FRRP-EDDnp and Abz-FRAPR-EDDnp with Pro at P'2 (rat high molecular weight kininogen sequence) are susceptible to rat tissue kallikrein but not to tissue kallikreins from horse and pig. Arg at P'3 increased the susceptibility of the Arg-Ala bond to rat tissue kallikrein. These data explain the release of bradykinin by rat tissue kallikrein and of kallidin by tissue kallikreins from other animal species. Abz-FRLV-EDDnp and Abz-FRLVR-EDDnp (T-kininogen sequence) are good substrates for T-kininogenase but not for tissue kallikrein. Arg at the leaving group (at either P'1, P'2, or P'3) lowers the Km values of T-kininogenase while Val at P'2 increases its kcat values.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
New specific assays for tonin and tissue kallikrein activities in rat submandibular glands. Assays reveal differences in the effects of sympathetic and parasympathetic stimulation on proteinases in saliva.
At least fourteen separate bands of proteinase activity, labelled A-N, were identified by an enzyme overlay membrane technique, using oligopeptide-7-amino-4-trifluoromethylcoumarin (AFC) substrates in rat submandibular gland extracts fractionated on pH 4-6.5 isoelectric focusing gels. The proteinases were eluted into an ammonium bicarbonate buffer pH 9.8 containing 0.1% Triton X-100 and the relative contribution of each band to total activity evaluated using D-Val-Leu-Arg-AFC (DVLR-AFC) and Z-Val-Lys-Lys-Arg-AFC (ZVKKR-AFC) as substrates. Immunoblotting of band eluants run on sodium dodecyl sulphate gels with antibodies showed that band A was identical with tonin and bands K-N contained tissue kallikrein. Tonin was found to hydrolyse ZVKKR-AFC but not DVLR-AFC. Estimates of the Km values of tissue kallikrein for DVLR-AFC and tonin for ZVKKR-AFC were found to be similar (approx. 20 microM) yet the former enzyme hydrolysed its substrate five times faster. Tonin was inhibited by soybean trypsin inhibitor (SBTI) but not by aprotinin. Tissue kallikrein, on the other hand, was inhibited by aprotinin but was considerably more resistant to inhibition by SBTI. In tissue extracts 95% of the ZVKKR-AFC lytic activity in the presence of 1 microM aprotinin is due to tonin and a similar percentage of the DVLR-AFC hydrolysing activity in the presence of 10 microM SBTI is due to tissue kallikrein. These findings were used for the specific measurement of these two proteinases in submandibular gland extracts and in saliva without prior purification. Using these inhibitor based assays we revealed qualitative differences in the composition of proteinases secreted into saliva during parasympathetic versus sympathetic stimulation of the submandibular gland. The distribution of proteinases in sympathetic saliva is very similar to that found in submandibular extracts but on parasympathetic stimulation, although much less proteinase is released, the contributions of the more acidic isomers of tissue kallikrein are increased and that of tonin and other proteinases dramatically decreased. The data suggest that parasympathetic and sympathetic nerves induce proteinase secretion via different pathways.
Amino Acid Sequence↗
Determinants of tissue kallikrein cleavage specificity in the limited proteolysis of kininogens.
Further evidence for interactions at tissue kallikrein extended binding sites, as determinants of the kininogen cleavage specificities is presented. Differences in the cleavage sites in kininogen hydrolysis by rat and other tissue kallikreins is related to subsite S1' specificity, while the low susceptibility of rat kininogen to horse tissue kallikrein is explained by the difference in their subsite S3'.
Amino Acid Sequence↗
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↗
A common nomenclature for members of the tissue (glandular) kallikrein gene families.
Explore the source record for details and available documents.
Animals↗
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↗
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↗