Biomedical subjects

S F Luo

Publications and source records attributed to S F Luo.

At least 19 recordsLinked to original sources

PubMed2001

Interleukin-1beta enhances bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilization in canine tracheal smooth-muscle cells: involvement of the Ras/Raf/mitogen-activated protein kinase (MAPK) kinase (MEK)/MAPK pathway.

Elevated levels of several cytokines including interleukin-1beta (IL-1beta) have been detected in airway fluid of asthmatic patients. Inhalation of IL-1beta induced a bronchial hyper-reactivity to contractile agonists. However, the implication of IL-1beta in the pathogenesis of bronchial hyper-reactivity is not completely understood. Therefore, we investigated the effect of IL-1beta on bradykinin (BK)-induced inositol phosphate [Ins(X)P] accumulation and Ca2+ mobilization, and up-regulation of BK receptor density in canine cultured tracheal smooth-muscle cells (TSMCs). Treatment of TSMCs with IL-1beta potentiated BK-induced Ins(X)P accumulation and Ca2+ mobilization. However, there was no effect on the Ins(X)P response induced by endothelin-1, 5-hydroxytryptamine or carbachol. Treatment with platelet-derived growth factor B-chain homodimer (PDGF-BB) also enhanced the BK-induced Ins(X)P response. These enhancements by IL-1beta and PDGF-BB might be due to an up-regulation of BK B(2) receptor density (B(max)), since [(3)H]BK binding to TSMCs was inhibited by the B(2)-selective agonist and antagonist, BK and Hoe 140, but not by B(1)-selective reagents. The enhancing effects of IL-1beta and PDGF-BB on Ins(X)P accumulation, Ca2+ mobilization and B(max) were attenuated by PD98059 [an inhibitor of activation of mitogen-activated protein kinase (MAPK) kinase, MEK] and cycloheximide (an inhibitor of protein synthesis), suggesting that IL-1beta may share a common signalling pathway with PDGF-BB via protein synthesis. Furthermore, overexpression of dominant negative mutants, H-Ras-15A and Raf-N4, significantly suppressed the up-regulation of BK receptors induced by IL-1beta, indicating that Ras and Raf may be required for activation of these kinases. These results suggest that the augmentation of BK-induced responses produced by IL-1beta might be, at least in part, mediated through activation of the Ras/Raf/MEK/MAPK pathway in TSMCs.

Animals↗

PubMed2001

Tumour necrosis factor-alpha enhances bradykinin-induced signal transduction via activation of Ras/Raf/MEK/MAPK in canine tracheal smooth muscle cells.

Inhalation of tumour necrosis factor-alpha (TNF-alpha) induced a bronchial hyperreactivity to contractile agonists. However, the mechanisms of TNF-alpha involved in the pathogenesis of bronchial hyperreactivity were not completely understood. Therefore, we investigated the effect of TNF-alpha on bradykinin (BK)-induced inositol phosphate (IP) accumulation and Ca(2+) mobilization, and up-regulation of BK receptor density in canine cultured tracheal smooth muscle cells (TSMCs). Pretreatment of TSMCs with TNF-alpha potentiated BK-induced IP accumulation and Ca(2+) mobilization. However, there was no effect on the IP response induced by endothelin-1 (ET-1), 5-hydroxytryptamine (5-HT), and carbachol. Pretreatment with PDGF B-chain homodimer (PDGF-BB) also enhanced BK-induced IP response. These enhancements induced by TNF-alpha and PDGF-BB might be due to an increase in BK B(2) receptor density (B(max)), since [3H]BK binding to TSMCs was inhibited by the B(2) selective agonist and antagonist, BK and Hoe 140, but not by the B(1) selective reagents. The enhancing effects of TNF-alpha and PDGF-BB were attenuated by PD98059 (an inhibitor of activation of MAPK kinase, MEK) and cycloheximide (an inhibitor of protein synthesis), suggesting that TNF-alpha may share a common signalling pathway with PDGF-BB via protein(s) synthesis in TSMCs. Furthermore, overexpression of dominant negative mutants, H-Ras-15A and Raf-N4, significantly suppressed p42/p44 mitogen-activated protein kinase (MAPK) activation induced by TNF-alpha and PDGF-BB and attenuated the effect of TNF-alpha on BK-induced IP response, indicating that Ras and Raf may be required for activation of these kinases. These results suggest that the augmentation of BK-induced responses produced by TNF-alpha might be, at least in part, mediated through activation of Ras/Raf/MEK/MAPK pathway in TSMCs.

Animals↗

PubMed2001

Correlation of acoustic velocity of synovial fluid with markers of inflammation in arthritic patients.

Analysis of synovial fluid is important in the evaluation and treatment of arthritic conditions. This study measured the acoustic velocity of synovial fluids in patients with degenerative joint diseases, crystal arthropathy, and other inflammatory arthropathies using the ultrasonic pulse-echo method. The measured acoustic velocities of these bio-fluids were then correlated with clinical parameters including the synovial white blood cell count (WBC), clarity, viscosity, string test, erythrocyte sedimentation rate (ESR), and serum C-reactive protein (CRP). Results showed that the acoustic velocities were correlated with ESR (p = 0.0016) and CRP (p = 0.0001). The mean acoustic velocity of inflammatory synovial fluids, defined as synovial fluids with WBCs of more than 2000/mm3, was greater than that of synovial fluids with WBCs of less than 2000/mm3 (1550 +/- 4.5 m/s vs 1544 +/- 1.5 m/s, p = 0.007). This study demonstrated that the acoustic velocity of synovial fluid correlates well with severity of inflammation. These findings suggest that measurement of acoustic velocity may be useful in the clinical evaluation and management of arthritic conditions.

Arthritis↗

PubMed2000

P2Y(2) receptor-mediated proliferation of C(6) glioma cells via activation of Ras/Raf/MEK/MAPK pathway.

1. Extracellular purine and pyrimidine nucleotides have been implicated in the regulation of several cellular functions including mitogenesis. In this study, experiments were conducted to characterize the P2Y receptor on C(6) glioma cells responsible for stimulating cell proliferation associated with mitogen-activated protein kinase (MAPK) activation. 2. UTP and ATP produced a similar effect on [(3)H]-thymidine incorporation in a time- and concentration-dependent manner, suggesting the involvement of P2Y(2) receptor in mediating proliferation of C(6) glioma cells. 3. In response to UTP, both p42 and p44 MAPK were activated in a time- and concentration-dependent manner using Western blot analysis with an anti-phospho-p42/p44 MAPK antibody. The phosphorylation reached maximal levels after 5 min and declining by 30 min. 4. Pretreatment with pertussis toxin (PTX) did not change these responses to UTP. Both DNA synthesis and phosphorylation of MAPK in response to UTP were attenuated by tyrosine kinase inhibitors, genistein and herbimycin A, protein kinase C (PKC) inhibitors, staurosporine and GF109203X, and removal of Ca(2+) by addition of BAPTA/AM plus EGTA. 5. UTP-induced [(3)H]-thymidine incorporation and p42/p44 MAPK phosphorylation was completely inhibited by PD98059 (an inhibitor of MEK1/2). Furthermore, we showed that overexpression of dominant negative mutants of Ras (RasN17) and Raf (Raf-301) completely suppressed MEK1/2 and p42/p44 MAPK activation induced by ATP and UTP. 6. These results conclude that the mitogenic effect of UTP mediated through a P2Y(2) receptor that involves the activation of Ras/Raf/MEK/MAPK pathway. UTP-mediated MAPK activation was modulated by Ca(2+), PKC, and tyrosine kinase associated with cell proliferation in cultured C(6) glioma cells.

Adenosine Triphosphate↗

PubMed2000

Tumour necrosis factor-alpha- and interleukin-1beta-stimulated cell proliferation through activation of mitogen-activated protein kinase in canine tracheal smooth muscle cells.

The elevated levels of inflammatory cytokines such as tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) have been found in the fluid of airways in symptomatic asthmatics. These cytokines have been considered as mitogens to stimulate cell proliferation in tracheal smooth muscle cells (TSMCs). We therefore investigated the effects of TNF-alpha and IL-1beta on cell proliferation and activation of p42/p44 mitogen-activated protein kinase (MAPK) in these cells. TNF-alpha and IL-1beta induced [(3)H]-thymidine incorporation in a time- and concentration-dependent manner. The maximal stimulation of [(3)H]-thymidine incorporation induced by TNF-alpha and IL-1beta was seen 12 h after incubation with these cytokines. In response to TNF-alpha and IL-1beta, p42/p44 MAPK was activated with a concentration-dependent manner in TSMCs. Pretreatment of TSMCs with pertussis toxin did not change DNA synthesis and phosphorylation of MAPK induced by TNF-alpha and IL-1beta. These responses were attenuated by a tyrosine kinase inhibitor herbimycin, a phosphatidyl choline (PC)-phospholipase C (PLC) inhibitor D609, a phosphatidyl inositide (PI)-PLC inhibitor U73122, a protein kinase C inhibitor staurosporine, and removal of Ca(2+) by addition of BAPTA/AM plus EGTA. TNF-alpha- and IL-1beta-induced [(3)H]-thymidine incorporation and phosphorylation of p42/p44 MAPK was completely inhibited by PD98059 (an inhibitor of MEK1/2), indicating that activation of MEK1/2 was required for these responses. These results suggest that the mitogenic effects of TNF-alpha and IL-1beta were mediated through the activation of MEK1/2 and p42/p44 MAPK pathway. TNF-alpha- and IL-1beta-mediated responses were modulated by PLC, Ca(2+), PKC, and tyrosine kinase associated with cell proliferation in TSMCs.

Animals↗

PubMed2000

Lipopolysaccharide enhances bradykinin-induced signal transduction via activation of Ras/Raf/MEK/MAPK in canine tracheal smooth muscle cells.

Bacterial lipopolysaccharide (LPS) was found to induce inflammatory responses and to enhance bronchial hyperreactivity to several contractile agonists. However, the implication of LPS in the pathogenesis of bronchial hyperreactivity was not completely understood. Therefore, in this study, we investigated the effect of LPS on mitogen-activated protein kinase (MAPK) activation associated with potentiation of bradykinin (BK)-induced inositol phosphates (IPs) accumulation and Ca(2+) mobilization in canine cultured tracheal smooth muscle cells (TSMCs). LPS stimulated phosphorylation of p42/p44 MAPK in a time- and concentration-dependent manner using a Western blot analysis against a specific phosphorylated form of MAPK antibody. Maximal stimulation of the p42 and p44 MAPK isoforms occurred after 7 min-incubation and the maximal effect was achieved with 100 microg ml(-1) LPS. Pretreatment of TSMCs with LPS potentiated BK-induced IPs accumulation and Ca(2+) mobilization. However, there was no effect on the IPs response induced by endothelin-1, 5-hydroxytryptamine, and carbachol. In addition, pretreatment with PDGF-BB enhanced BK-induced IPs response. These enhancements by LPS and PDGF-BB might be due to an increase in BK B(2) receptor density (B(max)) in TSMCs, characterized by competitive inhibition of [(3)H]-BK binding using B(1) and B(2) receptor-selective reagents. The enhancing effects of LPS and PDGF-BB were attenuated by PD98059, an inhibitor of MAPK kinase (MEK), suggesting that the effect of LPS may share a common signalling pathway with PDGF-BB in TSMCs. Furthermore, overexpression of dominant negative mutants, H-Ras-15A and Raf-N4, significantly suppressed p42/p44 MAPK activation induced by LPS and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. These results suggest that the augmentation of BK-induced responses produced by LPS might be, at least in part, mediated through activation of Ras/Raf/MEK/MAPK pathway in TSMCs.

Animals↗

PubMed2000

The clinical features and prognosis of lupus with disease onset at age 65 and older.

Systemic lupus erythematosus (SLE) in the elderly is uncommon and rarely reported with disease onset at age 65 and older. The aim of this study is to retrospectively analyze the influence of age at disease onset on the clinical features and prognosis of SLE. From 1988 to 1998, we encountered 21 lupus patients with disease onset at age 65 and older (all are included in group A). For comparison, 21 lupus patients with disease onset between 50-64 years of age (group B) and 152 lupus patients with disease onset before 50 years of age (group C) were obtained by a simple random sampling method from the hospital registry. Clinical features as included in the 1982 ARA revised criteria for classification of SLE and survival rate were analyzed and compared among these three groups. Group A had a smaller female to male ratio, longer duration from disease onset to diagnosis, less malar rash, more discoid lupus, and shorter survival rate that group C. There was no statistically significant difference in clinical features and survival between groups A and B, as well as between female and male patients of these two groups. The main cause of death in group A was septic shock. In conclusion, the clinical features and prognosis of SLE were influenced by the age at disease onset. However, clinical features and prognosis of SLE were similar in both late-onset lupus groups.

Adult↗

PubMed1999

Bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilization in canine cultured tracheal epithelial cells.

1. Experiments were designed to differentiate the mechanisms and subtype of kinin receptors mediating the changes in intracellular Ca2+ concentration ([Ca2+]i) induced by bradykinin (BK) in canine cultured tracheal epithelial cells (TECs). 2. BK and Lys-BK caused an initial transient peak of [Ca2+]i in a concentration-dependent manner, with half-maximal stimulation (pEC50) obtained at 7.70 and 7.23, respectively. 3. Kinin B2 antagonists Hoe 140 (10 nM) and [D-Arg0, Hyp3, Thi5,8, D-Phe7]-BK (1 microM) had high affinity in antagonizing BK-induced Ca2+ response with pKB values of 8.90 and 6.99, respectively. 4. Pretreatment of TECs with pertussis toxin (100 ng ml(-1)) or cholera toxin (10 microg ml(-1)) for 24 h did not affect the BK-induced IP accumulation and [Ca2+]i changes in TECs. 5. Removal of Ca2+ by the addition of EGTA or application of Ca2+-channel blockers, verapamil, diltiazem, and Ni2+, inhibited the BK-induced IP accumulation and Ca2+ mobilization, indicating that Ca2+ influx was required for the BK-induced responses. 6. Addition of thapsigargin (TG), which is known to deplete intracellular Ca2+ stores, transiently increased [Ca2+]i in Ca2+-free buffer and subsequently induced Ca2+ influx when Ca2+ was re-added to this buffer. Pretreatment of TECs with TG completely abolished BK-induced initial transient [Ca2+]i, but had slight effect on BK-induced Ca2+ influx. 7. Pretreatment of TECs with SKF96365 and U73122 inhibited the BK-induced Ca2+ influx and Ca2+ release, consistent with the inhibition of receptor-gated Ca2+-channels and phospholipase C in TECs, respectively. 8. These results demonstrate that BK directly stimulates kinin B2 receptors and subsequently phospholipase C-mediated IP accumulation and Ca2+ mobilization via a pertussis toxin-insensitive G protein in canine TECs. These results also suggest that BK-induced Ca2+ influx into the cells is not due to depletion of these Ca2+ stores, as prior depletion of these pools by TG has no effect on the BK-induced Ca2+ influx that is dependent on extracellular Ca2+ in TECs.

Animals↗

PubMed1999

Ankylosing spondylitis with severe chondrocalcinosis: case report.

The term chondrocalcinosis is often used to describe the radiological or pathological features of calcified joint cartilage, and it usually indicates the deposition in cartilage of calcium pyrophosphate dihydrate. The prevalence of chondrocalcinosis increases with age, with dramatic increases occurring in the decades past age 60. In younger patients with chondrocalcinosis, either clinical evidence of associated metabolic diseases leading to the chondrocalcinosis or familial disease occurrence usually can be detected. We report a 42-year-old Chinese woman with ankylosing spondylitis and arthritis involving multiple peripheral joints. Severe chondrocalcinosis was detected incidentally in this patient, however, subsequent studies revealed no associated metabolic disease or familial susceptibility and the clinical features of this patient were different from those of ankylosing chondrocalcinosis (pseudoankylosing spondylitis). The cause of chondrocalcinosis in this patient remains unknown, but joint damage and repair could have been initiating or aggravating factors of the chondrocalcinosis.

Adult↗

PubMed1999

[Hypothyroid myopathy: a case report].

Though proximal muscle weakness is characteristic of polymyositis, other agents may also lead to proximal muscle weakness, such as drugs, endocrine diseases, or infections. Here, we report a thirty year-old female suffering from proximal weakness which initially was thought to be a case of polymyositis with high serum creatine phosphokinase level. Very low thyroid hormone levels were found as the history inquiry revealed constipation, hoarseness and cold intolerance. The muscle biopsy showed no obvious inflammation. After thyroxine therapy, her muscle weakness recovered. Up to now, hypothyroid myopathy with muscle mitochondrial abnormalities shown by electron microscopic examination has not been reported in the medical literature in Taiwan. Hypothyroid myopathy, though it is rare, may be misdiagnosed as polymyositis clinically. Therefore, it is recommended that hypothyroid myopathy should be considered in the differential diagnosis of proximal muscle weakness.

Adult↗

PubMed1998

Salmonella septic arthritis in systemic lupus erythematosus and other systemic diseases.

Salmonella infection is an important problem in immunocompromised patients. The synovium is a particular metastatic focus of Salmonella infection and can result in many disabilities of life. Systemic lupus erythematosus (SLE) patients were highly susceptible to Salmonella infection. In the past 6 years, 41 patients with Salmonella septic arthritis have been treated in our hospital. Eleven patients had an underlying systemic disease of SLE which presented with a distinctive clinical course. Alcoholic liver disease (six cases) was another common underlying systemic disease. The most frequent predisposing articular factor was avascular necrosis (16 cases). The hip joint was the most commonly involved site. Salmonella group B was the most common serotype (30/41). Seventy-three per cent (8/11) of the SLE group had involvement of two or more joints compared with only three out of 30 patients in the non-SLE group. The sex differentiation shows a predominance of young females (10/11) in the SLE group and middle-aged males in the non-SLE group. Moreover, in the SLE group, all 11 patients shared the risk of lupus nephritis and steroid use. Ten patients had Salmonella group B bacteraemia and five had urinary tract infections simultaneously. In the non-SLE group, there were 10 patients with a history of steroid use, three with antecedent enteritis, 12 with bacteraemia, and three with necrotising fasciitis. Seven patients in each of the groups had a recurrent course. However, three patients in the non-SLE group had died during the episode of septic arthritis.

Adolescent↗

PubMed1998

Cloning of the American cockroach Cr-PII allergens: evidence for the existence of cross-reactive allergens between species.

BACKGROUND: Previously, we have identified the 28 and 32 kd proteins as additional important allergens from the American cockroach (Periplaneta americana) Cr-PII allergenic fraction. OBJECTIVE: The aim of this study was the cloning of P. americana Cr-PII allergens. METHODS: A lambdagt22A cDNA library constructed from P. americana mRNA was packaged into Escherichia coli Y1090 (r-), and clones recognized by murine anti-Cr-PII monoclonal antibodies and human IgE antibodies were isolated, sequenced, and subcloned into pET 21 and expressed in E. coli BL21(DE3). RESULTS: Six Cr-PII-positive clones recognized by human IgE antibodies were isolated. Two clones, C6 and C17, were sequenced, and we found encoding proteins of 228 and 274 amino acids with no cysteine or any potential N-glycosylation site, with predicted masses of 25.8 and 31.14 kd, respectively. Both molecules contain internal repeated sequences with a 94% identity between them. C6 and C17 showed 59% and 77.3% skin reactivities, respectively, on 22 cockroach-sensitive atopic patients. Both clones were found to have 28.9% to 31.8% identities to ANG12 protein, a precursor of the African malaria mosquito (Anopheles gambiae) and 82.7% to 85.1% identity to a nucleotide sequence of the German cockroach (Blattella germanica) Bla g Bd90K allergen. The anti-C6 and anti-C17 antibodies were able to recognize Cr-PII, recombinant proteins, five commercial American extracts, and two German cockroach extracts. Moreover, the binding of anti-C6 and anti-C17 antibodies to recombinant protein can be inhibited by B. germanica crude extract. Furthermore, Northern blot analyses have shown that B. germanica mRNAs could be detected by both cDNA probes. CONCLUSION: Our findings provide the first evidence of antigenic cross-reactivity between P. americana and B. germanica allergens on molecular levels. The results will be a great aid in facilitating the epitope mapping and improving diagnostic and therapeutic reagents for both cockroach species.

Allergens↗

PubMed1998

Uncoupling of bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilization by phorbol ester in canine cultured tracheal epithelial cells.

1. Regulation of the increase in inositol phosphates (IPs) production and intracellular Ca2+ concentration ([Ca2+]i by protein kinase C (PKC) was investigated in canine cultured tracheal epithelial cells (TECs). Stimulation of TECs by bradykinin (BK) led to IPs formation and caused an initial transient [Ca2+]i peak in a concentration-dependent manner. 2. Pretreatment of TECs with phorbol 12-myristate 13-acetate (PMA, 1 microM) for 30 min attenuated the BK-induced IPs formation and Ca2+ mobilization. The maximal inhibition occurred after incubating the cells with PMA for 2 h. 3. The concentrations of PMA that gave half-maximal (pEC50) inhibition of BK-induced IPs accumulation and an increase in [Ca2+]i were 7.07 M and 7.11 M, respectively. Inactive phorbol ester, 4alpha-phorbol 12,13-didecanoate at 1 microM, did not inhibit these responses. Prior treatment of TECs with staurosporine (1 microM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. 4. In parallel with the effect of PMA on the BK-induced IPs formation and Ca2+ mobilization, the translocation and down-regulation of PKC isozymes were determined. Analysis of cell extracts by Western blotting with antibodies against different PKC isozymes revealed that TECs expressed PKC-alpha, betaI, betaII, gamma, delta, epsilon, theta and zeta. With PMA treatment of the cells for various times, translocation of PKC-alpha, betaI, betaII, gamma, delta, epsilon and theta from cytosol to the membrane was seen after 5 min, 30 min, 2 h, and 4 h treatment. However, 6 h treatment caused a partial down-regulation of these PKC isozymes. PKC-zeta was not significantly translocated and down-regulated at any of the times tested. 5. Treatment of TECs with 1 microM PMA for either 30 min or 6 h did not significantly change the KD, and Bmax receptor for BK binding (control: KD=1.7+/-0.3 nM; Bmax=50.5+/-4.9 fmol/mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. 6. In conclusion, these results suggest that activation of PKC may inhibit the phosphoinositide hydrolysis and consequently attenuate the [Ca2+]i increase or inhibit independently both responses to BK. The translocation of pKC-alpha, betaI, betaII, delta, epsilon, gamma, and theta induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca2+ mobilization in TECs.

Animals↗

PubMed1998

Unusual coronary artery ectasia and stenosis in a patient with systemic lupus erythematosus and acute myocardial infarction.

Acute myocardial infarction is an important and sometimes fatal complication of systemic lupus erythematosus (SLE). We describe a case of acute myocardial infarction in a 23-year-old woman with SLE. Angiography revealed coronary ectasia in the left main and proximal circumflex coronary arteries, as well as a stenotic lesion in the left anterior descending artery. The possible pathophysiology is discussed.

Adult↗

PubMed1998

Total hip arthroplasty in Salmonella coxitis: four cases report.

Suppurative coxitis induced by salmonella is rarely reported in the literature. Furthermore, reconstruction with a total hip arthroplasty (THA) in such patients has never been reported. However, in this study, we present 4 cases; 2 normal male patients and 2 female patients with underlying systemic lupus erythematosus (SLE). The ages range from 26 to 61 years. All 4 cases revealed growth of group B salmonella from the cultures of synovial fluid. They all received THA after the infections were eradicated. The functional assessment of pain, range of motion, walking ability together with the radiographic assessment were used according to the new hip score system. After a minimum follow-up of 2 years, all of the reconstructed hips showed good to excellent results since no infections recurred during this period. Although the long-term results are uncertain, the short-term clinical advantage of THA in the treatment of quiescent salmonella coxitis is encouraging.

Adult↗

PubMed1997

Purinoceptor-stimulated phosphoinositide hydrolysis in Madin-Darby canine kidney (MDCK) cells.

Extracellular nucleotides, acting through P2-purinoceptors, have been implicated in the regulation of ion transport in epithelia, including Madin-Darby canine kidney (MDCK) cells. In this study, experiments were conducted to characterize the P2-purinoceptor subtype on MDCK cells responsible for stimulating inositol phosphate (IP) accumulation using a range of nucleotide analogues. In Ca2+- and Mg2+-free Krebs-Henseleit solution (KHS), ATP, UTP, and ATPgammaS caused an increase in IP accumulation as a function of concentration with comparable kinetics. The order of potency for the nucleotide analogues was UTP = ATPgammaS > ATP = 2-chloro ATP (Cl-ATP) >> alpha,beta-methylene ATP (alpha,beta-MeATP) = 2-methylthio ATP (2MeSATP). Selective agonists for P1-, P2X- and P2Y-purinoceptors, such as N6-cyclopentyl adenosine, AMP, alpha,beta-MeATP, and 2MeSATP, had little effect. Stimulation of MDCK cells with maximally effective concentrations of ATP and UTP showed no additive effect and furthermore, ATP, UTP, and ATPgammaS induced cross-desensitization of the IP response, suggesting that ATP and UTP act upon a common nucleotide receptor, i.e. a P2U-purinoceptor. In Ca2+- and Mg2+-containing KHS, the concentration-response curves of ATP, UTP, and ATPgammaS were shifted to the right of those obtained in Ca2+- and Mg2+-free buffer, and asymptotic maxima were not reached, indicating that ATP4- and not MgATP2- or CaATP2- was the active agonist. Pretreatment of MDCK cells with pertussis toxin (PTX) inhibited ATP- and UTP-induced IP accumulation in a concentration-dependent fashion but did not completely abolish the IP accumulation, indicating that a PTX-sensitive G protein was partially involved in the IP response. In conclusion, ATP- and UTP-stimulated IP accumulation in MDCK cells appears to be mediated through the activation of P2U-purinoceptors coupled to a G protein that is partially sensitive to PTX. A form of nucleotide uncomplexed with divalent ions such as ATP4- seems to be the preferential agonist form for the purinoceptors on MDCK cells.

Adenosine Triphosphate↗

PubMed1997

Sequencing and immunochemical characterization of the American cockroach per a 3 (Cr-PI) isoallergenic variants.

Two additional members of the American cockroach (Periplaneta americana) Per a 3 (Cr-PI) allergen, C13 and C28, were isolated and sequenced. They encoded proteins of 470 and 393 amino acids with two and no potential N-glycosylation sites, respectively. The molecular weights for C13 and C28 cloned proteins are 56,200 and 46,7000, with PI values of 7.06 and 6.54. C13 and C28 display 95.4% identity with several overlapping predicted central antigenic determinants. Both allergens were also found to have a 95% sequence homology with previously cloned C20 and share similar antigenic determinants, as defined by the structural prediction and ELISA analysis. However, the recombinant C13 and C28 allergens showed 26.3 and 94.7% skin reactivities on asthmatic patients while C20 elicited 47.4%. While no sequence similarity was found to other known allergens, these two aromatic amino acid-rich allergens were highly related to insect hemolymph proteins (28.7-36.5%), as with C20 cloned protein. Results suggest that these two are isoallergenic variants of C20. Sequence variations among isoforms, resulting a significant difference in skin reactivities, will be useful in elucidating the allergenic determinants.

Adolescent↗

PubMed1997

Effect of forskolin on bradykinin-induced calcium mobilization in cultured canine tracheal smooth muscle cells.

The effects of increases in intracellular adenosine 3':5'-cyclic monophosphate (cyclic AMP) on bradykinin (BK)-induced generation of inositol phosphates (IPs) and Ca2+ mobilization were investigated in canine cultured tracheal smooth muscle cells (TSMCs). Pretreatment of TSMCs with either forskolin or dibutyryl cyclic AMP attenuated BK-stimulated responses. The inhibitory effects of these agents produced both a depression of the maximal response and a shift to the right of the concentration-response curves of BK. The water-soluble forskolin analogue L-858051, 7-deacetyl-7 beta-(r-N-methylpiperazino)-butyryl forskolin, significantly attenuated BK-stimulated IPs accumulation, while 1,9-dideoxy forskolin, an inactive forskolin, had little effect on IPs response. Moreover, SQ-22536, 9-(tetrahydro-2-furanyl)-9-H-purin-6-amine, an inhibitor of adenylate cyclase, and both H-89, N-(2-aminoethyl)-5-isoquinolinesulfonamide, and HA-1004, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide, inhibitors of cyclic AMP-dependent protein kinase (PKA), reversed the ability of forskolin to attenuate BK-stimulated IPs accumulation. The KD and Bmax, values of the BK receptor for [3H]BK binding were not significantly changed by forskolin treatment for 30 min and 4 h. The AlF4(-)-induced IPs accumulation was attenuated by forskolin, indicating that G protein(s) are directly activated by AlF4- and uncoupled to phospholipase C by forskolin treatment. These results suggest that activation of cyclic AMP/PKA might inhibit the BK-stimulated PI breakdown and consequently reduce the [Ca2+]i increases or inhibit independently both responses, which is distal to the BK receptor in canine cultured TSMCs.

Adenine↗