The mechanism mediating regenerative intercellular Ca2+ waves in the blowfly salivary gland.
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Intercellular Ca2+ signaling in intact salivary glands of the blowfly Calliphora erythrocephala was studied by fluorimetric digital imaging combined with microinjection of putative messenger molecules. Iontophoretic injection of D-myo-inositol 1,4, 5-trisphosphate (InsP3) into salivary gland cells evoked regenerative intercellular Ca2+ waves that spread through the impaled cell and several rows of surrounding cells. Ca2+ increases induced by microinjection of Ca2+ ions were confined to the injected cells and their nearest neighbors. Depletion of intracellular Ca2+ stores by thapsigargin pre-treatment did not alter the time course of the Ca2+ increase caused by Ca2+ injection. However, activation of Ca2+ release became clearly evident when Ca2+ was injected in the presence of serotonin (5-HT). Under these conditions, injection of Ca2+ triggered intercellular Ca2+ waves that consecutively passed through >10 cells. The phospholipase C inhibitor U73122 blocked 5-HT-induced Ca2+ increases but did not affect InsP3-dependent Ca2+ spiking and intercellular Ca2+ wave propagation. The results demonstrate that propagation of agonist-evoked Ca2+ waves in the blowfly salivary gland requires supra-basal [InsP3] but does not depend on feedback activation of phospholipase C. We conclude that the intra- and intercellular transmission of these Ca2+ waves is mediated by diffusion of Ca2+ and Ca2+-induced Ca2+ release via the InsP3 receptor channel. (+info)
Clinical follow up study of 87 patients with sicca symptoms (dryness of eyes or mouth, or both).
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OBJECTIVE: To assess the prognosis of patients with sicca symptoms and to identify the clinical and immunological factors that most sensitively predict the later development of primary Sjogren's syndrome (SS) or other connective tissue diseases. METHODS: Eighty seven patients (72 female, 15 male) with sicca symptoms were re-evaluated after a median follow up time of 11 years (range 8-17). The clinical examination included ophthalmological examination (Schirmer's test, break up time and Rose-Bengal staining). Labial salivary gland biopsy was performed and histological findings graded according to the Chisholm-Mason scale. The immunoserological tests included determination of rheumatoid factor (RF), antinuclear antibodies (ANA), anti-extractable nuclear antigen-antibodies (ENA), serum immunoglobulins IgA, IgG, and IgM, and serum beta2-microglobulin (beta2m). RESULTS: At follow up 31 patients (36%) fulfilled modified Californian criteria (salivary flow measurements were not performed and Chisholm-Mason grades 3-4 were regarded as diagnostic histological findings) for possible or definite SS. Likewise, a significant progression of the histological findings was observed. Labial salivary gland re-biopsy was performed in 42 patients with grade 0-2 findings at baseline, progression to grades 3-4 being observed in 21 (50%) at follow up. The patients who later developed SS were at baseline significantly older (mean (SD) 52 (9) v 44 (14) years, p+info)
Etoposide-induced activation of c-jun N-terminal kinase (JNK) correlates with drug-induced apoptosis in salivary gland acinar cells.
(19/2115)
We have examined the ability of etoposide to induce apoptosis in two recently established rat salivary acinar cell lines. Etoposide induced apoptosis in the parotid C5 cell line as evidenced by the appearance of cytoplasmic blebbing and nuclear condensation, DNA fragmentation and cleavage of PARP. Etoposide also induced activation of c-jun N-terminal kinase (JNK) in parotid C5 cells by 4 h after treatment, with maximal activation at 8 - 10 h. Coincident with activation of JNK, the amount of activated ERK1 and ERK2 decreased in etoposide-treated parotid C5 cells. In contrast to the parotid C5 cells, the vast majority of submandibular C6 cells appeared to be resistant to etoposide-induced apoptosis. Likewise, activation of JNKs was not observed in etoposide-treated submandibular C6 cells, and the amount of activated ERK1 and ERK2 decreased only slightly. Etoposide treatment of either cell line had no effect upon the activation of p38. Treatment of the parotid C5 cells with Z-VAD-FMK, a caspase inhibitor, inhibited etoposide-induced activation of JNK and DNA fragmentation. These data suggest that etoposide may induce apoptosis in parotid C5 cells by activating JNKs and suppressing the activation of ERKs, thus creating an imbalance in these two signaling pathways. (+info)
Protein kinase C delta is essential for etoposide-induced apoptosis in salivary gland acinar cells.
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We have previously shown that parotid C5 salivary acinar cells undergo apoptosis in response to etoposide treatment as indicated by alterations in cell morphology, caspase-3 activation, DNA fragmentation, sustained activation of c-Jun N-terminal kinase, and inactivation of extracellular regulated kinases 1 and 2. Here we report that apoptosis results in the caspase-dependent cleavage of protein kinase C-delta (PKCdelta) to a 40-kDa fragment, the appearance of which correlates with a 9-fold increase in PKCdelta activity. To understand the function of activated PKCdelta in apoptosis, we have used the PKCdelta-specific inhibitor, rottlerin. Pretreatment of parotid C5 cells with rottlerin prior to the addition of etoposide blocks the appearance of the apoptotic morphology, the sustained activation of c-Jun N-terminal kinase, and inactivation of extracellular regulated kinases 1 and 2. Inhibition of PKCdelta also partially inhibits caspase-3 activation and DNA fragmentation. Immunoblot analysis shows that the PKCdelta cleavage product does not accumulate in parotid C5 cells treated with rottlerin and etoposide together, suggesting that the catalytic activity of PKCdelta may be required for cleavage. PKCalpha and PKCbeta1 activities also increase during etoposide-induced apoptosis. Inhibition of these two isoforms with Go6976 slightly suppresses the apoptotic morphology, caspase-3 activation, and DNA fragmentation, but has no effect on the sustained activation of c-Jun N-terminal kinase or inactivation of extracellular regulated kinase 1 and 2. These data demonstrate that activation of PKCdelta is an integral and essential part of the apoptotic program in parotid C5 cells and that specific activated isoforms of PKC may have distinct functions in cell death. (+info)
Defective secretion of saliva in transgenic mice lacking aquaporin-5 water channels.
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Aquaporin-5 (AQP5) is a water-selective transporting protein expressed in epithelial cells of serous acini in salivary gland. We generated AQP5 null mice by targeted gene disruption. The genotype distribution from intercross of founder AQP5 heterozygous mice was 70:69:29 wild-type:heterozygote:knockout, indicating impaired prenatal survival of the null mice. The knockout mice had grossly normal appearance, but grew approximately 20% slower than litter-matched wild-type mice when placed on solid food after weaning. Pilocarpine-stimulated saliva production was reduced by more than 60% in AQP5 knockout mice. Compared with the saliva from wild-type mice, the saliva from knockout mice was hypertonic (420 mosM) and dramatically more viscous. Amylase and protein secretion, functions of salivary mucous cells, were not affected by AQP5 deletion. Water channels AQP1 and AQP4 have also been localized to salivary gland; however, pilocarpine stimulation studies showed no defect in the volume or composition of saliva in AQP1 and AQP4 knockout mice. These results implicate a key role for AQP5 in saliva fluid secretion and provide direct evidence that high epithelial cell membrane water permeability is required for active, near-isosmolar fluid transport. (+info)
The murine cytomegalovirus chemokine homolog, m131/129, is a determinant of viral pathogenicity.
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Chemokines are important mediators of the early inflammatory response to infection and modify a wide range of host immune responses. Functional homologs of cellular chemokines have been identified in a number of herpesviruses, suggesting that the subversion of the host chemokine response contributes to the pathogenesis of these viruses. Transcriptional and reverse transcription-PCR analyses demonstrated that the murine cytomegalovirus (MCMV) chemokine homolog, m131, was spliced at the 3' end to the adjacent downstream open reading frame, m129, resulting in a predicted product of 31 kDa, which is significantly larger than most known chemokines. The in vivo impact of m131/129 was investigated by comparing the replication of MCMV mutants having m131/129 deleted (Deltam131/129) with that of wild-type (wt) MCMV. Our studies demonstrate that both wt and Deltam131/129 viruses replicated to equivalent levels during the first 2 to 3 days following in vivo infection. However, histological studies demonstrated that the early inflammatory response elicited by Deltam131/129 was reduced compared with that of wt MCMV. Furthermore, the Deltam131/129 mutants failed to establish a high-titer infection in the salivary glands. These results suggest that m131/129 possesses proinflammatory properties in vivo and is important for the dissemination of MCMV to or infection of the salivary gland. Notably, the Deltam131/129 mutants were cleared more rapidly from the spleen and liver during acute infection compared with wt MCMV. The accelerated clearance of the mutants was dependent on NK cells and cells of the CD4(+) CD8(+) phenotype. These data suggest that m131/129 may also contribute to virus mechanisms of immune system evasion during early infection, possibly through the interference of NK cells and T cells. (+info)
Lymphoma development in Sjogren's syndrome: novel p53 mutations.
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OBJECTIVE: Sjogren's syndrome (SS) is a chronic autoimmune disease characterized by lymphocytic infiltrations of the exocrine glands. Disease progression may lead to uncontrolled clonal proliferation of B lymphocytes and development of lymphoma. This study was undertaken to examine the possible involvement of the cell cycle checkpoint genes p53 and p21 in the pathophysiology of the syndrome. METHODS: Protein expression of p53 and p21 was studied, by immunohistochemistry and Western blot analysis, in minor salivary gland (MSG) biopsy specimens from 7 patients with SS and 5 control subjects. In addition, sequence analysis of the p53 gene was performed on DNA samples obtained from MSG biopsy samples of the same 7 patients with SS and from 4 patients with SS and in situ non-Hodgkin's lymphoma (NHL). RESULTS: The study revealed increased protein expression of p53 and p21 in MSG biopsy specimens from patients as compared with controls, while sequence analysis showed that the p53 gene was of the wild type. Furthermore, sequence analysis of the p53 gene from patients with SS and in situ NHL revealed 2 novel mutations in exon 5 of the p53 gene. These mutations are single-base substitutions and appear to be functional since exon 5 is included in the coding region of the p53 gene. CONCLUSION: This is the first report on wild-type p53 gene activation in SS. Our findings indicate a probable role for the DNA damage response genes in the pathogenesis of this syndrome. The novel mutations of the p53 gene implicate dysregulation of this tumor suppressor gene as a possible mechanism for lymphoma development in SS. (+info)
1.3 kilobases of the lung type I cell T1alpha gene promoter mimics endogenous gene expression patterns during development but lacks sequences to enhance expression in perinatal and adult lung.
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The T1alpha gene is one of few markers for the type I cell phenotype in the adult mammalian lung. Type I cells form a large, thin epithelial layer that facilitates gas exchange and transport of fluids between the air spaces and capillaries. The T1alpha gene has a complex pattern of developmental expression in lung and brain; in vitro studies indicate that expression is regulated in part by thyroid transcription factor 1, forkhead proteins, and Sp1/Sp3 proteins. To explore the mechanisms that confine T1alpha expression in intact adult animals to alveolar type I and choroid plexus epithelial cells, we generated mice bearing a 1.3-kb T1alpha promoter-chloramphenicol acetyltransferase (CAT) gene. In situ hybridization and RNase protection assays show that the 1.3-kb promoter confers a pattern of CAT expression that largely matches the endogenous T1alpha in embryos and mid-term fetuses in lung and central nervous system. However, the 1.3-kb promoter lacks elements important for perinatal up-regulation of T1alpha in the lung and maintenance of that expression in the adult lung and brain. The final adult pattern of T1alpha expression may be directed by elements outside the 1.3-kb fragment, perhaps those 5' to the 1.3-kb fragment as we show herein, or in 3' and intronic regions. Dev Dyn 1999;215:319-331. (+info)