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Selected AbstractsProteasome-driven turnover of tryptophan hydroxylase is triggered by phosphorylation in RBL2H3 cells, a serotonin producing mast cell lineFEBS JOURNAL, Issue 19 2002Yoshiko Iida We previously demonstrated in mast cell lines RBL2H3 and FMA3 that tryptophan hydroxylase (TPH) undergoes very fast turnover driven by 26S-proteasomes [Kojima, M., Oguro, K., Sawabe, K., Iida, Y., Ikeda, R., Yamashita, A., Nakanishi, N. & Hasegawa, H. (2000) J. Biochem (Tokyo) 2000, 127, 121,127]. In the present study, we have examined an involvement of TPH phosphorylation in the rapid turnover, using non-neural TPH. The proteasome-driven degradation of TPH in living cells was accelerated by okadaic acid, a protein phosphatase inhibitor. Incorporation of 32P into a 53-kDa protein, which was judged to be TPH based on autoradiography and Western blot analysis using anti-TPH serum and purified TPH as the size marker, was observed in FMA3 cells only in the presence of both okadaic acid and MG132, inhibitors of protein phosphatase and proteasome, respectively. In a cell-free proteasome system constituted mainly of RBL2H3 cell extracts, degradation of exogenous TPH isolated from mastocytoma P-815 cells was inhibited by protein kinase inhibitors KN-62 and K252a but not by H89. Consistent with the inhibitor specificity, the same TPH was phosphorylated by exogenous Ca2+/calmodulin-dependent protein kinase II in the presence of Ca2+ and calmodulin but not by protein kinase A (catalytic subunit). TPH protein thus phosphorylated by Ca2+/calmodulin-dependent protein kinase II was digested more rapidly in the cell-free proteasome system than was the nonphosphorylated enzyme. These results indicated that the phosphorylation of TPH was a prerequisite for proteasome-driven TPH degradation. [source] A case of mucosal leishmaniasis: beneficial usage of polymerase chain reaction for diagnosisINTERNATIONAL JOURNAL OF DERMATOLOGY, Issue 12 2001Hironori Onuma MD A 36-year-old woman, who had emigrated from Japan to Paraguay as a 4-year-old child before returning to Japan in 1991, visited our clinic on November 10, 1997. She had suffered from a persistent ulcer on her forearm as a 6-year-old child and received intravenous injections for a few months, although she did not remember the details of therapy. Since May 1997, she had been aware of redness and swelling on her nose and had been treated with topical corticosteroid, but no improvement had been noted. Physical examination revealed erythematous plaque with crust from the left internal naris to nasolabial region (Fig. 1a). The atrophic plaque that had resulted from prolonged ulceration was found on the right forearm (Fig. 1b). In a biopsy specimen from the erythematous plaque on the nasolabial region, mononuclear dermal infiltrate, consisting of lymphocytes and histiocytes, was seen (Fig. 2a). The histiocytes were filled with Leishman-Donovan (L-D) bodies on a Giemsa staining sample (Fig. 2b). Fiberscopic examination revealed white plaque in the pharynx. The biopsy from the affected mucosa showed the same histopathological finding as with the skin. Figure 1. (a) Erythematous plaque with crust from the left internal naris to nasolabial region. (b) Atrophic plaque on the right forearm Figure 2. (a) In the biopsy specimen from the erythematous plaque on the nasolabial region, a mononuclear dermal infiltrate consisting of lymphocytes and histiocytes was seen. (Hematoxylin-Eosin stain, × 100) (b) The histiocytes were filled with Leishman-Donovan bodies. (Giemsa staining, × 400) Total DNA was purified from the skin biopsy specimen for polymerase chain reaction (PCR) analysis using a specific primer for L (V) braziliensis.1,2 A 70-bp product was amplified (Fig. 3a); furthermore, the specificity of the PCR product was confirmed by Southern hybridization with the probe for L (V) braziliensis (Fig. 3b) and DNA sequence analysis (data not shown). From December 2, 1997, the patient received 20 mg/kg/day sodium stibogluconate (PentostamTM) intravenously for 20 days. After 5 days of treatment, the redness and swelling of the skin lesion was improved, and faint erythema remained at the end of 20 days' treatment. After a 2-week interval, since the erythema remained, another 20-day treatment was performed. All of the skin lesion became scar tissue and L-D bodies could not be found in a skin biopsy specimen. However, L-D bodies were still found in a biopsy from the pharyngeal mucosa that had a normal appearance. Though another additional treatment was planned, the patient refused it. Figure 3. (a) The results of PCR. 70-bps bands appear in lanes 2 and 6. Lane 1, a size marker (pUC19/HapII); lane 2, DNA extracted from the formalin-fixed patient's sample; lane 3, DNA extracted from a formalin-fixed control sample; lane 4, DNA (,); lane 5, DNA extracted from L (V) tropica; lane 6, DNA extracted from L (V) braziliensis. (b) Results of Southern blotting using the PCR products. The PCR products were transferred from agarose gel as shown in Fig. 3 (a). Specific probes were hybridized with 70-bps bands on lanes 2 and 6 [source] The increase in the frequency of MICA gene A6 allele in oral squamous cell carcinomaJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 6 2002Liu Chung-Ji Abstract Background: Oral squamous cell carcinoma (OSCC) was reported to be associated with immune function. The MICA (MHC class I chain-related gene A) is expressed by keratinocytes and other epithelial cells, and its encoded protein interacts with ,/, T cells localized in submucosa. The MICA also influences the heat shock protein function. We speculated that the alterations of MICA might influence the pathogenesis of OSCC through the aberration in presenting tumor antigens or heat shock protein. MICA gene has a triplet repeat (GCT) polymorphism in the transmembrane domain, resulting in five distinctive allelic patterns. Methods: We analysed this MICA polymorphism in 67 OSCC patients and 351 randomly selected unrelated controls. By using the ABI Prism 377,18 DNA sequencer (Applied Biosystems, Foster City, CA, USA) to analyse the sample DNA PCR products. The number of micro-satellite repeats was estimated with Genescan 672 software (Applied Biosystems) with a standard size marker of GS-350 TAMRA (N,N,N,N-tetramethyl-6-carbohydroxyl rhodamine; Applied Biosystems). Results: The phenotype frequency of allele A6 of MICA in subjects with OSCC was significantly higher than that in controls (RR = 3.46, 95% CI = 1.73,6.94, P = 0.0002), as was the frequency of allele (RR = 2.64, 95% CI = 1.39,5.02, P = 0.002). Conclusion: The results suggest that allele A6 in MICA might confer the risk of OSCC. [source] DNA fragment analysis by an affordable multiple-channel capillary electrophoresis systemELECTROPHORESIS, Issue 1-2 2003Ming S. Liu Abstract We are demonstrating a cost-effective multichannel capillary electrophoresis system for a high-efficiency double-stranded DNA (dsDNA) fragments analysis. This bench-type high-performance DNA analysis (HDAÔ) system uses fluorescence-type detection with inexpensive solid-state light sources and nonmoving integrated emission collection micro-optics. DNA samples are analyzed simultaneously by using a multiple usage and disposable multicapillary cartridge, which contains integrated capillary channels, optical fibers and an integrated sieving gel reservoir. Using commercially available dsDNA size markers as indicators, the HDAÔ system provides high resolving power in 7 min separations. The system can hold a total of 192 samples in two 96-well polymerase chain reaction (PCR) plates, which can be automatically analyzed within 2.5 h. This affordable system can be used in laboratories to replace slab gel electrophoresis for routine and high-throughput dsDNA analysis. [source] | ||||||||||