Divergent expression and localization of aquaporin 5, an exocrine-type water channel,in the submandibular gland of Sprague-Dawley rats

By Western blot analysis, the expression level of aquaporin (AQP) 5 in the submandibular gland (SMG) was found to be different among individual rats of the Sprague-Dawley (SD) strain. Such differences were observed for AQP5 but not for AQP1 and consequently the SD strain was divided into two groups, one expressing a high level of AQP5 and the other a low one. The difference in average intensity of expression between the two groups was more than twofold. Immunohistochemical analysis of the SMG demonstrated that the AQP5 protein was localized in the basal and apical/lateral plasma membrane of acinar cells in rats expressing the high level of AQP5. In the rat expressing the low level, however, this channel protein was localized strongly in the apical/lateral plasma membrane, but only very weakly in the basal membrane of the acinar cells. Such a diverse localization of AQP5 was confirmed by Western blotting as well. Breeding between brother and sister was repeated for two times within high expressers and low expressers to obtain the third generation progenies (F2); the AQP5 level of the SMG in the third generation (F2 rats) from high expressers was significantly higher than the F2 from low expressers. Our present study suggests the existence of genetic variation in the expression of a water channel protein, AQP5, in rats.     

Polymerization mechanism of N-carboxy-α-amino acid anhydride by organometallic compounds. II.. Reaction with organozinc compounds

Reactions of N-carboxy-α-amino acid anhydride (NCA) with dialkylzinc or related organozinc compounds were studied to elucidate the polymerization mechanism of NCA by dialkylzinc as initiator. The first stage of initiation reaction is a hydrogen abstraction reaction of dialkylzinc from NH group of α-amino acid NCA resulting in the formation of an activated NCA. The second stage of initiation is a reaction between two molecules of the activated NCA forming a zinc carbamate group. Propagation reaction is a carbonyl addition of the zinc carbamate group to the activated NCA to form a mixed anhydride which changes into an amide group releasing carbon dioxide. Regeneration of the activated NCA is supposed to be done by the reaction of free α-amino acid NCA with the zinc atom bonded to nitrogen atom at the growing chain end.     

Functional roles of MCP-1 in Propionibacterium acnes-induced, T cell-mediated pulmonary granulomatosis in rabbits

The immunological manifestation of granuloma formations in humans largely depends on the delayed-type hypersensitivity response. We investigated the involvement of monocyte chemoattractant protein-1 (MCP-1) in a rabbit model of T cell-mediated pulmonary granulomatosis. Intravenous injection of Propionibacterium acnes (P. acnes) into sensitized rabbits induced massive and diffuse pulmonary granulomas. Levels of MCP-1 in sera and bronchoalveolar lavage fluids (BALF) peaked before the granuloma formation reached the peak (on days 1 and 3 after challenge, respectively). Chemotactic activities toward monocytes and T cells in BALF were inhibited by anti-MCP-1 IgG by 80 and 36%, respectively. The phenotypic analysis of the migrating T cells revealed that activated and memory T cells rather than naive cells were preferentially attracted to the BALF. Administration of anti-MCP-1 antiserum inhibited the development of granuloma formation in both size and number, the numbers of infiltrating leukocytes in BALF, the expression of adhesion molecules on peripheral monocytes/T cells, and on macrophages/T cells in BALF, and the production of TNF-alpha in the lung. Anti-MCP-1 resulted in a trend toward decreased level of IL-1beta in the lung. The inhibition of the production of these cytokines appeared to be induced indirectly through the inhibition of the recruitment of macrophages that produce these cytokines. The results suggest important roles of MCP-1 in the development of granuloma formation in this model through the attraction and activation of specific types of cells.     

ATP regulation of ciliary beat frequency in rat tracheal and distal airway epithelium

Ciliary beat frequency (CBF) was measured by video-optical microscopy in rat tracheal and distal airway ciliary cells using a slice preparation. In tracheal ciliary cells (tracheal slice), ATP or 2-methylthio ATP (MeSATP) increased CBF, which was inhibited by suramin (100 microm, an inhibitor of purinergic receptor). Ionomycin (5 microm) or thapsigargin (2 microm) increased CBF similarly. Ca2+-free solution or addition of Ni2+ (1 mm) decreased CBF gradually by approximately 25% and subsequent stimulation with ATP (10 microm) increased CBF transiently. The purinergic agonist experiments demonstrated that ATP increases CBF in tracheal ciliary cells via both P2X and P2Y receptors. ATP increased the intracellular calcium concentration ([Ca2+]i) in tracheal ciliary cells. However, in distal airway ciliary cells (lung slice), ATP did not increase CBF and [Ca2+]i, although a Ca2+-free solution decreased CBF, and ionomycin (5 microm) or thapsigargin (2 microm) increased it. Moreover, acetylcholine (100 microm) did not increase CBF in distal airway ciliary cells, although it increased CBF in tracheal ciliary cells. Terbutaline (10 microm), a selective beta2-adrenergic agonist, increased CBF in both tracheal and distal airway ciliary cells. These observations suggest that the Ca2+-mobilization mechanisms via purinergic or muscarinic receptors of the distal airway ciliary cell may be different from those of the tracheal ciliary cell. In conclusion, the CBF increase is differently regulated in the tracheal and distal airway epithelia of the rat.     

Effect of Bcl-2 antisense oligonucleotide on drug-sensitivity in association with apoptosis in undifferentiated thyroid carcinoma

Although attempts have been made to treat undifferentiated thyroid carcinoma using multidisciplinary therapeutic procedures including surgery, radiotherapy, and chemotherapy, the prognosis of undifferentiated thyroid carcinoma remains quite poor. New approaches to increase the sensitivity of patients to anticancer drugs and radiation will be needed to improve the survival rate for undifferentiated thyroid carcinoma. We examined the effect of Bcl-2 antisense oligonucleotide on drug-sensitivity in association with apoptosis in the 8305C undifferentiated thyroid carcinoma cell line. The drug sensitivity was evaluated by MTT assay for 48 h, while apoptosis was assessed according to the formation of internucleosomal DNA ladders. The Bcl-2 antisense was introduced into 8305C cells by using a 18-mer phosphorothioate oligonucleotide by lipopolyamine-mediated transfection twice for 12 h. The expression of apoptosis genes was assessed by Western blotting. The 8305C cells were sensitive to adriamycin (ADM), mitomycin (MMC), docetaxel (TXT), and paclitaxel (TXL), showing mean IC50 values of 0.72, 1.1, 1.3, and 4.1 microM, respectively. In contrast, the 8305C cells were resistant to cisplatin (CDDP) and 5-fluorouracil (5-FU), with mean IC50 values of 42.0 and 48.0 microM, respectively. Treatment with Bcl-2 antisense suppressed the protein level of Bcl-2 in 8305C cells in a dose-dependent manner up to 1.0 microM. Drug-sensitivity was increased by pretreatment with Bcl-2 antisense as assessed by the IC50 (x-fold): 0.48 (1.5-fold) in ADM; 0.42 (2.6-fold) in MMC, 0.56 (2.3-fold) in TXT, 1.5 (2.7-fold) in TXL, 8.6 (4.9-fold) in CDDP, and 25.0 (1.9-fold) in 5-FU, respectively. The increased drug-sensitivity was associated with the induction of apoptosis-related proteins, Fas, caspase 8, cytochrome c, caspase 3, and to subsequent apoptosis, as determined by the formation of internucleosomal DNA ladders and PARP in the treated cells. Susceptibility in apoptotic cell death following treatment with anticancer drugs was associated with induction of apoptosis-related genes in undifferentiated thyroid carcinoma cells, and induction of apoptosis was enhanced by pretreatment with Bcl-2 antisense oligonucleotide. These results imply a potential new strategy targeting an antiapoptotic protein, Bcl-2, by its antisense oligonucleotide for enhancement of chemotherapeutic efficacy in undifferentiated thyroid carcinomas.     

Ultrastructural localization of laminin-5 (gamma2 chain) in the rat peri-implant oral mucosa around a titanium-dental implant by immuno-electron microscopy

Laminin-5 (Ln-5) is an important molecule associated with epithelial cell adhesion and migration. In the gingiva around the tooth, Ln-5 localizes within basement membranes between the junctional epithelium (JE) and the tooth or connective tissue. Recently, we reported that in the oral mucosa around a dental implant, Ln-5 is expressed within the basement membranes at the implant-peri-implant epithelium (PIE) interface, and at the PIE-connective tissue interface. However, the ultrastructural localization of Ln-5 within or along the PIE has not yet been reported. Therefore, peri-implant oral mucosa was treated with anti-Ln-5 (gamma2 chain) antibody and examined using immuno-electron microscopy. Ln-5 was localized in the cells of the innermost-third layer and basal layer of the PIE. A 100-nm-wide Ln-5-positive internal basal lamina (basement membrane) and hemidesmosomes as adhesion structures were formed at the apical portion of the implant-PIE interface. However, at the upper-middle portion of the interface, these adhesion structures were not observed. Furthermore, at the PIE-connective tissue interface, the Ln-5-positive external basal lamina (basement membrane) and hemidesmosomes were partially deficient. Judging from these findings, we concluded that Ln-5 contributes to the attachment of the PIE to the titanium surface, and that PIE attached to titanium at the apical portion of the dental implant-PIE interface.     

Pneumocystis carinii carriage in immunocompromised patients with and without human immunodeficiency virus infection

Eighty-one bronchoalveolar lavage (BAL) specimens obtained from 26 HIV-infected, 45 non-HIV immunosuppressed and 10 immunocompetent patients with primary pulmonary diseases were analysed for the presence of Pneumocystis carinii by staining and by P. carinii 5S rDNA determined by PCR. P. carinii was observed by staining of BAL specimens from HIV-infected patients significantly more frequently than those from immunocompromised hosts without HIV infection (57.7% versus 20.0%, respectively). P. carinii 5S rDNA was detected by PCR assay in seven (26.9%) HIV-infected individuals, which was significantly more frequent than for four (8.9%) immunosuppressed patients without HIV infection, for whom staining was negative. None of these patients developed P. carinii pneumonia (PCP) within the follow-up period. BAL specimens from 10 immunocompetent patients with pulmonary disorders were negative for PCP by both staining and PCR assay.     

Depletion of mitochondrial DNA in HIV-1-infected patients and its amelioration by antiretroviral therapy

Mitochondrial DNA (mtDNA) of peripheral blood mononuclear cells (PBMCs) collected from Human immunodeficiency virus 1 (HIV-1)-infected patients and healthy controls were measured longitudinally using real-time polymerase chain reaction to evaluate the effects of antiretroviral agents on mtDNA synthesis in vivo and to assess the value of monitoring mtDNA in PBMCs to predict adverse events amongst these patients. MtDNA levels in PBMCs were significantly decreased in treatment-naive HIV-1-infected patients compared with healthy people. MtDNA levels were not only significantly correlated with CD4(+) T-cell count, but also inversely correlated with HIV-1 viral load. MtDNA levels in untreated patients and healthy controls were stable during the period of observation. On the other hand, amongst patients treated with regimens containing AZT/3TC or d4T/3TC, mtDNA increased during treatment and recovered to levels comparable to healthy controls. In contrast, mtDNA decreased immediately after the initiation of an AZT/ddC-containing regimen. We did not find a correlation between mtDNA levels and changes in clinical parameters. There was no significant difference in mtDNA levels between patients with and those without lipoatrophy. Furthermore, there was no obvious difference in mtDNA levels amongst those patients exhibiting signs and symptoms of peripheral neuropathy. In conclusion, the decrease in mtDNA levels in PBMCs amongst HIV-1-infected patients and its amelioration by antiretroviral therapy may suggest the influence of direct effects on mitochondria or mtDNA by HIV-1 infection. Further investigations are needed to elucidate the mechanisms contributing to decreased mtDNA and the value of mtDNA measurement in the care of HIV-1-infected individuals.