We previously reported three histidine-rich conserved motifs containing HX3H, HX2H and HX2HH in the deduced primaty amino acid sequence of human sterol C-5 desaturase (SC5D), which introduces a double bond at the C5, 6 positions of ͥͥΔ7-sterols to form Δ5,7-sterols (Nishi, S. et al. (2000) Biochim. Biophys. Acta 1490, l06-l08). In the present study, the role of the 18 His residues contained in the enzyme was investigated by means of site-directed mutagenesis and expression of the mutated enzymes in an SC5D-deficient yeast strain (erg3). The activity of the recombinant SC5Ds to allow the yeast to convert ergosta-7,22-dien-3,β-ol to ergosterol (ergosta5,7,22-trien—3,β-ol) was examined directly in the erg3 strain in vivo.Individual replacements of each of the ten conserved His residues with Arg completely eliminated the desaturase activity in each case, except for one, His190. The conserved His-containing clusters have a consistent positioning with respect to the putative membrane spanning domains, and we propose that they are located on the cytoplasmic face of the endoplasmic reticulum. In contrast,mutation of eight nonconserved flanking His residues to Arg did not affect the ability to complement the erg3 phenotype. Therefore, these non-conserved His residues are not essential for the catalysis, but may contribute to the activity through conformational or other effects.Western blot analysis confirmed that the steady-state expression levels were equivalent for the wild-type human SC5D and all the mutants, supporting the idea that the conserved His residues are essential for catalytic function. A possible role for these His residues could be to act as ligands for the iron atom(s) contained in the enzyme.Hydropathy analyses indicated that the enzyme contains at least four membrane-spanning domains. Topologically, SC5D is predicted to have a C-terminal cytoplasmic domain and an N-terminal region located in the luminal side of the endoplasmic reticulum, since the N-terminal sequence was resistant to proteinase K proteolysis, but the C-terminal sequence was not.
The cellular expression of neurokinin 1 (NK1) receptor in the rat duodenal villi was studied at the levels of protein and mRNA by use of immunohistochemistly and in situ hybridization. Transmission electron microcopy was also performed to investigate the functional significance of the cells expressing NK1 receptor. Both the NK1 receptor-immunoreactivity and hybridization signals for its mRNA were localized in the fibroblast-like cells beneath the villous epithelium, indicating that NK1 receptor is produced and allocated in the cells. These cells connected with each other by their slender processes to form a well-developed network, which is probably provided with a contractile potential by the cellular characteristics including an abundance of cytoplasmic myofilaments and gap junctions between the processes, throughout the lamina propria of the villi. They were intimately associated with blood vessels and nerve fibers containing substance P. The present findings suggest that the contractile network of the fibroblast-like cells may regulate the blood flow of the vascular network in the villi by interaction between substance P released from the nerve terminals and its receptor on the cells.
Cytotoxic T lymphocytes (CTLs) play a central role in a broad spectrum of tumor immunity. Such CTLs generally recognize processed antigenic fragments in association with class I major histocompatibility complex (MHC) molecules. Thus, it is important to identify naturally processed antigens associated with class I MHC molecules to generate and activate antigen-specific CTLs. Those processed antigens fitted in the groove of class I MHC molecules are fixed by the β2-microglobulin. Mild acid elution is one method used to isolate antigenic fragments from class I MHC molecules on tumor cells by unfastening a clasp of β2-microglobulin, a critical component for stabilizing class I MHC molecules on the cell surface. Indeed, after the mild acid treatment, the expression of class I MHC molecules was temporarily down-modulated and a strong antigenic fraction for CTL recognition was obtained. To our surprise, such down- modulation of class I MHC molecule expression was also observed when the tumor cells were irradiated. Therefore, using HIV-1 gpl60 env gene transfectants, we examined the effect of X-irradiation on releasing the loaded antigenic fragments. Functional extracts were obtained from X-irradiated cell supernatants that sensitized syngeneic fibroblasts for specific CTL recognition, suggesting that X-irradiation extracts would also contain known antigenic epitopes. These results indicate that, in addition to the conventional mild acid elution treatment, X-irradiation method shown in this paper may provide a new approach for CTL-based vaccine development via isolating antigenic molecules from various tumors or virally infected cells.
Telomerase activity plays an important role in the regulation of cell senescence by limiting the number of divisions. Significant levels of telomerase activity have been detected in 85% to 90% of human cancers, however none or negligible amounts of telomerase activity were observed in benign tumors and healthy tissue. This study investigated the levels of telomerase activity in three cancer cell lines and a non-tumorigenic cell line after exposure to two endogenous metabolites respectively, namely 2-methoxyestradiol (2ME) and prostaglandin A2 (PGA2). Cells were exposed to either 1 μM 2ME or 20 μg/ml PGA2 and their relative telomerase activity (RTA) was measured by means of a combined method including the polymerase chain reaction and an enzyme—linked immunosorbent assay. PGA2 exposure caused a statistically significant decrease in the RTA of the three cancer cell lines (HeLa, WHCO3, MCF-7) (P < 0.05). In contrast, the RTA of the non-tumorigenie MCF-12A cell line was statistically significantly increased after exposure to PGA2 (P < 0.00005). The RTA of HeLa, WHCO3 and MCF-12A cells were significantly decreased after 2ME exposure (P < 0.05). These results confirm that PGA2 and 2ME could be of potential value as antitumor agents, since both exert an inhibitory activity on the tumour-associated function of the telomerase enzyme involved in cancer cell immortality.
We previously reported that overexpression of the HOXD3 homeobox gene in human erythroleukemia HEL cells gave rise to changes in cell adhesiveness. In the present study, we examined the consequences of over- or underexprcssion of HOXD3 in HEL cells for colony morphology, the ability to adhere to fibronectin, and expression of cell adhesion molecules in order to furtlicr confirm the effects attributed to varied levels of HOXD3 expression. Cells overexpressing HOXD3, which formed compact colonies composed of condensed cells, had a greater affinity for fibronectin and expressed higher levels of β3 integrin mRNA than the control cells transfected with an empty vector. Cells which did not express any HOXD3 mRNA failed to form colonies, had a low affinity for fibronectin, and expressed low levels of β3 integrin mRNA. Instead, the cells underexpressing HOXD3 exhibited higher levels of cadherin 4mRNA than the control and cells overexpressing HOXD3. These results demonstrate that the ability to form colonies and attach to fibronectin in HEL cells was dependent on levels of HOXD3 expression, and that HOXD3 served as a switch from cadherin 4 to β3 integrin gene expression.
The aims of the study were to evaluate whether inhalation anesthetics have the same mechanisms of negative inotropic action, and to compare the relationship between the calcium homeostasis and cardiac depression for each anesthetic. The effects of inhalation anesthetics on left ventricular contraction, calcium content of the sarcoplasmic reticulum (SR; as measured by caffeine-induced calcium release), and calcium content of the left ventricular muscle (as measured by atomic absorption spectroscopy) were evaluated in the isolated perfused rat heart. Inhalation anesthetics (i.e., halothane, enflurane, isoflurane, and sevoflurane) reduced both left ventricular contraction and calcium content of the SR. For all four drugs, there was a relationship between the negative inotropic effect and the decrease in calcium content of the SR. Sevoflurane maintained the calcium content of both the SR and the left ventricular muscle at a significantly higher level as compared with other anesthetics. These data suggest that, although inhalation anesthetics increase the calcium permeability of cardiac SR, their mechanisms of myocardial contractile depression are different.
Alendronate, one of the bisphosphonates, is known to have an inhibitory effect on bone resorption. The purpose of study was to investigate the effects of alendronate on ectopic bone graft resotption and to determine the optimal dose in the mouse. In control group, segments of chopped tarsal bone were immersed in saline solution and auto-grafted. in experimental groups, the same bone segments were immersed in alendronate ranging in concentration from 10-4 to 10-6 M prior to transplantation. The grafted bone in the control group disappeared due to resorption by osteoclasts within 5 weeks. In the experimental groups, the area of bone tissue decreased by only 20-40% at 5 weeks post-operatively. The transplanted bone volume in alendronate-treated groups exhibited a tendency of dose-dependent increase, being highest in the 10-4 M group. The number of osteoclasts was significantly less in the alendronate-treated groups than in the controls. These results suggested that the alendronate inhibits resorption of ectopic bone graft at a concentration of 10-4 to 10-6 M.