Sarcoglycan subcomplex in normal human smooth muscle: An immunohistochemical and molecular studyAnastasi, Giuseppe ; Cutroneo, Giuseppina ; Sidoti, Antonina ; Santoro, Giuseppe ; D'Angelo, Rosalia ; Rizzo, Giuseppina ; Rinaldi, Carmen ; Giacobbe, Oddone ; Bramanti, Placido ; Navarra, Giuseppe ; Amato, Aldo ; Favaloro, Angelo
doi: 10.3892/ijmm.16.3.367pmid: N/A
The sarcoglycans are transmembrane components of the dystrophin-glycoprotein complex, which links the cytoskeleton to the extracellular matrix in adult muscle fibers. Sarcoglycans seem to be functionally and pathologically as important as dystrophin. In the skeletal and cardiac muscle, the sarcoglycan subcomplex is a heterotetrameric unit composed of the transmembrane glycoproteins α-, β-, γ- and δ-sarcoglycan. A fifth sarcoglycan with significant homology to α-sarcoglycan, ε-sarcoglycan, has been identified; this sarcoglycan is expressed in both muscle and non-muscle cells. It is hypothesized that ε-sarcoglycan might replace α-sarcoglycan in smooth muscle, forming a novel sarcoglycan subcomplex consisting of ε-, β-, γ-, and δ-sarcoglycan. Recently, ζ-sarcoglycan, a novel sarcoglycan highly related to γ-sarcoglycan and δ-sarcoglycan, has been identified. On this basis, growing evidence suggests that there are two types of sarcoglycan complex; one, in skeletal and cardiac muscle, consisting of α-, β-, γ- and δ-sarcoglycan; and the other, in smooth muscle, containing β-, δ-, ζ- and ε-sarcoglycan. ε-sarcoglycan may be substituted for α-sarcoglycan in a subset of striated muscle complexes. Our results, obtained with immunofluorescence semi-quantitative analysis and molecular methods on smooth muscle biopsies of human adult gastroenteric tract, show for the first time that α-sarcoglycan fluorescence is also always detectable in smooth muscle, although its staining pattern is lower than ε-sarcoglycan. Normal α-sarcoglycan staining was detected at times, whereas there was reduced, but clearly detectable staining for ε-sarcoglycan. Moreover, γ-sarcoglycan staining is always detectable in all analyzed biopsies. On the basis of our results, we would be able to hypothesize the existence of a pentameric or, considering ζ-sarcolgycan, a hexameric arrangement of the sarcoglycan subcomplex. The hexameric sarcoglycan subcomplex, in conformity with a larger or lower expression of single sarcoglycans, could characterize skeletal, cardiac or smooth muscle, or distinct parts of gastroenteric tract. It is intriguing to integrate these results with other vascular and urogenital smooth muscle, skeletal and cardiac muscle, while also analyzing ζ-sarcoglycan.
Mucin expression and proliferating cell index of esophageal Barrett's adenocarcinomaYamamoto, Soichiro ; Kijima, Hiroshi ; Hara, Tadashi ; Chino, Osamu ; Shimada, Hideo ; Tanaka, Makiko ; Inokuchi, Sadaki ; Makuuchi, Hiroyasu
doi: 10.3892/ijmm.16.3.375pmid: N/A
Barrett's esophagus is a premalignant condition associated with gastroesophageal reflux disease, and consists of mucosa with a metaplastic columnar epithelium (specialized columnar epithelium). In this study, we examined the expression of mucin and the Ki-67 labeling index (LI) in 15 cases of esophageal Barrett's adenocarcinoma, and clarified the significance of incomplete intestinal metaplasia of Barrett's mucosa as a premalignant lesion. Gastric mucin (MUC5AC, HGM, and/or MUC6) was detected in 93.3% of the adenocarcinomas, while MUC2 and CD10 (markers of intestinal phenotypes) were detected in 73.3% and 46.2%, respectively. The Ki-67 LI was 34.1% in Barrett's adenocarcinoma. In all cases, gastric mucin was found in the non-neoplastic Barrett's mucosa around the adenocarcinoma. MUC2 was detected in 86.7% of proximal non-neoplastic mucosa and 100% of distal non-neoplastic mucosa, while CD10 was found in 20.0% of proximal non-neoplastic mucosa and 40.0% of distal non-neoplastic mucosa of Barrett's adenocarcinoma. In conclusion, Barrett's esophageal mucosa with intestinal metaplasia and a high Ki-67 LI is suggested to be more important as a premalignant lesion, and predominantly found in the proximal rather than distal region of Barrett's esophagus.
Modification of thermosensitivity by amrubicin or amrubicinol in human lung adenocarcinoma A549 cells and the kinetics of apoptosis and necrosis inductionHayashi, Sachiko ; Hatashita, Masanori ; Matsumoto, Hideki ; Jin, Zhao-Hui ; Shioura, Hiroki ; Kano, Eiichi
doi: 10.3892/ijmm.16.3.381pmid: N/A
The effects of amrubicin (AMR) and its active metabolite, amrubicinol (AMROH), on the sensitivity of human lung adenocarcinoma A549 cell line to hyperthermia at 44°C were investigated. The cell phase response as well as the kinetics of apoptosis and necrosis induction were also analyzed. The cytocidal effects of 44°C hyperthermia on A549 cells exhibited low thermosensitivity with a T0 value of 12 min. The slope of the survival curve in the exponential phase, described semilogarithmically, in 44°C hyperthermia combined treatment with AMROH (0.02 µg/ml) was approximately parallel to 44°C hyperthermia alone. The initial shoulder shape portion of the survival curve from 44°C hyperthermia alone, indicating the repair of sublethal thermal damage (SLTDR), was reduced with the sequential combined treatment of AMR or AMROH. Sequential treatments with AMR or AMROH prior to 44°C hyperthermia resulted in additive thermo-enhancement effect by reducing not only survival but was shoulder wide. Furthermore, like AMR and AMROH, adriamycin (ADM) and etoposide (VP-16) are DNA topoisomerase II inhibitors, and the effects of these 4 agents on 44°C hyperthermia were compared. All 4 agents exhibited comparable thermo-enhancement effects. Using synchronized A549 cells, AMR or AMROH did not elicit cell phase responses, irrespective of the concentration. The induction of apoptosis was investigated at 48 and 72 h after AMROH treatment, 44°C hyperthermia or the combined treatment, in which apoptosis was not significantly induced after any treatment. Furthermore, the incidence of necrosis was examined as well as apoptosis. The incidence of necrosis at 48 and 72 h after AMROH was 2.4 and 4.3%, respectively; after 44°C hyperthermia was 3.3 and 4.0%, respectively; and after the combined treatment it was 10.7 and 8.7%, respectively. The necrosis induced after the combined treatment was circa 3 times higher than that in either of the single treatments.
Deletion of the kinase domain from death-associated protein kinase attenuates p53 expression in chronic obstructive uropathyYukawa, Kazunori ; Hoshino, Katsuaki ; Kishino, Masanori ; Tsubota, Yuji ; Owada-Makabe, Kyoko ; Maeda, Masanobu ; Bai, Tao ; Tanaka, Tetsuji ; Akira, Shizuo
doi: 10.3892/ijmm.16.3.389pmid: N/A
Death-associated protein kinase (DAPK) is a Ca2+/calmodulin-dependent serine/threonine kinase that is thought to mediate apoptosis. We previously showed that the kinase domain of DAPK is crucial for the induction of renal tubular cell apoptosis in chronic obstructive uropathy (COU) caused by a unilateral ureteral ligation. Here, we used DAPK-mutant mice, generated by the deletion of 74 amino acids from the catalytic kinase domain, to investigate the role of the DAPK kinase domain in regulating the p53 level following COU. The p53 expression levels in obstructed kidneys of wild-type and mutant mice were determined during the course of COU. Western blot analysis revealed that the p53 protein levels were significantly increased at 5 days after a ureteral ligation. This increase in the p53 level was significantly attenuated in mutant kidneys compared to wild-type kidneys. The obstructed kidneys of DAPK-mutant mice showed a significantly lower number of p53-expressing renal tubule cells than wild-type mice. These results are consistent with the hypothesis that DAPK stabilizes p53 protein in response to apoptosis-inducing stimuli. Thus, the present results suggest that the DAPK kinase domain is crucial for stabilizing p53 protein in renal tubular cell apoptosis in a mouse model of COU.
Transport of maternal immunoglobulins through the human placental barrier in normal pregnancy and during inflammationBen-Hur, Herzl ; Gurevich, Pavel ; Elhayany, Asher ; Avinoach, Ilana ; Schneider, David F.; Zusman, Itshak F.
doi: 10.3892/ijmm.16.3.401pmid: N/A
We studied the effect of ascending infections of the birth canal on the transport of maternal immunoglobulins (Igs) through the placental barrier in humans. The study was performed on 41 human placentas obtained from embryos (n=21) and fetuses (n=20) who had died from different causes, including those connected with ascending infections of the birth canal, and seven placentas obtained after normal delivery at term. Different morphological and immunohistochemical methods were used. The transfer of Igs through the placental barrier is a complex process that involves tissues (trophoblast, stroma of the trophoblastic villi, and capillaries), cells (monocytes and erythroblasts) and molecular components (at least six types of transfer receptors and biologically active components). We found that the intensification of transfer of different types of maternal Igs (IgG, IgA, IgM) is accompanied by certain morphological and functional changes in the placental barrier. In normal development without infection, the transfer of IgG is steady and the process most intensive, while the transfer of IgA was evaluated in 75% of the cases, and of IgM in only 10%. Inflammation of the birth canal was accompanied by an increase in the transport of IgG in early embryogenesis, which was maintained throughout intrauterine development. In cases with moderate infection, transfer of IgG and IgA was found in all cases studied, while transfer of IgM was seen in 45% of the cases. In cases with massive infection, transfer of all three types of Igs was seen, the most intensive being of IgG and the least of IgM. Ascending infection of the birth canal changes dramatically the transport of Igs through the placenta and can be dangerous and even fatal for the embryo or fetus.
Co-expression of the complement regulatory proteins human DAF and CD59 with an IRES-mediated dicistronic mammalian vector enhances their cell protective effectsXu, Li ; Shao, Huanjie ; Wu, Wenlan ; Cao, Zhijian ; Zhao, Zhouzhou ; Liu, Hui ; Jiang, Dahe ; Mao, Xin ; Li, Wenxin
doi: 10.3892/ijmm.16.3.409pmid: N/A
The human complement regulatory proteins (hCRPs) decay accelerating factor (DAF/CD55) and protectin CD59 transfected into non-human cells could confer protection against human complement. The combination of DAF and CD59 would be an effective strategy to help overcome host complement-induced hyperacute rejection in xenotransplantation. We constructed a dicistronic mammalian expression vector pcDNA3-CD59IRESDAF by using the internal ribosomal entry sites (IRES) of the encephalomyocarditis virus (EMCV). RT-PCR, Western blotting and immunofluorescence microscopic analysis demonstrated that the EMCV IRES allowed for efficient co-expression of hCD59 and hDAF on the surface of NIH/3T3 cells transfected stably with pcDNA3-CD59IRESDAF. Human complement-mediated cytolysis assays showed that co-expressed DAF and CD59 proteins could provide more significant protection against complement-mediated cytolysis than either hCD59 or hDAF alone. These results suggest that IRES containing polycistronic vector should improve the efficiency and effectiveness of multi-gene delivery and that the construct pcDNA3-CD59IRESDAF vector has potential therapeutic value for effectively controlling complement activation and for preventing hyperacute rejection in clinical gene therapy.
Ex vivo detection of apoptotic trophoblast cells applying flow cytofluorometry and immunocytochemistry using M30 antibody directed to the cytokeratin 18 neo-epitopeKrol, Janna ; Mengele, Karin ; Öttl-Mantchenko, Irina ; Welk, Anita ; Wasilewitsch, Irina ; Pildner von Steinburg, Stephanie ; Schneider, Karl-Theodor M.; Schmitt, Manfred M.
doi: 10.3892/ijmm.16.3.415pmid: N/A
Apoptosis of placental trophoblast cells has become the subject of intensive research. Recently, a monoclonal antibody (M30) directed against a neo-epitope of cytokeratin 18, that is formed after cleavage of this cytoskeletal protein by caspases, was shown to be of advantage over other tests for the detection of trophoblast cell apoptosis. In the present study, we describe a method for the enrichment of highly pure villous trophoblast cells based on the proteolytic digestion of placental tissue, density gradient separation of dissected cells, and immunoelimination of contaminating, non-trophoblast cells employing an antibody to the HLA class I antigen. The high purity (94-99%) of the trophoblast cell preparation was shown by antibody staining for cytokeratin 7 and absence of vimentin. Furthermore, we demonstrate that after a simple permeabilization and fixation step with 90% methanol and using the M30 CytoDeath, FITC-conjugated antibody, apoptotic trophoblast cells could be distinguished from non-apoptotic cells by flow cytofluorometry in a highly quantitative and sensitive fashion. Our protocol is an improvement over previously used methods such as immunocytochemistry as it allows to differentiate rapidly between competent and apoptotic trophoblast cells by the quantitative method of flow cytofluorometry.
Effect of soy sauce on the antioxidative capacity of the gelatin gel food ‘Nikogori’ measured using the chemiluminescence methodNagatsuka, Norie ; Harada, Kazuki ; Ando, Mami ; Nagao, Keiko
doi: 10.3892/ijmm.16.3.427pmid: N/A
Oxygen-related free radicals have been suggested as a cause of aging and various diseases, for example, various cancers and rheumatoid arthritis. Because of this a radical scavenger as an antioxidant has been sought in many foods. ‘Nikogori‘ gelatin gel from cooked fish is a traditional food in Japan. Recently, ‘Nikogori’ has been noted as a readily consumed food for the elderly and those with medical problems associated with swallowing. In this study we report that the ‘Nikogori’ gelatin gels made from the collagen in various meats have a high peroxyl radical scavenging capability as the antioxidative capacity using the chemiluminescence method and the order of the strength of antioxidative capacity (IC50 value) was the Japanese common squid mantle meat (0.163%) > flatfish (marbled sole) meat (0.534%) > chicken wing meat (0.585%) > beef shin meat (0.655%) > yellowtail meat (0.659%) > chub mackerel meat (0.789%). Furthermore, when soy sauce was added to the ‘Nikogori’ gelatin gel, the antioxidative capacity increased markedly to 0.098% of IC50 value of yellowtail, 0.165% of chicken, 0.182% of flatfish and 0.252% of chub mackerel.