Antigen retrieval (AR) and ultra-super sensitive immunohistochemistry (ultra-IHC) have been established for application to archival human pathology specimens. The original ultra-IHC was the ImmunoMax method or the catalyzed signal amplification system (ImmunoMax/CSA method), comprising the streptavidin-biotin complex (sABC) method and catalyzed reporter deposition (CARD) reaction with visualization of its deposition. By introducing procedures to diminish non-specific staining in the original ultra-IHC method, we developed the modified ImmunoMax/CSA method with AR heating sections in an AR solution (heating-AR). The heating-AR and modified ImmunoMax/CSA method visualized expression of the predominantly simple present form of HTLV-1 proviral DNA pX region p40Tax protein (Tax) in adult T-cell leukemia/lymphoma (ATLL) cells in archival pathology specimens in approximately 75% of cases. The simple present form of Tax detected exhibited a close relation with ATLL cell proliferation. We also established a new simplified CSA (nsCSA) system by replacing the sABC method with the secondary antibody- and horse radish peroxidase-labeled polymer reagent method, introducing the pretreatments blocking non-specific binding of secondary antibody reagent, and diminishing the diffusion of deposition in the CARD reaction. Combined with AR treating sections with proteinase K solution (enzymatic-AR), the nsCSA system visualized granular immunostaining of the complex present form of Tax in a small number of ATLL cells in most cases, presenting the possibility of etiological pathological diagnosis of ATLL and suggesting that the complex present form of Tax-positive ATLL cells were young cells derived from ATLL stem cells. The heating-AR and ultra-IHC detected physiological expression of the p53 protein and its probable phosphorylation by Tax in peripheral blood mononuclear cells of peripheral blood tissue specimens from HTLV-1 carriers, as well as physiological and pathological expression of the molecules involved with G1 phase progression and G1–S phase transition (E2F-1, E2F-4, DP-1, and cyclin E) in ATLL and peripheral T-cell lymphoma cells. The ultra-IHC with AR is useful for etiological pathological diagnosis of ATLL since HTLV-1 pathogenicity depends on that of Tax, and can be a useful tool for studies translating advanced molecular biology and pathology to human pathology.
The objectives of this study were to investigate the time-course of influence of compression of bone tunnel wall in ligament reconstruction on tissue around the bone tunnel and to histologically examine the mechanism of preventing the complication of bone tunnel dilation, using rabbit tibia. A model in which the femoral origin of the extensor digitorum longus tendon was cut and inserted into a bone tunnel made proximal to the tibia was prepared in the bilateral hind legs of 20 Japanese white rabbits. In each animal, a tunnel was made using a drill only in the right leg, while an undersized bone tunnel was made by drilling and then dilated by compression using a dilator to the same tunnel size as that in the right leg. Animals were sacrificed at 0, 2, 4, 8 and 12 weeks after surgery (4 animals at each time point). Observation of bone tunnels by X-ray radiography showed osteosclerosis in the 2- and 4-week dilation groups. Osteosclerosis appeared as white lines around the bone tunnel on X-ray radiography. This suggests that dilation promotes callus formation in the bone tunnel wall and prevents the complication of bone tunnel enlargement after ligament reconstruction.
Mouse Trmt112, the homologous gene of yeast Trm112 (tRNA methyltransferase 11-2), was initially cloned from RIKEN with uncertain function. The yeast TRM112 is now known to play important roles in RNA methylation. Here, we studied the expression of Trmt112 by in situ hybridization and quantitative real-time RT-PCR (QRT-PCR). A higher expression level of Trmt112 was observed in the brain and nervous system by whole mount in situ hybridization from embryonic day 10.5 (E10.5) to E11.5. At later developmental stages E13.5 and E16.5, abundant expression was prominently found in various organs and tissues including developing brain, nervous system, thymus, lung, liver, intestine, kidney, and cartilage. Furthermore, Trmt112 was persistently expressed from E9.5 to E18.5 on whole embryos and highly expressed in multiple organs at E12.5, E15.5 and E18.5 by QRT-PCR. These results showed that Trmt112 gene was highly and ubiquitously expressed during mouse embryonic development, implying that it might be involved in the morphogenesis of diverse organs and tissues and numerous physiological functions.
A novel alanine:glyoxylate aminotransferase (AGT) mutation involved in primary hyperoxaluria type 1 (PH1) was studied in Japanese patients. Two mutations in exon 7, c.751T>A and c.752G>A, lead to a W251K amino acid substitution. Proband 1 (patient 1) was homozygous for the W251K mutation allele (DDBJ Accession No. AB292648), and AGT-specific activity in the patient’s liver was very low. To reveal the cause of the low enzymatic activity, the intracellular localization of AGT (W251K) was studied using immunohistochemistry and immunoelectron microscopy. The latter analysis showed that patient 2 had only one-fifth of the normal AGT expression per catalase, suggesting impairment of AGT (W251K) dependent transport into peroxisomes. Peroxisomal transport of human AGT is believed to be dependent on the presence of the type 1 peroxisomal targeting sequence. The C-terminal tripeptide of AGT, KKL is necessary for peroxisomal targeting. In cultured cells, EGFP-AGT (W251K) localized both in the peroxisome and cytosol. These results were consistent with the data obtained from liver analysis of patient 2. The subcellular distribution of AGT (W251K) and the results from a random mutagenesis study suggest that KKL is necessary for peroxisomal targeting of human AGT, but additional signal other than KKL may be necessary.
Alveolar macrophages are known to express a variety of growth factors and neurotrophins. Fibroblast growth factor-1 (FGF-1) is abundantly present in the lung and has mitogenic and neurotrophic activities similarly to neurotrophins. In order to determine whether FGF-1 associates with neurotrophins in alveolar macrophages, we investigated the immunocytochemical colocalization of FGF-1 with neurotrophins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3), in mouse alveolar macrophages. The results showed that 34% of macrophages were immunoreactive for FGF-1, 10% for NGF, 9% for BDNF, and 17% for NT-3. Of FGF-1-immunoreactive (IR) macrophages, 16% were immunoreactive for NT-3, but only small percentages were immunoreactive for NGF (0.8%) and for BDNF (0.3%). FGF-1 and neurotrophins were all localized in the intracellular vesicles. In the vesicles, FGF-1 and NT-3 were frequently colocalized. All macrophages expressed lysosome-associated protein-2 (LAMP-2), a late endosomal and lysosomal marker, and early endosomes antigen 1 (EEA1), an early endosomal marker. FGF-1 and NT-3 were predominantly colocalized with LAMP-2 rather than with EEA1, whereas NGF and BDNF were colocalized with EEA1 rather than with LAMP-2. These results indicate that FGF-1 and NT-3 are substantially expressed in mouse alveolar macrophages and colocalized in vesicles, predominantly in late endosomes and lysosomes.
Aquaporin 2 (AQP2) is a membrane water channel protein that traffics between the intracellular membrane compartment and the plasma membrane in a vasopressin-dependent manner in the renal collecting duct cell to control the amount of water reabsorption. We examined the relation between AQP2 internalization from the plasma membrane and caveolin-1, which is a major protein in membrane microdomain caveolae, in Mardin-Darby canine kidney cells expressing human AQP2 (MDCK-hAQP2 cells). Double-immunofluorescence microscopy showed that AQP2 is colocalized with caveolin-1 in the apical plasma membrane by stimulating the intracellular signaling cascade of vasopressin with forskolin. After washing forskolin, both AQP2 and caveolin-1 were internalized to early endosomes and then separately went back to their individual compartments, which are subapical compartments and the apical membrane, respectively. Double-immunogold electron microscopy in ultrathin cryosections confirmed the colocalization of AQP2 with caveolin-1 at caveolar structures on the apical plasma membrane of forskolin-treated cells and the colocalization within the same intracellular vesicles after washing forskolin. A co-immunoprecipitation experiment showed the close interaction between AQP2 and caveolin-1 in forskolin-treated cells and in cells after washing forskolin. These results suggest that a caveolin-1-dependent and possibly caveolar-dependent pathway is a candidate for AQP2 internalization in MDCK cells.
Many malignant epithelial tumors show increased expression of glucose transporter-1 (GLUT-1) and hexokinase II (HK-II), both of which are involved in glucose metabolism. GLUT-1 levels are often correlated with prognosis in these tumors. The current retrospective study was conducted to evaluate the importance of GLUT-1 and HK-II expression in leiomyosarcoma (LMS), a malignant uterine non-epithelial tumor with a poor prognosis. The subjects were 23 patients with stage I LMS. Expression of GLUT-1 and HK-II was evaluated immunohistochemically in samples removed surgically, and the MIB-1 index was evaluated as a measure of cell proliferation. The association of these results with prognosis was examined. Twenty samples of leiomyoma (LOM), a benign non-epithelial tumor, were used as controls. Immunohistochemical expression was defined as negative staining (–), weak to sporadic staining (1+), and strong staining (2+) per microscopic field, respectively. Malignancy was evaluated in 2000 cells and the MIB-1 index was calculated. Overall survival for LMS was estimated using the Kaplan-Meier method. Of the LMS cases, 12 were GLUT-1-positive (52.2%; 2+: 2, 1+: 10) and 15 were HK-II-positive (65.2%; 2+: 1, 1+: 14). GLUT-1 expression in LMS was significantly correlated with the MIB1 index. The 10-year survival rates were 90.9% and 58.3% in GLUT-1-negative and GLUT-1-positive cases, respectively, and 75.0% and 73.3% in HK-II-positive and HK-II-negative cases, respectively. GLUT-1 expression was significantly correlated with prognosis. Cases of stage I LMS showed a significant correlation between the expression level of GLUT-1 and the MIB-1 index, an indicator of malignancy. GLUT-1-negative cases had a better prognosis than GLUT-1-positive cases, suggesting that GLUT-1 expression is an effective prognostic marker.