Calcium pump expression in human bone and soft tissue tumours

Ninety-one cases of human bone and soft tissue tumours were studied for calcium pump expression by strepto-avidin-biotin immunohistochemical staining with a monoclonal antibody against sarcoplasmic reticulum calcium-ATPase (mAb6F5). Two out of 5 cases of embryonal rhabdomyosarcoma, 1 out of 5 cases of biphasic synovial sarcoma, 4 of 4 cases of chordoma and all of 3 chondrosarcoma cases were positive for mAb6F5. Although this novel monoclonal antibody can be used as a marker of myogenic tumours, the present positive result for endoplasmic reticulum calcium-ATPase (calcium pump) in other tumours including chordoma, chondrosarcoma and synovial sarcoma indicates a wider immunoreactivity. The findings further suggest that intracellular calcium may play an important role in cell proliferation and/or differentiation.     

Enhancement of NF-kappaB activity by resveratrol in cytokine-exposed mesangial cells

Resveratrol, a natural polyphenolic phytoalexin, has been considered as a potential anti-inflammatory agent because of its suppressive effect on nuclear factor-kappaB (NF-kappaB). However, we recently found that treatment of glomerular mesangial cells with resveratrol significantly and dose-dependently enhanced NF-kappaB activation triggered by proinflammatory cytokines. This finding was evidenced by different reporter assays as well as by expression of an endogenous NF-kappaB-dependent gene, intercellular adhesion molecule-1. The NF-kappaB promoting effect of resveratrol was also observed in renal tubular LLCPK1 cells, but not in HepG2 hepatoma cells. In all cell types tested, treatment with resveratrol alone did not affect NF-kappaB activity. The enhanced activation of NF-kappaB by resveratrol progressed for at least 24 h and was accompanied by sustained down-regulation of an endogenous NF-kappaB inhibitor, IkappaBbeta, but not IkappaBalpha. Although expression of inducible nitric oxide synthase was suppressed by resveratrol, nitric oxide, a negative regulator of NF-kappaB, was not involved in the regulation of NF-kappaB by resveratrol. These data elucidated, for the first time, that resveratrol may enhance activation of NF-kappaB under certain circumstances.     

CD8+ T cells from feline immunodeficiency virus (FIV) infected cats suppress exogenous FIV replication of their peripheral blood mononuclear cells in vitro

Summary.  Feline immunodeficiency virus (FIV) isolates from domestic cats have been classified into five subtypes, designated A, B, C, D and E. Although many FIV-infected cats may have frequent contact with multiple strains of FIV, they usually become infected with a single FIV subtype. In the present study, we demonstrate that peripheral blood mononuclear cells (PBMC) of FIV infected cats were resistant to exogenous FIV (second virus) replication in vitro and that the resistance of these PBMC was mediated by CD8⁺ T cells. In cats with a low anti-FIV activity of CD8⁺ T cells, the proviral DNA of the second virus inoculated into PBMC was detected intracellularly, and both the second and the originally infecting strain (original virus) were produced in the culture supernatant. In contrast, in cats with a high anti-FIV activity of CD8⁺ T cells, both the proviral DNA of the second virus and the original virus were detected in PBMC intracellularly, but neither virus was produced in the culture supernatant. However, when PBMCs from these cats were depleted of CD8⁺ T cells, the RNA of both viruses was detected in the culture supernatant. These results suggest that CD8⁺ T cells inhibit the late phase of FIV replication after viral integration. Moreover, the inhibition was also effective against FIV strains of different subtypes from that of the original strain. It appears that the CD8⁺ T cell-mediated immune response plays important roles in the maintenance of an asymptomatic state in FIV-infected cats and their resistance to superinfection. Received December 11, 2001; accepted March 22, 2002 Published online June 21, 2002     

Beta amyloid is focally deposited within the outer basement membrane in the amyloid angiopathy of Alzheimer''s disease. An immunoelectron microscopic study.

The fine structure of cerebral amyloid angiopathy, especially in small and presumably early deposits, was examined by immunolabeling of the beta/A4 protein in semithin and ultrathin sections from brains with Alzheimer's disease. The following findings emerged: 1) in large leptomeningeal arteries, small, focal amyloid deposits appear to consist of clusters of delicate (approximately 8 nm diameter) amyloid fibrils, not previously described, in the outermost part of the basement membrane (BM) at the media-adventitia junction; 2) in small leptomeningeal arteries and perforating cortical arterioles, small foci of delicate amyloid fibrils were observed within the BM. They appeared mostly in the outer portion of the BM, around intact smooth muscle cells, rather than in the subendothelial region. In larger and presumably more advanced deposits, coarse amyloid fibrils (approximately 10 nm) occupied the abluminal BM, and adjacent smooth muscle cells showed degeneration; and 3) in capillaries, small amounts of delicate (approximately 8 nm) amyloid fibrils, not previously described, were seen within the BM in the smallest discernible deposits. The BM at these sites was abnormally folded and layered. In larger deposits, amyloid fibrils appeared to extravasate from the outer BM of the capillary into the neuropil and were surrounded by astrocytic foot processes and/or microglia. Our results suggest that vascular amyloid fibrils may first be formed within the abluminal vascular BM, that is, outside of cells. The BM may trap degradative intermediates of the amyloid precursor protein that contain the beta/A4 region, and local proteases may then cleave them further to yield amyloidogenic fragments.     

An ambulatory respirometer (Hotmate) as a tool for screening of sleep apnea syndrome

The gold standard diagnostic method for sleep apnea syndrome(SAS) is overnight polysomnography(PSG), but is costly in terms of time and money. We studied the usefulness of a 24-hour ambulatory respirometer equipped with oximeter(Hotmate) for screening of SAS. Seventy-six cases of suspected SAS were enrolled(68 males and 8 females, mean age 51). The correlation between data from Hotmate and PSG was evaluated in 24 cases who underwent both of the tests for the final diagnosis of SAS. There was a good correlation between the two parameters of the data obtained by Hotmate(H) (H-apnea index(AI) vs H-desaturation index(DI)). Among 24 cases who underwent both Hotmate and PSG, there was a good correlation between the data from PSG and Hotmate(PSG-AI vs H-AI: r = 0.80, p < 0.001). Both sensitivity and specificity were highest when screening criteria of H-DI > 15 was utilized(sensitivity = 91.7%, specificity = 66.7%). Our findings suggest that the respiromonitor with oximeter is useful for the screening the patients with SAS.     

SF-1/Ad4BP transforms primary long-term cultured bone marrow cells into ACTH-responsive steroidogenic cells

Bone marrow stem cells develop into haematopoietic and mesenchymal lineages, but have not been known to participate in steroidogenic cell production. Steroidogenic factor 1 (SF-1), also designated adrenal 4 binding protein (Ad4BP), is an essential orphan nuclear receptor for steroidogenesis as well as for adrenal and gonadal gland development. In the present study, we revealed that the adenovirus-mediated forced expression of SF-1 can transform cultured primary long-term cultured bone marrow cells into steroidogenic cells, showing the de novo synthesis of multiple steroid hormones in response to adrenocorticotropic hormone (ACTH). This finding may provide an initial step in innovative autograft cell transfer therapy for steroid hormone deficiencies.     

Analysis of mRNA with microsomal fractionation using a SAGE-based DNA microarray system facilitates identification of the genes encoding secretory proteins

In the regulation of host defense responses such as inflammation and immunity, the secretory proteins, including membrane proteins, play central roles. Although many secretory proteins have been identified by using methods such as differential display, random screening, or the signal sequence trap method, each method suffers from poor reproducibility, low sensitivity, or time-consuming or laborious work. Therefore, the strategy for facilitating the selection of the genes encoding the secretory proteins is desired. In this paper, we describe a system for isolating the genes encoding secretory proteins by analyzing mRNAs with microsomal fractionation on serial analysis of gene expression (SAGE)-based DNA microarray system. This system succeeded in discriminating the genes encoding secretory proteins from ones encoding nonsecretory proteins with 80% accuracy. We applied this system to human T lymphocytes. As a result, we were able to identify the genes that are not only encoding secretory proteins but also expressing selectively in a specific subset of T lymphocytes. The SAGE-based DNA microarray system is a promising system to identify the genes encoding specific secretory proteins.