Characterization of htAKR, a novel gene product in the aldo-keto reductase family specifically expressed in human testis

In human testis, expression of a novel member of the aldo-keto reductase family was identified. Based on its testis-specific expression, we termed this protein human testis aldo-keto reductase (htAKR). In addition to four major isoforms, the existence of multiple alternatively spliced products of htAKR was detected using RT-PCR followed by nested PCR. htAKR was a homologue of mouse liver keto-reductase, AKR1E1, with close similarity in their genomic organizations. htAKR4, the longest isoform, was expressed as a non-fused native form. It exhibited a limited activity toward 9,10-phenanthrenequinone, while no activity toward the steroids or prostaglandins was demonstrated. Using the laser capture microdissection technique and RT-PCR, expression of htAKR was detected in testicular germ cells as well as in interstitial cells. The levels of htAKR mRNA in the tissues obtained from seminoma were much lower than those in normal testes. A significant decline in the htAKR expression was observed when NEC8, a cell line originated from a human testicular germ cell tumour, was exposed to phorbol 12-myristate 13-acetate or 5alpha-dihydrotestosterone. These results indicate that the expression of htAKR, down-regulated in the testicular tumour, is possibly controlled by mitogenic and hormonal signals.     

Immunologic immaturity, but high IL-4 productivity, of murine neonatal thymic CD4 single-positive T cells in the last stage of maturation

To determine the levels of maturation and differentiation ofmurine CD4 single-positive (SP) T cells, we compared the secondaryresponses of staphylococcal enterotoxin A (SEA)-induced neonatalthymic, adult thymic and adult splenic CD4 SP T cell blastsprepared from whole or heat-stable antigen^low CD4 SP T cells.Proliferative responses upon re-stimulation with SEA were strongin adult splenic CD4 SP T cell blasts, but quite weak in neonatalthymic and adult thymic CD4 SP T cell blasts. SEA-induced IL-2production was weaker in neonatal thymic blasts than in theadult splenic CD4 SP T cell blasts. In contrast, SEA-inducedIL-4 production was high in neonatal thymic CD4 SP T cell blasts,and low in adult splenic and thymic CD4 SP T cell blasts. Expressionof GATA-3, that directs production of IL-4 in T cells, examinedat protein and mRNA levels, was higher in neonatal thymic cellsthan in adult thymic and splenic cells. These results suggestthat neonatal and adult thymic CD4 SP T cells in the final stageof maturation are relatively immature compared with adult splenicCD4 SP T cells. The cytokine production profile of neonatalthymic CD4 SP T cells suggests that they are inclined towardsa T_h2 response.     

Prevention of apoptosis of mammalian cells by the CED-3-cleaved form of CED-9

CED-9 prevents apoptosis in embryonic cells of Caenorhabditis elegans but not in mammalian cells. We show here that the prevention of apoptosis in mammalian cells requires a CED-3-cleaved form (68-280) of CED-9 which is localized in the inner mitochondrial membrane. The viability of PC12 and HeLa cells was significantly increased after death stimuli when truncated CED-9 was expressed in these cells but full-length CED-9 did not. The truncated CED-9 expressed in these cells was largely localized to the inner mitochondrial and the endoplasmic reticulum membranes, while full-length CED-9 was detected mainly in endoplasmic reticulum fractions. Moreover, truncated CED-9 in purified mitochondria was resistant to trypsin digestion, but full-length CED-9 was not. These results suggest that the CED-3-cleaved form of CED-9 prevents apoptosis in mammalian cells by localizing to the inner mitochondrial membrane.     

Immunohistochemical examination of lung cancers using monoclonal antibodies reacting with sialosylated Lewisx and sialosylated Lewisa

Summary In order to explore the relationship between the expression of cancer-associated glycolipids such as sialylated Le^x (SLEX) and sialylated Le^a (SLEA) and the histological subtypes of lung cancers, 30 cases of small cell carcinoma (SCC) and 47 cases of non-small cell carcinoma (non-SCC) were examined immunohistochemically using monoclonal antibodies reacting with SLEX and SLEA. The forty-seven cases of non-SCC included 20 cases of adenocarcinoma, 20 of squamous cell carcinoma and 7 of large cell carcinoma. Tumour cells of most non-SCCs expressed SLEX and SLEA. In adenocarcinomas, the number of tumour cells having SLEX and SLEA was more than that of squamous cell carcinomas, large cell carcinomas and SCC. In SCC, 14 of the 30 cases were found to be positive for both antigens. Although the cancer cells of 11 cases of 17 intermediate cell type SCC had both antigens, the cells of only 3 of 13 oat cell tumours expressed SLEX and SLEA. The present study shows that SLEX and SLEA are useful markers for lung adenocarcinomas, that most cases of intermediate cell type of SCCs have characteristics similar to non-SCC but that many oat cell tumours lack them.     

Accumulation of prion protein in muscle fibers of experimental chloroquine myopathy: in vivo model for deposition of prion protein in non-neuronal tissues

Prion protein (PrP) is known to accumulate in some non-neuronal tissues under conditions unrelated to prion diseases. The biochemical and biological nature of such accumulated PrP molecules, however, has not been fully evaluated. In this study, we established experimental myopathy in hamsters by long-term administration of chloroquine, and we examined the nature of the PrP molecules that accumulated. PrP accumulation was immunohistochemically demonstrated in autophagic vacuoles in degenerated muscle fibers, and this was accompanied by the accumulation of other molecules related to the neuropathogenesis of prion diseases such as clathrin, cathepsin B, heparan sulfate, and apolipoprotein J. Accumulated PrP molecules were partially insoluble in detergent solution and were slightly less sensitive to proteinase K digestion than normal cellular PrP. Muscle homogenates containing these PrP molecules did not cause disease in inoculated hamsters. The findings indicate that the PrP molecules that accumulated in muscle fibers have distinct biochemical and biological properties. Therefore, experimental chloroquine myopathy is a novel and useful model to investigate the mechanism of deposition of PrP in non-neuronal tissues and might provide new insights in the pathogenesis of prion diseases.     

Repair of infarcted myocardium mediated by transplanted bone marrow-derived CD34+ stem cells in a nonhuman primate model

Rodent and human clinical studies have shown that transplantation of bone marrow stem cells to the ischemic myocardium results in improved cardiac function. In this study, cynomolgus monkey acute myocardial infarction was generated by ligating the left anterior descending artery, and autologous CD34(+) cells were transplanted to the peri-ischemic zone. To track the in vivo fate of transplanted cells, CD34(+) cells were genetically marked with green fluorescent protein (GFP) using a lentivirus vector before transplantation (marking efficiency, 41% on average). The group receiving cells (n = 4) demonstrated improved regional blood flow and cardiac function compared with the saline-treated group (n =4) at 2 weeks after transplant. However, very few transplanted cell-derived, GFP-positive cells were found incorporated into the vascular structure, and GFP-positive cardiomyocytes were not detected in the repaired tissue. On the other hand, cultured CD34(+) cells were found to secrete vascular endothelial growth factor (VEGF), and the in vivo regional VEGF levels showed a significant increase after the transplantation. These results suggest that the improvement is not the result of generation of transplanted cell-derived endothelial cells or cardiomyocytes; and raise the possibility that angiogenic cytokines secreted from transplanted cells potentiate angiogenic activity of endogenous cells.     

Notch-RBP-J signaling is involved in cell fate determination of marginal zone B cells

RBP-J is a key mediator of Notch signaling that regulates cell fate determination in various lineages. To investigate the function of Notch-RBP-J in mature B cell differentiation, we generated mice that selectively lacked B cell RBP-J expression using conditional mutagenesis. Absence of RBP-J led to the loss of marginal zone B (MZB) cells with a concomitant increase in follicular B cells; in contrast, B1 cells in the peritoneal cavity were unaffected. Lack of RBP-J caused no defects in B cells maintenance, survival, plasma cell differentiation or activation. It is therefore likely that Notch-RBP-J signaling regulates the lineage commitment of mature B cells into follicular versus MZB cells. In addition, in mice with RBP-J-deficient B cells, had no obvious changes in immunoglobulin production in response to Ficoll, lipopolysaccharide or chicken gammaglobulin. In contrast, these mice exhibited increased mortality rates after blood-borne bacterial infection, which indicates that MZB cells play pivotal roles in the clearance of these bacteria.     

Streptococcus sobrinus antigens that react to salivary antibodies induced by tonsillar application of formalin-killed S. sobrinus in rabbits

We previously found that tonsillar application of antigen induces a strong antibody response to Streptococcus sobrinus in saliva and blood plasma. Rabbits immunized against S. sobrinus by tonsillar application were highly resistant to experimental dental caries triggered by oral inoculation of living S. sobrinus organisms with sucrose. In the present study, we examined the reaction of S. sobrinus antigens to the antibodies induced by the tonsillar application of S. sobrinus AHT-k in rabbits and compared them to those antibodies induced by intramuscular injection. In an enzyme-linked immunosorbent assay using ultrasonic fragments from mutans group streptococci, the saliva and blood plasma selectively reacted to S. sobrinus AHT-k (serotype g) and serologically related streptococci (serotypes a, d, and h) in the sixth week after tonsillar application, whereas the blood plasma in the sixth week after intramuscular injection reacted to the unrelated streptococci (serotypes b, c, e, and f) in addition to the aforementioned streptococci. The antibody reactivity induced after tonsillar application was not lost after treatment of the antigen with heat or proteinase digestion, whereas these treatments resulted in a 70% decrease of the antibody reactivity induced by intramuscular injection. The inhibition by haptenic sugars and the decrease in immunoreactivity by heat treatment and proteinase digestion suggested that 80% of the antibodies induced by tonsillar application reacted to saccharides. These saccharide antigens appeared to be involved in a specific reaction with S. sobrinus-specific streptococci and a selective reaction with serologically related streptococci. These antigens are probably involved in anticaries reactions in experimental dental caries.     

Mucosal defense against gastrointestinal nematodes: responses of mucosal mast cells and mouse mast cell protease 1 during primary strongyloides venezuelensis infection in FcRgamma-knockout mice

A possible role for the gamma subunit of immunoglobulin Fc receptors (FcR) in mucosal defenses against intestinal nematode parasites was studied using age-matched FcRgamma-knockout (FcRgamma(-/-)) and wild-type (FcRgamma(+/+)) C57BL/6 mice. Mice were infected subcutaneously with 3,000 infective larvae of Strongyloides venezuelensis, and the degree of infection was monitored by daily fecal egg counts and adult worm recovery on days 8 and 13 postinfection. Mucosal mast cell (MMC) responses were assayed by in situ intestinal mast cell counts in stained histological sections of the jejunum and by measuring mouse mast cell protease 1 (MMCP-1) release in serum using sandwich enzyme-linked immunosorbent assay. FcRgamma(-/-) mice had significantly higher egg counts (P<0.01) and numbers of adult worms (P<0.05) than FcRgamma(+/+) mice, but mastocytosis and serum MMCP-1 release were comparable. It was concluded that MMCP-1 release may be spontaneous, does not depend on mast cell degranulation via the FcRgamma signaling system, and appears to play no role in the expulsion of S. venezuelensis. The delay in worm expulsion in the FcRgamma(-/-) mice might be related to inability of the MMC to degranulate and release effector molecules other than MMCP-1, since FcRgamma deletion abrogates mast cell degranulative responses.