Bisphenol A induces apoptosis and G2-to-M arrest of ovarian granulosa cells

We investigated the impact of bisphenol A (BPA) on murine ovarian granulosa cells. Ovarian granulosa cells were cultured with 100 fM to 100 microM BPA for 24 h to 72 h. BPA decreased granulosa cell viability in a dose- and time-dependent manner. The lowest concentration that induced a significant decrease was 100 pM (89.2 +/- 4.0% of the control). TUNEL analysis demonstrated that treatment with BPA increased apoptosis of granulosa cells in a dose- and time-dependent manner. In addition, flow cytometry analyses revealed that treatment with BPA resulted in G2-to-M arrest, which was most prominent at 48 h. BPA increased the expression of Bax and concomitantly decreased the expression of Bcl2 at both protein and mRNA levels of granulosa cells. These findings suggest that low, presumably environmentally relevant doses of BPA, decrease the viability of granulosa cells by inducing apoptosis and G2-to-M arrest. Up-regulation of Bax and down-regulation of Bcl2 were suggested to be involved in this apoptotic effect.     

Establishment by the rat lymph node method of epitope-defined monoclonal antibodies recognizing the six different α chains of human type IV collagen

A group of rat monoclonal antibodies recognizing the six different chains of human type IV collagen have been established by our novel method. The method is designated the rat lymph node method in which enlarged medial iliac lymph nodes of a rat injected with an antigen emulsion via hind footpads are used as a source of B cells for cell fusion to produce hybridomas. The immunogens used were synthetic peptides having non-consensus amino acid sequences near the carboxyl termini of type IV collagen chains. Hybridomas were screened both by ELISA with synthetic peptides and by indirect immunofluorescence with cryostat sections of human kidneys. Because the epitopes of all antibodies were determined by multipin-peptide scanning, they were confirmed to be isoform-specific. They are useful for identification of chains of type IV collagen at the protein level in normal and abnormal conditions. The combined use of synthetic peptides as immunogens, the rat lymph node method as making monoclonal antibodies, and the multipin-peptide scanning as epitope mapping is found to be a strong tool for identification of peptides and proteins whose amino acid sequences are known or have been deduced.     

Effect of hepatocyte growth factor on long term hematopoiesis of human progenitor cells in transgenic-severe combined immunodeficiency mice

Hepatocyte growth factor (HGF), which was originally isolated as a liver generating factor, enhances hematopoiesis. To study the effect of HGF on hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs), we generated severe combined immunodeficiency (SCID) mice producing human (h) HGF and/or stem cell factor (SCF) by transferring the relevant genes to fertilized eggs, and then transplanted hematopoietic progenitors from human cord blood into the transgenic (Tg) SCID mice. Six months after transplantation, a significantly larger number of human cells were found in the Tg SCID mice than in non-Tg controls. Characteristically, the recipient SCID mice producing h HGF (HGF-SCID) had a significantly increased number of h CD41+ cells, whereas the SCF-SCID recipients had more CD11b+ cells. Significantly large numbers of CD34+ progenitors were found in the SCID mice transferred with both h HGF and h SCF genes (HGF/SCF-SCID) when compared with HGF-SCID or SCF-SCID mice. These results imply that HGF supports the differentiation of progenitors in megakaryocyte lineage, whereas SCF supports that in myeloid lineage. The results also imply that HGF acts on HSCs/HPCs as a synergistic proliferative factor combined with SCF. We have demonstrated the advantage of the human cytokine-producing animal in the maintenance of human HSCs.     

Titin mutations as the molecular basis for dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a heterogeneous cardiac disease characterized by ventricular dilatation and systolic dysfunction. Recent genetic studies have revealed that mutations in genes for cardiac sarcomere components lead to DCM. The cardiac sarcomere consists of thick and thin filaments and a giant protein, titin. Because one of the loci of familial DCM was mapped to the region of the titin gene, we searched for titin mutations in the patients and identified four possible disease-associated mutations. Two mutations, Val54Met and Ala743Val, were found in the Z-line region of titin and decreased binding affinities of titin to Z-line proteins T-cap/telethonin and alpha-actinin, respectively, in yeast two-hybrid assays. The other two mutations were found in the cardiac-specific N2-B region of titin and one of them was a nonsense mutation, Glu4053ter, presumably encoding for a truncated nonfunctional molecule. These observations suggest that titin mutations may cause DCM in a subset of the patients.     

Adipose-Derived Mesenchymal Stem Cells Restore Impaired Mucosal Immune Responses in Aged Mice

It has been shown that adipose-derived mesenchymal stem cells (AMSCs) can differentiate into adipocytes, chondrocytes and osteoblasts. Several clinical trials have shown the ability of AMSCs to regenerate these differentiated cell types. Age-associated dysregulation of the gastrointestinal (GI) immune system has been well documented. Our previous studies showed that impaired mucosal immunity in the GI tract occurs earlier during agingthan is seen in the systemic compartment. In this study, we examined the potential of AMSCs to restore the GI mucosal immune system in aged mice. Aged (>18 mo old) mice were adoptively transferred with AMSCs. Two weeks later, mice were orally immunized with ovalbumin (OVA) plus cholera toxin (CT) three times at weekly intervals. Seven days after the final immunization, when fecal extract samples and plasma were subjected to OVA- and CT-B-specific ELISA, elevated levels of mucosal secretory IgA (SIgA) and plasma IgG antibody (Ab) responses were noted in aged mouse recipients. Similar results were also seen aged mice which received AMSCs at one year of age. When cytokine production was examined, OVA-stimulated Peyer’s patch CD4⁺ T cells produced increased levels of IL-4. Further, CD4⁺ T cells from the lamina propria revealed elevated levels of IL-4 and IFN-γ production. In contrast, aged mice without AMSC transfer showed essentially no OVA- or CT-B-specific mucosal SIgA or plasma IgG Ab or cytokine responses. Of importance, fecal extracts from AMSC transferred aged mice showed neutralization activity to CT intoxication. These results suggest that AMSCs can restore impaired mucosal immunity in the GI tract of aged mice.     

β-Galactosidase from Ginkgo biloba seeds active against β-galactose-containing N-glycans: purification and characterization

In this study, we purified an acidic β-galactosidase to homogeneity from Ginkgo biloba seeds (β-Gal’ase Gb-1) with approximately 270-fold purification. A molecular mass of the purified β-Gal’ase Gb-1 was estimated about 35 kDa by gel filtration and 32 kDa by SDS-PAGE under non-reducing condition, respectively. On the other hand, β-Gal’ase Gb-1 produced a single band with a molecular mass of 16 kDa by SDS-PAGE under reducing condition. The N-terminal amino acid sequences of 32 kDa and 16 kDa molecules were the same and identified as H-K-A-N-X-V-T-V-A-F-V-M-T-Q-H-, suggesting that β-Gal’ase Gb-1 may function as a homodimeric structure in vivo. When complex-type N-glycans containing β-galactosyl residues were used as substrates, β-Gal’ase Gb-1 showed substantial activity for β1-4 galactosyl residue and modest activity for β1-3 galactosyl residue with an optimum pH near 5.0. Based on these results, the involvement of β-Gal’ase Gb-1 in the degradation of plant complex-type N-glycans is discussed.