Splenocyte proliferation and anaphylaxis induced by BSA challenge in a D-galactose-induced aging mouse model.

We previously found a cross-reactive autoantibody that bound to bovine serum albumin generated in a D-galactose-induced aging mouse model. Also, we confirmed that other reducing sugars (glucose and fructose) could induce the formation of autoantibody, and only following subcutaneous injection, not oral or intraperitoneal administration. Mice that had never been exposed to bovine serum albumin produced an anti-bovine serum albumin autoantibody following repeated subcutaneous injection of D-galactose (D-gal). In this study, we investigated the involvement of the adaptive immune system in the production of this autoantibody. In particular, we examined bovine serum albumin-induced splenocyte proliferation and bovine serum albumin-induced active cutaneous and systemic anaphylaxis in D-gal-treated mice. We find our results particularly interesting: bovine serum albumin stimulates splenocyte proliferation and induces both active cutaneous and systemic anaphylaxis in D-gal-treated mice. In summary, our results suggest that adaptive immune response participates in the autoantibody formation against bovine serum albumin in D-gal-treated mice.

The production of cross-reactive autoantibodies that bind to bovine serum albumin in mice administered reducing sugars by subcutaneous injection.

INTRODUCTION: In a previous study, we identified the formation of cross-reactive autoantibodies that bound to bovine serum albumin (BSA) in a D-galactose-induced aging mouse model. AIM OF THE STUDY: In this study, we investigated the effect of other reducing sugars (namely, glucose and fructose) on the formation of autoantibodies. The effects of concentration and route of administration on the formation of autoantibodies were examined in detail. MATERIAL AND METHODS: Three concentrations (100, 500, and 1,000 mg/kg) of reducing sugars were tested. The effects of different routes of administration (subcutaneous, oral, and intraperitoneal) on the formation of autoantibodies were also analysed. The immunoreactivities of serum samples from mice treated with reducing sugars were analysed by an enzyme-linked immunosorbent assay (ELISA) using BSA or mouse serum albumin antigens (MSA). RESULTS: Repeated subcutaneous administration of all reducing sugars lead to autoantibody formation in a concentration-dependent manner. However, these autoantibodies did not cross-react with MSA, and simultaneous treatment of aminoguanidine with reducing sugars did not show any inhibitory effects on the formation of autoantibodies. No autoantibodies were detected after oral or intraperitoneal administration of reducing sugars. Immunohistochemistry data showed that the target antigen(s) of the autoantibodies were present only in the skin tissue of mice treated with reducing sugars. CONCLUSIONS: Our results show that administration of reducing sugars by subcutaneous injection leads to the formation of autoantibodies that cross-react with BSA; the formation and target antigen(s) of the autoantibodies may originate from within the skin tissue treated with the reducing sugars.

Polycystic ovary syndrome (PCOS)-like phenotypes in the d-galactose-induced aging mouse model.

The D-galactose (D-gal)-induced animal model, which is established by consecutive subcutaneous d-gal injections for approximately 6weeks, has been frequently used for aging research. This animal model has been shown to accelerate aging of the brain, kidneys, liver, and blood cells. However, aging of the female reproductive organs in this animal model has not been reported. The aim of this study was to investigate changes in the ovary in the d-gal-induced aging mouse model. First, we evaluated anti-Müllerian hormone (AMH) as a marker of ovarian aging in blood plasma. We speculated there would be lower AMH levels in d-gal-treated mice because ovarian aging would be induced by d-gal, as reported for other tissues. However, the results showed that AMH levels in d-gal-treated mice were approximately four-fold higher than control mice. Abnormally high AMH levels are detected in ovarian cancer and polycystic ovary syndrome (PCOS) patients. Therefore, we examined PCOS-related markers in this mouse model. Total testosterone levels were high and abnormal estrous cycles were induced in d-gal-treated mice. These changes, including AMH levels, in d-gal-treated mice were inhibited by aminoguanidine treatment, an advanced glycation end product reducer. In addition, ovarian cysts were observed in some d-gal-treated mice. These results indicate that with respect to female reproduction, d-gal-treated mice are suitable for PCOS studies, rather than aging studies.

Bone marrow-derived circulating progenitor cells fail to transdifferentiate into adipocytes in adult adipose tissues in mice.

Little is known about whether bone marrow-derived circulating progenitor cells (BMDCPCs) can transdifferentiate into adipocytes in adipose tissues or play a role in expanding adipocyte number during adipose tissue growth. Using a mouse bone marrow transplantation model, we addressed whether BMDCPCs can transdifferentiate into adipocytes under standard conditions as well as in the settings of diet-induced obesity, rosiglitazone treatment, and exposure to G-CSF. We also addressed the possibility of transdifferentiation to adipocytes in a murine parabiosis model. In each of these settings, our findings indicated that BMDCPCs did not transdifferentiate into either unilocular or multilocular adipocytes in adipose tissues. Most BMDCPCs became resident and phagocytic macrophages in adipose tissues--which resembled transdifferentiated multilocular adipocytes by appearance, but displayed cell surface markers characteristic for macrophages - in the absence of adipocyte marker expression. When exposed to adipogenic medium in vitro, bone marrow cells differentiated into multilocular, but not unilocular, adipocytes, but transdifferentiation was not observed in vivo, even in the contexts of adipose tissue regrowth or dermal wound healing. Our results suggest that BMDCPCs do not transdifferentiate into adipocytes in vivo and play little, if any, role in expanding the number of adipocytes during the growth of adipose tissues.

Angiogenic role of LYVE-1-positive macrophages in adipose tissue.

Here we report the discovery of a characteristic dense vascular network (DVN) in the tip portion of epididymal adipose tissue in adult mice. The DVN is formed by angiogenesis rather than by vasculogenesis, and has functional blood circulation. This DVN and its subsequent branching may provide a new functional route for adipogenesis. The recruitment, infiltration, and accumulation of bone marrow-derived LYVE-1(+) macrophages in the tip region are crucial for the formation of the DVN. Matrix metalloproteinases (MMPs) and the VEGF-VEGFR2 system are responsible not only for the formation of the DVN, but also for the recruitment and infiltration of LYVE-1(+) macrophages into the epididymal adipose tissue tip region. SDF-1, but not the MCP-1-CCR2 system, is a critical factor in recruitment and ongoing retention of macrophages in this area. We also demonstrate that the tip region of epididymal adipose tissue is highly hypoxic, and thus provides a microenvironment conducive to the high expression and enhanced activities of VEGF, VEGFR2, MMPs, and SDF-1 in autocrine and paracrine manners, to create an ideal niche for the recruitment, retention, and angiogenic action of macrophages. These findings shed light on the complex interplay between macrophage infiltration, angiogenesis, and adipogenesis in the tip region of adult epididymal adipose tissue, and provide novel insight into the regulation of alternative outgrowth of adipose tissue.

Oocyte-based screening of cytokinesis inhibitors and identification of pectenotoxin-2 that induces Bim/Bax-mediated apoptosis in p53-deficient tumors.

In this study, we demonstrate that a loss of p53 sensitizes tumor cells to actin damage. Using a novel oocyte-based screening system, we identified natural compounds that inhibit cytokinesis. Among these, pectenotoxin-2 (PTX-2), which was first identified as a cytotoxic entity in marine sponges, which depolymerizes actin filaments, was found to be highly effective and more potent to activate an intrinsic pathway of apoptosis in p53-deficient tumor cells compared to those with functional p53 both in vitro and in vivo. Other agents that depolymerize or knot actin filaments were also found to be toxic to p53-deficient tumors. In p53-deficient cells, PTX-2 triggers apoptosis through mitochondrial dysfunction, and this is followed by the release of proapoptotic factors and caspase activation. Furthermore, we observed Bax activation and Bim induction only in p53-deficient cells after PTX-2 treatment. RNA interference of either Bim or Bax resulted in the inhibition of caspases and apoptosis induced by PTX-2. However, the small interfering RNAs (SiRNA) of Bim blocked a conformational change of Bax, but Bax SiRNA did not affect Bim expression. Therefore, these results suggest that Bim triggers apoptosis by activating Bax in p53-deficient tumors upon actin damage, and that actin inhibitors may be potent chemotherapeutic agents against p53-deficient tumors.

Subcutaneous administration of monosodium glutamate to pregnant mice reduces weight gain in pups during lactation.

Administering monosodium glutamate (MSG) to neonatal rodents induces obesity and type 2 diabetes. In addition, several studies have shown that MSG administered to pregnant animals can cross the placenta and reach the foetus. The present study was performed to investigate the effects of administering MSG to pregnant ICR mice on dam and neonatal growth. Pregnant mice were treated with 60 or 120 mg MSG once daily from day 5 of pregnancy to one day before parturition by subcutaneous injection. In addition, the body weights of the neonates were determined until nine weeks of age. The birth weights of neonates were not different between the control and MSG-treated groups. However, MSG treatment resulted in a lower body weight gain of neonates during lactation. In addition, this underweight of the MSG-treated group at weaning returned to normal compared with the control group at five weeks of age. Cross-fostering experiments indicated that the lower body weight gain of neonates in the MSG-treated group during lactation was due to its effects on the dam. Serum prolactin levels and mammary gland development of the mice were examined next to determine the reasons for this lactation problem. Although there were no differences in prolactin levels, morphological analyses of the mammary glands revealed apparent differences, including low numbers and altered phenotype of alveoli, between the control and MSG-treated groups. Taken together, our results show that treating pregnant mice with excess MSG induced lower neonate body weight gain during lactation.

A phenotypic study of murine oocyte death in vivo.

Most studies of oocyte apoptosis have been performed in vitro and have employed the method of artificial induction of apoptosis by an anti-cancer agent. However, the process of oocyte death in vivo has not been clearly identified. To investigate the death process in unfertilized oocytes in vivo, we examined the cytochemical change of oocytes collected by oviduct flushing at various intervals after hCG injection. At each collection time, the collected oocytes were phenotypically classified under the microscope into four groups: single-cell oocytes (non-activated and without a nucleus and cytokinesis), activated oocytes (single-, 2- or 4-cell with a nucleus), fragmented oocytes, and dead oocytes. The number of single oocytes decreased and dead oocytes increased with the lapse of time, but the number of activated oocytes or fragmented oocytes did not. Also, most of the dead oocytes observed were single cell. At each time point, single oocytes were stained with anti-tubulin antibody to examine their spindle status. At 24 h after hCG injection, all ovulated oocytes had a normal bipolar spindle, while at 64 h all single-cell oocytes had no spindle. From these observations, we concluded that most oocyte deaths in vivo occur in the single oocyte stage, not in activated or fragmented oocytes.

Oocyte-based screening system for anti-microtubule agents.

Taxol and vinblastine have been widely used in cancer chemotherapy as anti-microtubule agents. However, there are on-going efforts to find new anti-microtubule agents with fewer of the side effects associated with these drugs, such as toxicity or the development of resistance. The standard method used to identify anti-microtubule agents has been the in vitro microtubule polymerization assay. One limitation of this system is that the only compounds selected are those that act on tubulin. Novel compounds whose targets are upstream or are related unknown molecules are not detected. Therefore, many researchers have recently tried to develop novel, phenotype-based drug screening systems. In this study, we developed an oocyte-based screening system for anti-microtubule agents. Dramatic phenotypic changes in microtubules can easily be observed in ovulated oocytes treated with microtubule-stabilizing or -destabilizing agents, such as taxol or vinblastine. After culturing with test samples for 5 h, oocytes were analyzed with fluorescence microscopy after immunostaining. In the oocyte-based screening system, the effective dose (ED50) of taxol for microtubule polymerization is approximately 5 nM, and the ED50 of vinblastine for microtubule depolymerization is approximately 2.5 nM. In addition, taxol-like and vinblastine-like compounds can be evaluated simultaneously in a single assay using this system.

Cdk2-dependent phosphorylation of the NF-Y transcription factor and its involvement in the p53-p21 signaling pathway.

Recent studies have suggested that the NF-Y transcription factor is involved in transcription repression of the cell cycle regulatory genes in a response to p53 induction or DNA damage. Here we demonstrate the cdk2-dependent phosphorylation of NF-Y and its involvement in transcription repression by the p53-p21 signaling pathway. Cdk2 phosphorylates two serine residues near the DNA-binding domain of the YA subunit of NF-Y. Cyclin A-cdk2 appears to associate with NF-Y both in vitro and in vivo. Furthermore, YA protein is phosphorylated in parallel with a cell cycle-dependent activation of cdk2 kinase and cyclin A expression. YA phosphorylation is unnecessary for heterotrimer formation with the YB-YC dimer. However, NF-Y containing a phosphorylation-deficient mutant form of YA, YA-aa, has its DNA binding activity impaired. Consistently, YA-aa inhibits transcription activation of a NF-Y target promoter, cdc2, by cdk2. These results facilitate the elucidation of the regulatory mechanisms of cell cycle progression involving the p21-cdk2-NF-Y signaling pathway.

Bcl-xL and E1B-19K proteins inhibit p53-induced irreversible growth arrest and senescence by preventing reactive oxygen species-dependent p38 activation.

In this study, we describe novel functions of the anti-apoptotic Bcl-2 family proteins. Bcl-x(L) and E1B-19K were found to inhibit p53-induced irreversible growth arrest and senescence, but not to inhibit transient growth arrest, implying that Bcl-x(L) and E1B-19K are specifically involved in senescence without participating in growth arrest. We provide several lines of evidences showing that the functions of Bcl-x(L) and E1B-19K to prevent generation of reactive oxygen species (ROS) are important to inhibit senescence induction. First, we found that that ROS are increased during p53-induced senescence. Moreover, Bcl-x(L) and E1B-19K inhibit this p53-induced ROS generation. Second, antioxidants prevent the induction of senescence and ROS by p53, but not the persistence of the senescence phenotype. Third, the anti-senescence functions of Bcl-x(L) and E1B-19K were suppressed by adding exogenous ROS. These results suggest that Bcl-x(L) and E1B-19K inhibit senescence induction by preventing ROS generation. Furthermore, p38 kinase was found to be activated during p53-induced senescence, but not in cells expressing Bcl-x(L) or E1B-19K, or in cells treated with anti-oxidants. Consistently, a chemical inhibitor of p38 kinase, SB203580, was found to inhibit p53-induced senescence, but only when treated before the cellular commitment to senescence, implying that p38 kinase is necessary for senescence induction. Therefore, Bcl-x(L) and E1B-19K inhibit p53-induced senescence by preventing ROS generation, which in turn leads to the activation of p38 kinase. These results also suggest that the oncogenic potential of Bcl-2 is due to its ability to inhibit senescence as well as apoptosis.