In-vitro Culture with a Tilting Device in Chemically Defined Media During Meiotic Maturation and Early Development Improves the Quality of Blastocysts Derived from In-vitro Matured and Fertilized Porcine Oocytes

Under physiological conditions, mammalian oocytes and embryos appear to be stimulated not only chemically but also mechanically, such as by compression, shear stress and/or friction force in the follicle and female reproductive tract. The present study was undertaken to examine the effects of kinetic culture with a tilting device in chemically defined media during in vitro maturation (IVM) of porcine oocytes and in vitro culture (IVC) following in vitro fertilization (IVF) on the early developmental competence and quality of blastocysts. After culture in a chemically defined IVM medium, modified porcine oocyte medium (mPOM) containing gonadotropins and dibutyryl cAMP for 20 h, the mean diameter of the cumulus-oocyte complexes (COCs) was larger in the tilting culture than in the static controls, whereas the diameter of the oocytes did not differ. When culture of the COCs was continued additionally in a fresh medium without gonadotropins and dibutyryl cAMP for 24 h, the incidences of oocytes completing GVBD and developing to the metaphase-II stage did not differ between the tilting and static culture systems. Furthermore, the sperm penetration after IVF and developmental competence of the oocytes to the blastocyst stage were not different between the tilting and static systems during IVM and IVC. However, tilting culture during both IVM and IVC had a significant positive effect on the number of cells per blastocyst (P<0.05). These observations indicate that tilting culture during IVM and IVC in chemically defined media improves the quality of blastocyst, as determined by the number of cells per blastocyst, without any effects on penetrability and developmental competence

禅那院珍海已講の浄土教思想

早大学位記番号:新8450早稲田大

Expression of anti-fungal peptide, β-defensin 118 in oral fibroblasts induced by C. albicans β-glucan-containing particles

Objective: Although oral fibroblasts are thought to have the potential to enhance host defenses against Candida albicans , it is unknown whether they are able to recognize Candida cell components to increase the expression of antifungal peptides, such as defensin factors, against Candida infection. Methodology: We performed expression profiles of defensin genes induced by heat-killed C. albicans in oral immortalized fibroblasts (GT1) using cDNA microarray analysis. From those results, quantitative RT-PCR was used to examine the effects of Candida β-glucan-containing particles (β-GPs) on β-Defensin 118 (DEFB 118) expression in oral mucosal cells. Furthermore, the antifungal activities of recombinant DEFB 118 against C. albicans and C. glabrata were investigated using fungicidal assays. Results: Microarray analysis showed that DEFB118, β-Defensin 129 (DEFB129), and α-Defensin 1 (DEFA1) genes were induced by heat-killed C. albicans and that their mRNA expressions were also significantly increased by live as well as heat-killed C. albicans . Next, we focused on DEFB118, and found that GT1, primary fibroblasts, and RT7 (oral immortalized keratinocytes) constitutively expressed DEFB118 mRNA expression in RT-PCR. Furthermore, C. albicans β-GPs significantly increased the expression of DEFB118 mRNA in GT1 and primary fibroblasts. Although DEFB118 mRNA expression in RT7 was significantly induced by both live and heat-killed C. albicans, C. albicans β-GPs failed to have an effect on that expression. Finally, recombinant DEFB118 significantly decreased the survival of both strains of C. albicans in a dose-dependent manner, whereas no effects were seen for both C. glabrata strains. Conclusion: DEFB118, induced by C. albicans β-GPs from oral fibroblasts, may play an important role in oral immune responses against C. albicans infection

The Inhibitory Role of Rab11b in Osteoclastogenesis through Triggering Lysosome-Induced Degradation of c-Fms and RANK Surface Receptors

Rab11b, abundantly enriched in endocytic recycling compartments, is required for the establishment of the machinery of vesicle trafficking. Yet, no report has so far characterized the biological function of Rab11b in osteoclastogenesis. Using in vitro model of osteoclasts differentiated from murine macrophages like RAW-D cells or bone marrow-derived macrophages, we elucidated that Rab11b served as an inhibitory regulator of osteoclast differentiation sequentially via (i) abolishing surface abundance of RANK and c-Fms receptors; and (ii) attenuating nuclear factor of activated T-cells c1 (NFATc-1) upstream signaling cascades, following RANKL stimulation. Rab11b was localized in early and late endosomes, Golgi complex, and endoplasmic reticulum; moreover, its overexpression enlarged early and late endosomes. Upon inhibition of lysosomal function by a specific blocker, chloroquine (CLQ), we comprehensively clarified a novel function of lysosomes on mediating proteolytic degradation of c-Fms and RANK surface receptors, drastically ameliorated by Rab11b overexpression in RAW-D cell-derived osteoclasts. These findings highlight the key role of Rab11b as an inhibitor of osteoclastogenesis by directing the transport of c-Fms and RANK surface receptors to lysosomes for degradation via the axis of early endosomes-late endosomes-lysosomes, thereby contributing towards the systemic equilibrium of the bone resorption phase

Mechanical stretch stimulates integrin αVβ3-mediated collagen expression in human anterior cruciate ligament cells

Biomechanical stimuli have fundamental roles in the maintenance and remodeling of ligaments including collagen gene expressions. Mechanical stretching signals are mainly transduced by cell adhesion molecules such as integrins. However, the relationships between stress-induced collagen expressions and integrin-mediated cellular behaviors are still unclear in anterior cruciate ligament cells. Here, we focused on the stretch-related responses of different cells derived from the ligament-to-bone interface and midsubstance regions of human anterior cruciate ligaments. Chondroblastic interface cells easily lost their potential to produce collagen genes in non-stretched conditions, rather than fibroblastic midsubstance cells. Uni-axial mechanical stretches increased the type I collagen gene expression of interface and midsubstance cells up to 14- and 6-fold levels of each non-stretched control, respectively. Mechanical stretches also activated the stress fiber formation by shifting the distribution of integrin αVβ3 to the peripheral edges in both interface and midsubstance cells. In addition, integrin αVβ3 colocalized with phosphorylated focal adhesion kinase in stretched cells. Functional blocking analyses using anti-integrin antibodies revealed that the stretch-activated collagen gene expressions on fibronectin were dependent on integrin αVβ3-mediated cellular adhesions in the interface and midsubstance cells. These findings suggest that the integrin αVβ3-mediated stretch signal transduction might have a key role to stimulate collagen gene expression in human anterior cruciate ligament, especially in the ligament-to-bone interface

A diagnostic marker for superficial urothelial bladder carcinoma : lack of nuclear ATBF1 (ZFHX3) by immunohistochemistry suggests malignant progression

Background: Pathological stage and grade have limited ability to predict the outcomes of superficial urothelial bladder carcinoma at initial transurethral resection (TUR). AT-motif binding factor 1 (ATBF1) is a tumor suppressive transcription factor that is normally localized to the nucleus but has been detected in the cytoplasm in several cancers. Here, we examined the diagnostic value of the intracellular localization of ATBF1 as a marker for the identification of high risk urothelial bladder carcinoma. Methods: Seven anti-ATBF1 antibodies were generated to cover the entire ATBF1 sequence. Four human influenza hemagglutinin-derived amino acid sequence-tagged expression vectors with truncated ATBF1 cDNA were constructed to map the functional domains of nuclear localization signals (NLSs) with the consensus sequence KR[X10-12]K. A total of 117 samples from initial TUR of human bladder carcinomas were analyzed. None of the patients had received chemotherapy or radiotherapy before pathological evaluation. Results: ATBF1 nuclear localization was regulated synergistically by three NLSs on ATBF1. The cytoplasmic fragments of ATBF1 lacked NLSs. Patients were divided into two groups according to positive nuclear staining of ATBF1, and significant differences in overall survival (P = 0.021) and intravesical recurrence-free survival (P = 0.013) were detected between ATBF1+ (n= 110) and ATBF1− (n=7) cases. Multivariate analysis revealed that ATBF1 staining was an independent prognostic factor for intravesical recurrence-free survival after adjusting for cellular grading and pathological staging (P = 0.008). Conclusions: Cleavage of ATBF1 leads to the cytoplasmic localization of ATBF1 fragments and downregulates nuclear ATBF1. Alterations in the subcellular localization of ATBF1 due to fragmentation of the protein are related to the malignant character of urothelial carcinoma. Pathological evaluation using anti-ATBF1 antibodies enabled the identification of highly malignant cases that had been overlooked at initial TUR. Nuclear localization of ATBF1 indicates better prognosis of urothelial carcinoma