Abilities of cumulus and granulosa cells to enhance the developmental competence of bovine oocytes during in vitro maturation period are promoted by midkine; a possible implication of its apoptosis suppressing effects

We previously reported that when midkine (MK), a heparin-binding growth differentiation factor was used in in vitro maturation (IVM) culture of bovine cumulus-enclosed oocytes (CEOs), their developmental competence to the blastocyst stage after in vitro fertilization (IVF) was enhanced and the effect of MK might be mediated by its action upon mural granulosa cells and cumulus cells that closely surround the oocyte. In the present study, when denuded oocytes (DOs) were matured in IVM medium with or without MK (200 ng/ml) in the presence or absence of isolated cumulus cell masses and subjected to IVF, the enhancing effects of MK on the developmental competence of DOs to the blastocyst stage after IVF were exerted only in the presence of cumulus cells. In addition, we prepared the conditioned media of granulosa cells cultured with or without 200 ng MK/ml (CMMK+ or CMMK- respectively) and examined their effects on the IVM of DOs in terms of their developmental competence to the blastocyst stage after IVF. The supplementation of CMMK+ into IVM medium at 40% (v/v) significantly enhanced the blastocyst development compared with the no additive control and the CMMK- supplemented groups. Furthermore, the effects of MK during IVM of bovine CEOs on the cumulus cell apoptosis were investigated. CEOs were cultured up to 24 h in IVM medium without (control) or with 200 ng MK/ml. The genomic DNA was extracted from CEOs at 0, 6, 12, 18 and 24 h of IVM and subjected to ligation-mediated PCR (LM-PCR) to detect the apoptotic internucleosomal DNA fragmentation. DNA fragmentation was scarcely detected at the start of IVM, whereas it increased time-dependently as the IVM culture progressed. The degree of the fragmentation was significantly lower in the MK-treatment group compared with the control group at 18 and 24 h of IVM. The apoptosis-suppressing effect of MK on cumulus cells was further confirmed in situ by using TUNEL on CEOs. In conclusion, data from the present study further confirmed that MK enhances the developmental competence of bovine oocytes via cumulus and granulosa cells. It was also demonstrated that MK suppresses the apoptosis that occurs in cumulus cells during the period of IVM of bovine CEOs. The putative soluble factor(s) from cumulus cells was suggested from the experiment using CMMK+ . MK may promote the production of such factors in part by its anti-apoptotic effects on cumulus cells.     

In situ X-ray diffraction of surface oxide on type 430 stainless steel in breakaway condition using synchrotron radiation

Changes in the crystal structure of type 430 stainless steel and the oxides on its surface were studied in situ at 1373 K using a high-intensity synchrotron X-ray source provided by SPring-8 in Japan. The surface of the steel was initially covered with Cr₂O₃, which was then converted to FeCr₂O₄, and finally Fe₃O₄ and Fe₂O₃ formed on it. These results indicated that the reason for the breakaway oxidation in type 430 stainless steel is Cr depletion beneath Cr₂O₃ layer and the subsequent ionisation of Fe, not the simple mechanical failure of Cr₂O₃.     

Study of Densification Process and Microstructure for Highly Dense Doped MgB2 Bulk Samples

In order to give a contribution in the subject of MgB₂ processing technique to obtain low cost high density bulk samples with improved superconducting properties, studies on dilatometry and on the microstructures of in situ and ex situ MgB₂ bulk samples prepared in the presence of carbon compounds are presented in this paper. The ex situ densification was performed by isostatic pressure followed by high temperature treatment in air, which promoted high density samples compared to the common magnesium diboride in situ samples. It was observed that each carbon compound addition (hydrocarbon??C₈H₁₈, SiC and silicon oil??SiC₂H₆O), corresponds to one microstructure, which can be noticed even after the ex situ process for sintering the pellets. Among the compounds, the SiC showed to present the most irregular structure for the in situ samples. However, this microstructure showed to change completely and reaches higher density (90?%) very rapidly when heat treated in the ex situ process. The X ray diffraction (XRD) analyses also revealed that the presence of spurious phases??like Mg₂Si, Mg₃B₂O₆, MgO and others??for the ex situ samples treated in air at high temperature.     

Estimation of mechanochemical dechlorination rate of poly(vinyl chlorde)

Poly(vinyl chloride) (PVC) was ground in air with CaO in the presence of quartz powder as a grinding aid by a small-scale planetary ball mill to investigate the relation of the dechlorination rate of PVC with the impact energy of the balls calculated from a computer simulation based on the Discrete Element Method under various conditions. Mechanochemical dechlorination proceeds as the grinding progresses and is improved with an increase in both the mill speed and the amount of balls introduced into the mill. The same trend can be seen in the relation between the specific normal impact energy of the balls and the rotational speed. The relationship between the observed dechlorination rate and the computed normal impact energy of the balls is linear, with a correlation coefficient of 0.965. This relationship can be used to estimate the dechlorination rate of PVC in a large-scale planetary ball mill.     

Effect of Hydrogen Atmosphere on Pyrolysis of Cured Polycarbosilane Fibers

SiC-based fibers with various chemical compositions were synthesized using an irradiation-curing process. Polycarbosilane (PCS) fibers were cured by irradiation with an electron beam in a helium atmosphere. The cured PCS fibers were pyrolyzed at 1300°C under controlled hydrogen or argon atmospheres, and SiC fibers with C/Si of 0.84 to 1.56 were obtained. The fibers consisted of <1.0 wt% O, <0.2 wt% N, <0.1 wt% H, with the balance being Si and C. The mechanism of pyrolytic transformation of cured PCS to SiC-based ceramics was investigated using TG/DTA analysis. Greater mass losses were observed during pyrolysis in a hydrogen atmosphere than in argon. This result suggests that the hydrogen atmosphere suppresses H₂ evolution and helps to remove excess carbon as CH₄ during pyrolysis. The microstructure and mechanical properties of the resulting SiC-based fibers were found to be very dependent on their C/Si chemical compositions.     

Changes in serum lipoprotein (a) levels related to hyperlipidemia during pregnancy--comparing normal pregnancy and toxemia of pregnancy

The aim of this 2-year study funded by the English National Board for Nursing, Midwifery and Health Visiting was to examine the relationship between teaching, support, supervision and role modelling in student clinical learning within the context of Project 2000 courses. The research, using a two-stage case-study design, employing predominantly qualitative methods of data collection, investigated the perceptions of students, tutors and practitioners of their experiences of these processes in the clinical setting. This paper focuses on the findings obtained from the practitioner data, high-lighting not only the significant role played by practitioners in facilitating student learning, but also the implications for practitioners in undertaking this role. The preparation required for supervising clinical learning, the integration of theory and practice and the organization of patient care have been identified as particularly important to the development of effective clinical learning environments.     

Sorption of gaseous tritium on the surface of type 316 stainless steel

The sorption of gaseous tritium on the type 316 stainless steel was studied. The stainless steel was first contacted with gaseous tritium, and then the remaining tritium was evacuated. During a gradual etching from the surface by an acid solution, the tritium was released as HTO with a fraction of HT. They were radioassayed separately. The HTO mostly originates from the tritium present on the outer-most surface and about 90% of it could be released easily into water. However, the rest is sorbed tightly and remained in the surface layer. A fraction of the sorbed-tritium will diffuse atomically through the surface layer into the bulk of stainless steel and is released as HT by etching. The activation energy of the diffusion was determined as 32.8 kJ/mol.     

Effective adoptive immunotherapy by T-LAK cells retargeted with bacterial superantigen-conjugated antibody to MUC1 in xenografted severe combined immunodeficient mice

To reinforce cytotoxic activity and the targeting ability of lymphokine-activated killer cells with a T-cell phenotype (T-LAK) for adoptive immunotherapy against human bile duct carcinoma (BDC), staphylococcal enterotoxin A (SEA) was conjugated chemically with MUSE11 monoclonal antibody (MUSE11 mAb), directed to the MUC1 antigen, using N-succinimidyl 3-(2-pyridyldithio) propionate and 2-iminothiolane HCl. Both SEA-conjugated MUSE11 mAb (SEA-MUSE11) and the F(ab')2 of MUSE11 mAb (SEA-F(ab')2) showed significant enhancement of T-LAK cell tumor neutralization for MUC1 positive-target tumor cells, even with a concentration of 0.01 microg/ml at an E:T ratio of 5:1 in vitro. In this in vitro test, MUC1-positive BDC cells were observed to attach to surrounding T-LAK cells in the presence of SEA-MUSE11 or SEA-F(ab')2. Remarkable tumor growth inhibition was observed in BDC-grafted severe combined immunodeficient mice to which 2 x 10(7) T-LAK cells preincubated with 2 microg of SEA-MUSE11 or SEA-F(ab')2, together with recombinant interleukin 2 (500 IU), were administered i.v. for 4 consecutive days, when tumor size was 5 mm in diameter. These results point to a promising adoptive immunotherapy for patients with BDC.     

Characterization of brain-type ryanodine receptor permanently expressed in Chinese hamster ovary cells

To clarify a function of brain-type ryanodine receptor (RyR3) and its regulation, we established a stable cell line expressing rabbit RyR3 by transfection of Chinese hamster ovary cells (CHO cells) with the cDNA and investigated characteristics of the RyR3. Scatchard analysis of [3H]-ryanodine binding to the membrane from CHO cells expressing RyR3 showed two distinct binding sites. The Kd values of high and low affinity binding sites were 1.92 and 25.9 nM, respectively. [3H]-ryanodine binding to the membrane from CHO cells expressing RyR3 was dependent on pCa. Extracellular Ca2+ (2-10 mM) and high concentration (more than 30 mM) of caffeine activated the RyR3 in CHO cells and increased its intracellular Ca2+ concentration. The enhancement of [3H]-ryanodine binding to the membrane from CHO cells expressing RyR3 was observed by bromoeudistomin D (BED), a caffeine-like powerful Ca2+ releaser, at pCa 5.5. Stably expressed RyR3 in CHO is useful for characterization of its function.