Mayumi Umeno

Analysis of serum elements and the contaminations from devices used for serum preparation by inductively coupled plasma mass spectrometry

Two different samples of serum were prepared from a blood specimen by using two types of serum-separation tubes. Each serum sample was diluted by 100 times with deionized and sub-boiling distilled water. Thirty-six elements in the serum and exudate, from devices used for the serum preparation, were determined by inductively coupled plasma mass spectrometry (ICP-MS). From the comparison of elemental concentrations in two groups of 11 serum samples, it was found that the exudate from serum-separation tubes, as well as from disposable stainless steel needles, had serious effects on the elemental concentrations in the serum. Means for the concentrations of Li, Co, Ga, Cd, Sn, Sb, Ba, La, and Ce in the two serum groups were statistically different from each other. The concentrations of Cr, Mn, Fe, and Mo in both groups were apparently higher than those reported in the literature, suggesting contamination from the disposable stainless steel needle and spectral interferences due to molecular species produced in the argon plasma.

Proteasome Subunits mRNA Expressions Correlate With Male BMI: Implications for a Role in Obesity

Obesity as well as its associated chronic diseases and adverse health consequences such as type 2 diabetes mellitus, dyslipidemia, hypertension, and coronary artery disease are afflicting middle‐aged adults and an ever greater number of children globally. We planned to investigate new obesity‐related factors using proteomics approaches in a randomly selected three high and three low BMI samples of Epstein‐Barr‐transformed B (EBV‐B) lymphoblastoid cell lines prepared from two groups of young Japanese men with different BMI. To search novel obesity‐related factors, comparisons of protein expressions between high and low BMI groups were carried out by two‐dimensional gel electrophoresis (2‐DE). Gene transcripts of proteasome subunits found out from 2‐DE were further determined by quantitative real‐time PCR. Results from proteomics approach showed that the expression of proteasome α subunit type 5 (PSMA5) was significantly lower in the high BMI male group than in those with low BMI (P < 0.05). To validate these results, we expanded the study to include 20 more men and used real‐time PCR to quantify the mRNA expression level in their EBV‐B cells. Both PSMA5 and PSMA2 of EBV‐B cells showed negative correlation with BMI. Furthermore, the mRNA levels measured in the peripheral blood B lymphocytes for many proteasome subunits in 75 healthy men and women showed significant negative correlation with BMI in healthy men. Our findings suggest that proteasome expression may play a key role in obesity.

Molecular analysis of TBL1Y, a Y-linked homologue of TBL1X related with X-linked late-onset sensorineural deafness

AbstractRecent progress in sequencing the human Y chromosome has unveiled a series of X-Y homologous genes. In the present study, we focused on Transducin beta-like 1Y (TBL1Y), which is a Y-linked homologue of TBL1X that is related with X-linked late-onset sensorineural deafness. Recently, it has been shown that TBLR1, another homologue whose gene resides on chromosome 3, and TBL1X act as a corepressor/coactivator exchanger for several nuclear receptors and transcription factors. However, the expression pattern and function of TBL1Y remain unknown. The RT-PCR analysis of the TBL1 family revealed that TBL1Y was expressed in all 13 tissues examined but not in leukocytes. Among the cell lines tested, however, it was only expressed in NT2/D1 cells and in lymphoblasts transformed with Epstein Barr (EB) virus. To compare the functions of the TBL1 family, we generated a series of expression plasmids for GAL4DBD-fused proteins of the TBL1 family. We carried out dual luciferase assays using these plasmids in combination with a plasmid having a luciferase reporter gene harboring 5×GAL4 binding sites. Unlike the other constructs, GAL4DBD-fused TBL1Y did not repress the promoter activity. Moreover, we found three novel polymorphisms in the TBL1Y gene, IVS7+9G>A, G268C, and IVS7+1G>C, which is presumed to cause splicing error. These polymorphisms are found in males within Y-haplogroup O3 (XO3e), which is defined as the Y-haplogroup O3 excluding O3e, a branch of O3. The results show that TBL1Y differs from other members of the TBL1 family in expression and function, suggesting other roles in maleness.

Roscovitine in combination with calcium ionophore induces oocyte activation through reduction of M-phase promoting factor activity in mice

The aim of the present study was to determine oocyte activation and change in M-phase promoting factor (MPF) activity induced by treatment with calcium ionophore and roscovitine in comparison with those induced by treatment with roscovitine alone and treatment with calcium ionophore and puromycin in mice. Freshly ovulated oocytes obtained from 6-8-week-old mice were divided into five groups (no activation treatment; 5 μM calcium ionophore A23187; 50 μM roscovitine; 5 μM calcium ionophore and 10 μg/ml puromycin; and 5 μM calcium ionophore and 50 μM roscovitine) and were incubated for 6 h. Oocyte activation, assessed by morphological changes, and changes in MPF activity in the five groups at 0, 2, 4 and 6 h of incubation were examined. Activated oocytes were defined as oocytes with at least one pronucleus. Oocytes treated with roscovitine alone were not activated during the 6-h incubation period. All of the oocytes in the calcium ionophore with puromycin group and in the calcium ionophore with roscovitine group were activated. The percentage activity of MPF in oocytes treated with roscovitine alone was decreased after 2 h and increased after 4 h of incubation. The percentage activity of MPF in oocytes treated with calcium ionophore and roscovitine was significantly decreased with suppression of MPF activity being maintained for 6 h, and this change was similar to that in oocytes treated with calcium ionophore and puromycin. Roscovitine with calcium ionophore is effective for induction of oocyte activation through suppression of MPF activity in mice.