In vitro maturation and ultrastructural observation of cryopreserved minke whale (Balaenoptera acutorostrata) follicular oocytes

Minke whale (Balaenoptera acutorostrata) follicular oocytes were cryopreserved by a slow-step freezing procedure using ethylene glycol. The morphologically viable proportion of postthawed minke whale follicular oocytes was 39.7%. The maturity of the animals (immature and mature whales) or the presence or absence of cumulus cells (CC) did not affect the proportion of morphologically viable oocytes. Postthawed oocytes were examined for nuclear status after in vitro maturation. The presence of CC (29.1%) significantly enhanced (P < 0.05) the proportion of oocytes at metaphase I/anaphase I/telophase I stages compared to results with the absence of CC (13.5%). A total of 4 of 194 postthawed oocytes matured to the second metaphase stage after culture for 5.5 days with or without CC. The cryopreserved immature oocytes obtained from immature and mature whales were processed to examine the ultrastructure by transmission electron microscopy. Varying ultrastructural damage to the cytoplasm was observed as a result of the cryopreservation procedures. These results show that 20-30% of cryopreserved minke whale follicular oocytes can resume meiosis in vitro, but damage induced by the freezing and thawing procedures was observed.     

Induction of Manganese Superoxide Dismutase by Nuclear Translocation and Activation of SIRT1 Promotes Cell Survival in Chronic Heart Failure

Oxidative stress plays a pivotal role in chronic heart failure. SIRT1, an NAD⁺-dependent histone/protein deacetylase, promotes cell survival under oxidative stress when it is expressed in the nucleus. However, adult cardiomyocytes predominantly express SIRT1 in the cytoplasm, and its function has not been elucidated. The purpose of this study was to investigate the functional role of SIRT1 in the heart and the potential use of SIRT1 in therapy for heart failure. We investigated the subcellular localization of SIRT1 in cardiomyocytes and its impact on cell survival. SIRT1 accumulated in the nucleus of cardiomyocytes in the failing hearts of TO-2 hamsters, postmyocardial infarction rats, and a dilated cardiomyopathy patient but not in control healthy hearts. Nuclear but not cytoplasmic SIRT1-induced manganese superoxide dismutase (Mn-SOD), which was further enhanced by resveratrol, and increased the resistance of C2C12 myoblasts to oxidative stress. Resveratrol''s enhancement of Mn-SOD levels depended on the level of nuclear SIRT1, and it suppressed the cell death induced by antimycin A or angiotensin II. The cell-protective effects of nuclear SIRT1 or resveratrol were canceled by the Mn-SOD small interfering RNA or SIRT1 small interfering RNA. The oral administration of resveratrol to TO-2 hamsters increased Mn-SOD levels in cardiomyocytes, suppressed fibrosis, preserved cardiac function, and significantly improved survival. Thus, Mn-SOD induced by resveratrol via nuclear SIRT1 reduced oxidative stress and participated in cardiomyocyte protection. SIRT1 activators such as resveratrol could be novel therapeutic tools for the treatment of chronic heart failure.     

Anticancer susceptibility test method using general-purpose dissolved oxygen meter

Drug susceptibility of cell can be rapidly measured by continuously monitoring its metabolic changes. We focused on respiration volume as a signal of metabolic change, and developed a new dissolved oxygen measuring system which can detect respiration volume of cells. The main feature of the system is the use of a new type bare oxygen electrode which can easily detect the changing rate of dissolved oxygen concentration. In this study, single type electrode was used to evaluate this rapid anticancer drug susceptibility test. The result obtained was almost equivalent to that with MTT method, which is a conventional method for susceptibility test of HL-60 to various kinds of anticancer drugs. We have also developed multi-type electrode plate with oxygen electrodes embedded in the bottom of 96-well plate, with which clinical evaluation of this method can be easily made.     

Lung volume and collapsibility of the passive pharynx in patients with sleep-disordered breathing.

Lung volume dependence of pharyngeal airway patency suggests involvement of lung volume in pathogenesis of obstructive sleep apnea. We examined the structural interaction between passive pharyngeal airway and lung volume independent of neuromuscular factors. Static mechanical properties of the passive pharynx were compared before and during lung inflation in eight anesthetized and paralyzed patients with sleep-disordered breathing. The respiratory system volume was increased by applying negative extrathoracic pressure, thereby leaving the transpharyngeal pressure unchanged. Application of -50-cmH(2)O negative extrathoracic pressure produced an increase in lung volume of 0.72 (0.63-0.91) liter [median (25-75 percentile)], resulting in a significant reduction of velopharyngeal closing pressure of 1.22 (0.14-2.03) cmH(2)O without significantly changing collapsibility of the oropharyngeal airway. Improvement of the velopharyngeal closing pressure was directly associated with body mass index. We conclude that increase in lung volume structurally improves velopharyngeal collapsibility particularly in obese patients with sleep-disordered breathing.     

Single-molecule imaging of β-actin mRNAs in the cytoplasm of a living cell

β-Actin mRNA labeled with an MS2-EGFP fusion protein was expressed in chicken embryo fibroblasts and its localization and movement were analyzed by single-molecule imaging. Most β-Actin mRNAs localized to the leading edge, while some others were observed in the perinuclear region. Singe-molecule tracking of individual mRNAs revealed that the majority of mRNAs were in unrestricted Brownian motion at the leading edge and in restricted Brownian motion in the perinuclear region. The macroscopic diffusion coefficient of mRNA (D_MACRO) at the leading edge was 0.3 μm²/s. On the other hand, D_MACRO in the perinuclear region was 0.02 μm²/s. The destruction of microfilaments with cytochalasin D, which is known to delocalize β-actin mRNAs, led to an increase in D_MACRO to 0.2 μm²/s in the perinuclear region. These results suggest that the microstructure, composed of microfilaments, serves as a barrier for the movement of β-actin mRNA.     

Transgenic medaka enables easy oocytes detection in live fish

Easy oocyte detection in living specimens benefits various developmental biology and environmental toxicology studies. One of the earliest markers of sex differentiation in medaka (Oryzias latipes) is oocyte development. Within the field of toxicology, a simple detection method for induced oocyte in the testis (testis-ova) as a result of endocrine disruption is necessary. In this study we produced transgenic medaka whose oocytes were labeled with fluorescent proteins using the regulatory region of the 42Sp50 gene, an isoform of polypeptide elongation 1-alpha. Short (201 nt) 5'- and 3'-flanking regions were sufficient for reporter gene expression. GFP expression was first observed in female germ cells approximately 5 days post-hatching. In the mature ovaries, germ cells showed such intense fluorescence that the fluorescence was observed from outside the body wall. In contrast, very faint fluorescence was observed in the mature testes. Testis-ova, oocytes artificially induced in the testes, were also labeled with GFP. These findings indicate through the use of transgenic medaka, that detection of female germ cells was straightforward and this transgenic medaka model proves useful for tracking female germ cells in developmental and toxicological studies.     

Bmp2 and Bmp4 genetically interact to support multiple aspects of mouse development including functional heart development

Bone morphogenetic proteins (BMPs) have multiple roles during embryogenesis. Current data indicate that the dosage of BMPs is tightly regulated for normal development in mice. Since Bmp2 or Bmp4 homozygous mutant mice show early embryonic lethality, we generated compound heterozygous mice for Bmp2 and Bmp4 to explore the impact of lowered dosage of these BMP ligands. Genotyping pups bred between Bmp2 and Bmp4 heterozygous mice revealed that the ratio of adult compound heterozygous mice for Bmp2 and Bmp4 is much lower than expected. During embryogenesis, the compound heterozygous embryos showed several abnormalities, including defects in eye formation, body wall closure defects, and ventricular septal defects (VSD) in the heart. However, the ratio of the compound heterozygous embryos was the same as expected. Caesarean sections at E18.5 revealed that half of the compound heterozygotes died soon after birth, and the majority of the dead individuals exhibited VSD. Survivors were able to grow to adults, but their body weight was significantly lower than control littermates. They demonstrated progressive abnormalities in the heart, eventually showing a branched leaflet in atrioventricular valves. These results suggest that the dosage of both BMP2 and 4 is critical for functional heart formation during embryogenesis and after birth. genesis 47:374–384, 2009. © 2009 Wiley‐Liss, Inc.     

A Catechin‐rich Beverage Improves Obesity and Blood Glucose Control in Patients With Type 2 Diabetes

We investigated the effects of continuous ingestion of a catechin‐rich beverage in patients with type 2 diabetes who were not receiving insulin (Ins) therapy in a double‐blind controlled study. The participants ingested green tea containing either 582.8 mg of catechins (catechin group; n = 23) or 96.3 mg of catechins (control group; n = 20) per day for 12 weeks. At week 12, the decrease in waist circumference was significantly greater in the catechin group than in the control group. Adiponectin, which is negatively correlated with visceral adiposity, increased significantly only in the catechin group. Although the increase in Ins at week 12 was significantly greater in the catechin group than in the control group, no apparent difference was noted between the two groups in glucose and hemoglobin A_1c. In patients treated with insulinotropic agents, the increase in Ins at week 12 was significantly greater in the catechin group than in the control group. This significant increase in Ins levels was observed only in the catechin group. In the catechin group receiving other treatments, Ins levels remained unchanged. In addition, in patients treated with insulinotropic agents, the decrease in hemoglobin A_1c at week 12 was significantly greater in the catechin group than in the control group. These results suggest that a catechin‐rich beverage might have several therapeutic uses: in the prevention of obesity; in the recovery of Ins‐secretory ability; and, as a way to maintain low hemoglobin A_1c levels in type 2 diabetic patients who do not yet require Ins therapy.