New Evidence Confirms That the Mitochondrial Bottleneck Is Generated without Reduction of Mitochondrial DNA Content in Early Primordial Germ Cells of Mice

In mammals, observations of rapid shifts in mitochondrial DNA (mtDNA) variants between generations have led to the creation of the bottleneck theory for the transmission of mtDNA. The bottleneck could be attributed to a marked decline of mtDNA content in germ cells giving rise to the next generation, to a small effective number of mtDNA segregation units resulting from homoplasmic nucleoids rather than the single mtDNA molecule serving as the units of segregation, or to the selective transmission of a subgroup of the mtDNA population to the progeny. We have previously determined mtDNA copy number in single germ cells and shown that the bottleneck occurs without the reduction in germline mtDNA content. Recently one study suggested that the bottleneck is driven by a remarkable decline of mtDNA copies in early primordial germ cells (PGCs), while another study reported that the mtDNA genetic bottleneck results from replication of a subpopulation of the mtDNA genome during postnatal oocyte maturation and not during embryonic oogenesis, despite a detected a reduction in mtDNA content in early PGCs. To clarify these contradictory results, we examined the mtDNA copy number in PGCs isolated from transgenic mice expressing fluorescent proteins specifically in PGCs as in the aforementioned two other studies. We provide clear evidence to confirm that no remarkable reduction in mtDNA content occurs in PGCs and reinforce that the bottleneck is generated without reduction of mtDNA content in germ cells

Vitrification of Germinal Vesicle Stage Oocytes

In order to cryopreserve germinal vesicle (GV) stage oocytes, we first need to develop a novel container for keeping large quantities of GV oocytes, because of collecting them as cumulus oocytes complexes (COCs) that have bigger size and larger volume than oocytes themselves, and second modify a protocol for optimizing vitrification of them. In this mini-review, we describe our recent progress for attaining these objectives. When 65 bovine COCs having GV oocytes could be placed on a sheet of nylon mesh, and plunged directly into liquid nitrogen for vitrification, the recovery rate was significantly higher compared with that in 15 ones on the electron microscope (EM) grid as a control, followed by obtaining the resultant cleavage and developmental rates after in vitro fertilization and culture (IVFC) without significant difference. Using bovine and murine oocytes, we found that a step-wise manner to expose them with the vitrification solution increased rates of in vitro maturation, subsequent development to blastocysts and hatching/hatched blastocysts after IVFC. Our results show that nylon mesh is an alternative material for cryopreserving large quantities of bovine GV oocytes, and that a step-wise exposure to cryoprotectants may have befit for decreasing disadvantage during vitrification

Calcineurin-GATA-6 pathway is involved in smooth muscle–specific transcription

Intracellular calcium is one of the important signals that initiates the myogenic program. The calcium-activated phosphatase calcineurin is necessary for the nuclear import of the nuclear factor of activated T cell (NFAT) family members, which interact with zinc finger GATA transcription factors. Whereas GATA-6 plays a role in the maintenance of the differentiated phenotype in vascular smooth muscle cells (VSMCs), it is unknown whether the calcineurin pathway is associated with GATA-6 and plays a role in the differentiation of VSMCs. The smooth muscle–myosin heavy chain (Sm-MHC) gene is a downstream target of GATA-6, and provides a highly specific marker for differentiated VSMCs. Using immunoprecipitation Western blotting, we showed that NFATc1 interacted with GATA-6. Consistent with this, NFATc1 further potentiated GATA-6–activated Sm-MHC transcription. Induction of VSMCs to the quiescent phenotype caused nuclear translocation of NFATc1. In differentiated VSMCs, blockage of calcineurin down-regulated the amount of GATA-6-DNA binding as well as the expression of Sm-MHC and its transcriptional activity. These findings demonstrate that the calcineurin pathway is associated with GATA-6 and is required for the maintenance of the differentiated phenotype in VSMCs

Increased Rac1 activity and Pak1 overexpression are associated with lymphovascular invasion and lymph node metastasis of upper urinary tract cancer

<p>Abstract</p> <p>Background</p> <p>Lymphovascular invasion (LVI) and lymph node metastasis are conventional pathological factors associated with an unfavorable prognosis of urothelial carcinoma of the upper urinary tract (UC-UUT), but little is known about the molecular mechanisms underlying LVI and nodal metastasis in this disease. Rac1 small GTPase (Rac1) is essential for tumor metastasis. Activated GTP-bound Rac1 (Rac1 activity) plays a key role in activating downstream effectors known as Pak (21-activated kinase), which are key regulators of cytoskeletal remolding, cell motility, and cell proliferation, and thus have a role in both carcinogenesis and tumor invasion.</p> <p>Methods</p> <p>We analyzed Rac1 activity and Pak1 protein expression in matched sets of tumor tissue, non-tumor tissue, and metastatic lymph node tissue obtained from the surgical specimens of 108 Japanese patients with UC-UUT.</p> <p>Results</p> <p>Rac1 activity and Pak1 protein levels were higher in tumor tissue and metastatic lymph node tissue than in non-tumor tissue (both <it>P </it>< 0.0001). A high level of Rac1 activity and Pak1 protein expression in the primary tumor was related to poor differentiation (<it>P </it>< 0.05), muscle invasion (<it>P </it>< 0.01), LVI (<it>P </it>< 0.0001), and lymph node metastasis (<it>P </it>< 0.0001). Kaplan-Meier survival analysis showed that an increase of Rac1 activity and Pak1 protein was associated with a shorter disease-free survival time (<it>P </it>< 0.01) and shorter overall survival (<it>P </it>< 0.001). Cox proportional hazards analysis revealed that high Rac1 activity, Pak1 protein expression and LVI were independent prognostic factors for shorter overall and disease-free survival times (<it>P </it>< 0.01) on univariate analysis, although only Pak1 and LVI had an influence (<it>P </it>< 0.05) according to multivariate analysis.</p> <p>Conclusions</p> <p>These findings suggest that Rac1 activity and Pak1 are involved in LVI and lymph node metastasis of UC-UUT, and may be prognostic markers for this disease.</p

Current status of clinical background of patients with atrial fibrillation in a community-based survey: The Fushimi AF Registry

AbstractBackgroundAtrial fibrillation (AF) increases the risks of stroke and death, and the prevalence of AF is increasing significantly. Until recently, warfarin was the only oral anticoagulant for stroke prevention, but novel anticoagulants are now under development.Methods and resultsThe Fushimi AF Registry is a community-based survey of AF patients. We aimed to enroll all of the AF patients in Fushimi-ku, which is located at the southern end of the city of Kyoto. Fushimi-ku is densely populated with a total population of 283,000, and is assumed to represent a typical urban community in Japan. On the basis of the general prevalence of AF in the Japanese (0.6%), we estimated the total number of AF patients as 1700. A total of 76 institutions, a large proportion of which were private clinics, participated in the study. At present, we have enrolled 3183 patients from March 2011 to June 2012 (approximately 1.12% of total population). The mean age was 74.2±11.0 years, and 59.3% of subjects were male. The mean body weight was 58.5±13.2kg, and the proportions with a body weight of less than 50kg and 60kg were 25.7% and 55.0%, respectively. The type of AF was paroxysmal in 46.0%, persistent in 7.3%, and permanent in 46.7%. Major co-existing diseases were hypertension (60.6%), heart failure (27.9%), diabetes (23.2%), stroke (19.4%), coronary artery disease (15.0%), myocardial infarction (6.4%), dyslipidemia (42.4%), and chronic kidney disease (26.4%). The mean CHADS2 score was 2.09±1.35: 0 in 11.8% of patients, 1 in 27.1%, and 2 in 29.1%. Warfarin was prescribed in only 48.5% of patients, whereas anti-platelet drugs, mainly aspirin, were prescribed for more than 30% of the patients.ConclusionsThe Fushimi AF Registry provides a unique snapshot of current AF management in an urban community in Japan

Proximal nail fold flap for digital mucous cyst excision

The skin covering a digital mucous cyst is often very thin and is often excised with the cyst. Thus, transfer of a skin flap is needed for the defect. We have developed a proximal nail fold flap technique by which the thin skin covering the cyst can be preserved. We conducted a retrospective study to assess the effectiveness and reliability of this technique for digital mucous cyst excision. The study group comprised 26 patients treated for 28 digital mucous cysts. The flap was elevated on the nail matrix to expose the distal interphalangeal joint capsule. To preserve the skin in cases in which the skin covering the cyst was exceptionally thin, we did not excise the upper part of the cyst wall. Excision of the cyst and stalk was successful in all cases. Additional excision of the joint capsule or osteophyte(s) was achieved in 20 cases and 5 cases, respectively. No flap necrosis, skin defect or nail deformity resulted. Three of the cysts recurred and were treated successfully by reoperation involving the same flap elevation technique. We conclude that the proximal nail fold flap is useful for excision and reliable for wound coverage after digital mucous cyst excision