Mitochondrial dynamics and their intracellular traffic in porcine oocytes.

Meiotic maturation of oocytes requires a variety of ATP-dependent reactions, such as germinal vesicle breakdown, spindle formation, and rearrangement of plasma membrane structure, which is required for fertilization. Mitochondria are accordingly expected be localized to subcellular sites of energy utilization. Although microtubule-dependent cellular traffic for mitochondria has been studied extensively in cultured neuronal (and some other somatic) cells, the molecular mechanism of their dynamics in mammalian oocytes at different stages of maturation remains obscure. The present work describes dynamic aspects of mitochondria in porcine oocytes at the germinal vesicle stage. After incubation of oocytes with MitoTracker Orange followed by centrifugation, mitochondria-enriched ooplasm was obtained using a glass needle and transferred into a recipient oocyte. The intracellular distribution of the fluorescent mitochondria was then observed over time using a laser scanning confocal microscopy equipped with an incubator. Kinetic analysis revealed that fluorescent mitochondria moved from central to subcortical areas of oocytes and were dispersed along plasma membranes. Such movement of mitochondria was inhibited by either cytochalasin B or cytochalasin D but not by colcemid, suggesting the involvement of microfilaments. This method of visualizing mitochondrial dynamics in live cells permits study of the pathophysiology of cytoskeleton-dependent intracellular traffic of mitochondria and associated energy metabolism during meiotic maturation of oocytes.

Possible selection of viable human blastocysts after vitrification by monitoring morphological changes.

PURPOSE: Morphological assessment of human blastocysts has been effective for selecting embryos with high potential. However, they often show repeated shrinkage and expansion toward their hatching. Here we assessed whether capturing morphological changes over time of vitrified-warmed blastocysts could lead to a better selection of viable embryos from shrunken blastocysts. METHODS: The implantation rates of vitrified-warmed blastocysts that were shrunken or expanded (developing) at the time of loading for transfer were compared among 2,729 cycles that were subjected to single blastocyst transfer. Vitrified (107) and fresh blastocysts (17) were donated for the experimental study. To assess the relationship between morphology (expanded vs. shrunken) and the mitochondrial respiration of blastocysts, the oxygen consumption rate (OCR) was analyzed for 55 specimens using an uncoupler of oxidative phosphorylation. The remaining 69 blastocysts were used for recording morphological changes every 15 min for 48 h after warming. RESULTS: Because there were no surplus embryos, 7 % of the vitrified-warmed blastocysts were shrunken and transferred. The shrunken embryos had sufficient implantation ability (40 %). The OCR of the shrunken embryos was significantly lower than that of their expanded counterparts. Upon exposure to the uncoupler, the OCR of some shrunken embryos increased to levels similar to the expanded specimens. Time-lapse images revealed some shrunken embryos which formed blastocoel by 5 h following warming exhibited developmental competence to the hatched stage. CONCLUSIONS: Data of the present study suggest a group of shrunken blastocysts contains many viable and clinically available embryos and time-lapse observation of vitrified-warmed blastocysts is a potential method to distinguish viable embryos from shrunken blastocysts.

Successful delivery after the transfer of twice-vitrified embryos derived from in vitro matured oocytes: a case report.

We report here the first case of successful pregnancy and delivery after the blastocyst transfer of twice-vitrified embryos produced following in vitro maturation (IVM) and ICSI. The patient received 5000 IU hCG on day 12 of the treatment cycle, and oocyte retrieval was carried out 36 h after hCG injection. A total of 22 immature oocytes were obtained. Following incubation for 26 h in IVM medium, 15 oocytes (68.2%) reached metaphase II stage. In total, 13 oocytes (86.7%) were fertilized after ICSI with the husband's sperm, and 11 embryos at the pronuclear stage and two cleaved embryos on day 2 were vitrified because of thin endometrial thickness. Eight cryopreserved embryos at the pronuclear stage were warmed and cultured until the day 3 stage. Three embryos were transferred, and three embryos were twice vitrified. Unfortunately, these transferred embryos did not implant. Three twice-vitrified embryos were rewarmed and cultured until the day 5 stage, and two embryos were transferred. The second transfer attempt of twice-vitrified embryos resulted in the full-term delivery of a healthy infant. This case report demonstrates that twice-vitrified embryos, developed using an IVM protocol, retain the developmental competence for full-term, healthy infants.

Phase I/II study of cisplatin combined with weekly paclitaxel in patients with advanced non-small-cell lung cancer.

To determine the maximum-tolerated dose (MTD) and the recommended dose (RD) of paclitaxel administered weekly with a fixed dose of cisplatin, and to assess the toxicity and activity of this combination, we conducted a phase I/II trial in patients with advanced non-small-cell lung cancer (NSCLC). In this study, patients with stage IIIB/IV NSCLC were eligible. Paclitaxel, at a starting dose of 40 mg x m(-2) week(-1) on days 1, 8, and 15, was combined with a fixed dose of cisplatin 80 mg x m(-2) on day 1. Chemotherapy was given in a 4-week cycle. In this phase I/II study, 38 patients were enrolled. Dose-limiting toxicities (DLT) were neutropenia, fatigue, and omission of treatment due to leucopenia, thrombocytopenia, or febrile neutropenia. The MTD and RD were estimated to be 70 mg x m(-2). Of the 37 assessable patients, 23 had a partial response and one had a complete response. Overall response rate was 62.1% (95% confidence interval (CI): 46.5-77.7%). The progression-free survival, the median survival time, and the 1-year survival rate were 5.5 months, 13.7 months, and 56.9%, respectively. This regimen is tolerable and very active against advanced NSCLC, and its efficacy should be confirmed in a phase III study.

Inflammatory cytokines and antigen-responsive mononuclear cells in peripheral blood of cattle infected with Salmonella takoradi.

To determine the immunological response in lactating dairy cows infected with Salmonella (S.) Takoradi, the relationships among distributions of peripheral blood mononuclear cell (PBMC) subpopulations, endotoxin concentrations and dynamics of inflammatory cytokines in blood were investigated. The ratio of CD4+ T cells to CD8+ T cells was significantly lower in the affected cattle than in the control cattle (p<0.05) to decrease in the number of CD4+ T cells in the infected cattle. In contrast, the numbers of gammadeltaT cells, MHC class II-positive cells were significantly higher in the affected cattle than in the control cattle (p<0.01 respectively). Endotoxemia was found in all but one of the affected cattle. Serum IL-1 and IL-6 bioactivities were significantly higher in the affected cattle than in the control cattle (IL-1, p<0.05; IL-6. p<0.01). Serum TNF-alpha activities and levels were not detected in the control and affected cattle. The activities of proinflammatory cytokines determined by the bioassay are important to the relationships between concentration of endotoxin, cytokines and clinical signs. such as leukocytosis, leukopenia, fever or bacterial shedding. Serum IL-2 levels were lower in the affected cattle than in the control cattle. Serum IFN-y was not detected in the affected cattle except one. These results by the ELISA seemed to reflect the condition of subpopulation in the PBMCs from the shedding cattle. The present results suggest that cellular immunity is suppressed while the humoral immunity is activated in acute bovine salmonellosis.

Impact of pre-stent plaque debulking for chronic coronary total occlusions on restenosis reduction.

BACKGROUND: While stenting improves the long-term angiographic outcomes of successfully recanalized chronic coronary total occlusions (CTO), the restenosis rate still remains high. The massive plaque burden in CTO is considered to be one of the causes of in-stent restenosis. METHODS: We examined the pre-stent plaque debulking strategy with high-speed rotational atherectomy (RA) for 50 CTO (Thrombolysis in Myocardial Infarction flow grade 0; estimated occlusive duration, 3 months). Angiographic follow-up results were compared to those of 120 consecutive CTO recanalized with primary stenting in which RA could be indicated retrospectively. Angiographic restenosis was defined as diameter stenosis > 50% at 6-month follow-up. RESULTS: RA could be performed safely in all lesions without any major complications. Adjunctive ballooning and stenting could be performed without high-pressure dilatation (8.4 +/- 1.7 atmospheres). Follow-up angiography was performed in 48 lesions 184 +/- 61 days after the procedure. There were no significant differences in baseline characteristics between the two groups; however, the implanted stent type was different. Quantitative coronary angiography revealed that diameter stenosis was smaller at follow-up (36.2 +/- 20.0% versus 52.2 +/- 26.7%; p = 0.0003) as well as post-procedure (7.8 +/- 11.5% versus 17.8 +/- 13.6%; p < 0.0001) compared with the control group. Angiographic restenosis was also significantly reduced (29.2% versus 52.5%; p = 0.0061). CONCLUSIONS: RA is a safe procedure for plaque debulking of CTO in selected cases. Plaque debulking of CTO facilitates subsequent stent expansion and may reduce the restenosis rate.

Activation of signal transducer and activator of transcription 3 protects cardiomyocytes from hypoxia/reoxygenation-induced oxidative stress through the upregulation of manganese superoxide dismutase.

BACKGROUND: Mice with cardiac-specific overexpression of signal transducer and activator of transcription 3 (STAT3) are resistant to doxorubicin-induced damage. The STAT3 signal may be involved in the detoxification of reactive oxygen species (ROS). METHODS AND RESULTS: The effects of leukemia inhibitory factor (LIF) or adenovirus-mediated transfection of constitutively activated STAT3 (caSTAT3) on the intracellular ROS formation induced by hypoxia/reoxygenation (H/R) were examined using rat neonatal cardiomyocytes. Either LIF treatment or caSTAT3 significantly suppressed the increase of H/R-induced ROS evaluated by 2',7'-dichlorofluorescin diacetate fluorescence. To assess whether ROS are really involved in H/R-induced cardiomyocyte injury, the amount of creatine phosphokinase in cultured medium was examined. Both LIF treatment and caSTAT3 significantly decreased H/R-induced creatine phosphokinase release. These results indicate that the gp130/STAT3 signal protects H/R-induced cardiomyocyte injury by scavenging ROS generation. To investigate the mechanism of scavenging ROS, the effects of LIF on the induction of antioxidant enzymes were examined. LIF treatment significantly increased the expression of manganese superoxide dismutase (MnSOD) mRNA, whereas the expression of the catalase and glutathione peroxidase genes were unaffected. This induction of MnSOD mRNA expression was completely blocked by adenovirus-mediated transfection of dominant-negative STAT3. Moreover, caSTAT3 augmented MnSOD mRNA and its enzyme activity. In addition, the antisense oligodeoxyribonucleotide to MnSOD significantly inhibited both LIF and caSTAT3-mediated protective effects. CONCLUSIONS: The activation of STAT3 induces a protective effect on H/R-induced cardiomyocyte damage, mainly by inducting MnSOD. The STAT3-mediated signal is proposed as a therapeutical target of ROS-induced cardiomyocyte injury.

Bone morphogenetic protein-2 inhibits serum deprivation-induced apoptosis of neonatal cardiac myocytes through activation of the Smad1 pathway.

Bone morphogenetic protein (BMP)-2 has been shown to induce ectopic expression of cardiac transcription factors and beating cardiomyocytes in non-precardiac mesodermal cells, suggesting that BMP-2 is an inductive signaling molecule that participates in cardiac development. However, direct evidence of the effects of BMP-2 on cardiac myocytes has not been reported. To examine the role of BMP-2 and its receptors, we studied the ability of BMP-2 to promote survival of isolated neonatal rat cardiac myocytes. BMP receptors IA, IB, and II and activin receptor I were found to be expressed in myocytes, and BMP-2 phosphorylated Smad1 and p38 MAPK. Interestingly, BMP-2 promoted survival and inhibited apoptosis of serum-deprived myocytes, although it did not strongly induce hypertrophic growth. To explore the mechanisms for this protective effect, an adenovirus-based vector system was used. Similar to BMP-2, Smad1 promoted survival that was repressed by Smad6. Moreover, BMP-2 and Smad1 enhanced the expression of the anti-apoptotic molecule Bcl-x(L). Antisense oligonucleotides to bcl-x(L) attenuated the survival effected by BMP-2. Overall, our findings suggest that BMP-2 prevents apoptosis of myocytes by induction of Bcl-x(L) via a Smad1 pathway and might be a novel survival factor without any hypertrophic effect on myocytes.

Isolation of Encephalitozoon cuniculi using primary tissue culture techniques from a rabbit in a colony showing encephalitozoonosis.

Encephalitozoon spores were isolated in a primary tissue culture of the kidneys from an encephalitozoonosis-suspected rabbit in a municipal zoo in Hokkaido. The isolated spores were morphologically characteristic of microsporidial ones in chromotrope stain, and proven to be E. cuniculi by a polymerase chain reaction (PCR) with a species-specific primer set and by direct DNA sequencing of the PCR products.

Molecular typing and epidemiological study of Salmonella enterica serotype Typhimurium isolates from cattle by fluorescent amplified-fragment length polymorphism fingerprinting and pulsed-field gel electrophoresis.

One hundred twenty Salmonella enterica serotype Typhimurium strains, including 103 isolates from cattle gathered between 1977 and 1999 in the prefecture located on the northern-most island of Japan, were analyzed by using fluorescent amplified-fragment length polymorphism (FAFLP) and pulsed-field gel electrophoresis (PFGE) to examine the genotypic basis of the epidemic. Among these strains, there were 17 FAFLP profiles that formed four distinct clusters (A, B, C, and D). Isolates that belonged to cluster A have become increasingly common since 1992 with the increase of bovine salmonellosis caused by serotype Typhimurium. PFGE resolved 25 banding patterns that formed three distinct clusters (I, II, and III). All the isolates that belonged to FAFLP cluster A, in which all the strains of definitive phage type 104 examined were included, were grouped into PFGE cluster I. Taken together, these results indicate that clonal exchange of serotype Typhimurium has taken place since 1992, and they show a remarkable degree of homogeneity at a molecular level among contemporary isolates from cattle in this region. Moreover, we have sequenced two kinds of FAFLP markers, 142-bp and 132-bp fragments, which were identified as a polymorphic marker of strains that belonged to clusters A and C, respectively. The sequence of the 142-bp fragment shows homology with a segment of P22 phage, and that of the 132-bp fragment shows homology with a segment of traG, which is an F plasmid conjugation gene. FAFLP is apparently as well suited for epidemiological typing of serotype Typhimurium as is PFGE, and FAFLP can provide a source of molecular markers useful for studies of genetic variation in natural populations of serotype Typhimurium.

Effect of a 60 Hz magnetic field on the behavior of Paramecium.

Paramecium multimicronucleatum was used as a model cell to study the effects of 60 Hz magnetic fields on swimming behavior. When exposed to a vertical field of 0.6 T, the cells accumulated at the upper end of the cuvette. An analysis of the swimming behavior revealed that the exposure to the field increased the number of cells swimming upwards maximally at 1 min after onset of the exposure. This effect of the magnetic field was transient, disappearing within a few minutes during the exposure. It is suggested that the magnetic field may amplify to a large extent the negative gravitaxis of Paramecium. Effects of an induced electric field on the swimming behavior are also discussed.

Activation of prostacyclin synthesis by mechanical stimulation in the ciliated protozoan Tetrahymena thermophila.

We detected a HPLC peak corresponding to 6-keto-PGF(1alpha), a stable metabolite of prostacyclin (PGI(2)), in [1-(14)C]arachidonate metabolites from the ciliated protozoan Tetrahymena thermophila. Quantitative analysis of 6-keto-PGF(1alpha) by enzyme immunoassay revealed that the synthesis and release were rapidly activated by the mechanical stimulation of a short centrifugation. The activation was suppressed significantly by a cyclooxygenase inhibitor, ibuprofen, and was independent of the extracellular Ca(2+). External addition of PGI(2) and its stable analogue, beraprost, caused a transient increase in the tumbling frequency of swimming. Other prostanoids, PGE(2) and PGF(2alpha), have no effect on the swimming. These results indicate that a free-living ciliate, T. thermophila, synthesizes and has a specific sensitivity to PGI(2).

Prostaglandin I(2), a possible thermo-sensory mediator in Paramecium.

Thermo-sensory mechanisms are little understood. The protozoan, Paramecium multimicronucleatum, is sensitive and responsive to a cooling stimulus. We found that inhibitors of prostaglandin (PG) biosynthesis reduced the response to the cooling stimulus. Inversely, the response suppressed by the inhibitors was recovered by application of stable PGI(2) analogs, including iloprost. Paramecium cells showed binding activity specific for [(3)H]iloprost. Moreover, an arachidonic acid metabolite, possibly PGI(2), was rapidly increased in response to the cooling stimulus, suggesting that prostaglandin biosynthesis plays a crucial role in the cooling-sensory transduction. Paramecium may be a useful model for the investigation of the molecular basis of thermo-sensory mechanisms.

Differentiation of rat thymic myoid progenitor cell line established by coculture with human T-lymphotropic virus type-I producing human T cells.

A thymus-derived myoid precursor cell line (ST1), which differentiates to myoid cells in the growth arrest condition, was established by the cocultivation of F344 rat thymic cells with human T-lymphotropic virus type-I (HTLV-I)-producing human lymphoid cells. No integration of HTLV-I was detected in ST1 cells by Southern blot hybridization. In a differentiation culture condition such as confluent culture or serum starvation, ST1 cells began to fuse, creating multinuclear giant cells, with the induced expression of MyoD1 and various muscle-specific antigens, including alpha-sarcomeric actin, skeletal muscle myosin, myoglobin, desmin, and acetylcholine receptor. Ultrastructural investigation revealed that differentiated ST1B cells created aggregates of thick and thin filaments with Z-band-like composition, then formed sarcomeric structures and tubular honeycomb arrays. Finally, these cells spontaneously contracted with a frequency of 0.5-2.0 Hz and synchronized with adjoining cells. Transplantation of ST1B cells into nude mice produced a small tumor nodule, showing clear differentiation to skeletal muscle cells. ST1B cells did not indicate any colony-forming activities in soft agar, demonstrating that ST1B cells retain some of the physiologically normal phenotypes. This rare cell line is promising for use in various physiological and pathological investigations including functional research of thymic myoid cells and the pathological role in autoimmune diseases, as well as animal model experiments of cell therapy related to muscular degenerative disorders or regeneration of injured muscles.

Percutaneous myocardial ablation in double-chamber right ventricle.

Double-chamber right ventricle (DCRV) exhibits intracavitary outflow obstruction. We report the first case of percutaneous myocardial ablation of DCRV in a 73-year-old patient. An alcohol-induced conus branch occlusion provided the reduction of pressure gradient from 81 to 48 mm Hg and clinical improvement. This strategy may be an alternative therapy to surgery in the adult patients with DCRV. Cathet. Cardiovasc. Intervent. 49:97-101, 2000.

An assessment of oral self-care level among Japanese dental hygiene students and general nursing students using the Hiroshima University--Dental Behavioural Inventory (HU-DBI): surveys in 1990/1999.

PURPOSE: The purpose of this study was to determine the differences in oral self-care levels between Japanese dental hygiene students and general nursing students in 1999, and compare them with a similar study in 1990. METHODS: The examination was mainly based upon responses to a questionnaire titled "Hiroshima University--Dental Behavioural Inventory (HU-DBI)". Higher scores of the HU-DBI indicate better oral health attitudes/behaviour. RESULTS: The mean HU-DBI score of the 2nd-year dental hygiene students was significantly greater than that of the 2nd-year nursing students (9.56 and 6.65, respectively; p<0.001), and much higher than those of the 1st-year students at each course (dental hygiene course; 6.30, nursing course; 5.88). Except for the 2nd-year dental hygiene class, there were no significant differences in the HU-DBI score between the 1990 and 1999 subgroups of each class. About 1/3 of the 2nd-year nursing students reported a belief that they may eventually require false teeth, while 6% of the 2nd-year dental hygiene students held this belief. More than 60% of this study sample stated that they put off going to the dentist until they had toothache. The 2nd-year dental hygiene students were more likely to have this attitude than their 1990 peers. Flossing was not popular among the nursing students in either 1990 or 1999. CONCLUSION: Although the difference in the HU-DBI score across time was not major, the variation in HU-DBI and the favourable attitudes/behaviour toward oral health appeared to reflect the differences in schooling between student hygienists and student nurses.

Simple fluctuation of Ca2+ elicits the complex circadian dynamics of cyclic AMP and cyclic GMP in Paramecium.

The circadian dynamics of cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine 3',5'-monophosphate (cGMP) were simulated in Paramecium multimicronucleatum. The mathematical functions determined closely mimic the Ca2+ dependence of adenylate cyclase (AC) and guanylate cyclase (GC) activities as documented in P. tetraurelia. Patterns of cAMP concentration ([cAMP]), cGMP concentration ([cGMP ]), and the ratio [cGMP]/[cAMP] were calculated with respect to Ca2+ concentrations ([Ca2+]) fluctuating sinusoidally with a period of 24 hours at three different levels: low, medium, and high. The functions displayed varying patterns of [cAMP] characteristic for [Ca2+] fluctuating at each level, while patterns of [cGMP] and [cGMP]/[cAMP] almost paralleled [Ca2+] fluctuations. Similar patterns were observed for actual [cAMP] and [cGMP] measured during the light/dark cycle in P. multimicronucleatum, grown in axenic media additionally containing [Ca2+] at 25 (low), 100 (medium), or 400 (high) microM, respectively. The coincidence between simulated and measured fluctuations of [cAMP] and [cGMP] suggests that the circadian fluctuations of intracellular [Ca2+] primarily stimulate activities of AC and GC via their different degrees of Ca2+ dependence, which are ultimately responsible for the circadian spatiotemporal organization of various physiological functions in Paramecium.

Churg-Strauss syndrome diagnosed and followed with gastrointestinal fiberscopic studies and electroneuromyography.

Churg-Strauss syndrome (CSS) is a disorder characterized by hypereosinophilia and systematic vasculitis occurring in individuals with bronchial asthma. We present a case of 24-year-old woman with CSS diagnosed by and followed with gastrointestinal fiberscopic examination and electroneuromyography. The duodenal biopsy showed granulomatous angiitis with eosinophilic infiltration, and the electroneuromyography showed peripheral nerve dysfunction. After steroid treatment, the duodenal erosion and ulcer were almost completely resolved. There was no improvement in electroneuromyography, but the patient was able to walk independently after intensive rehabilitative training.

Cooling sensitive [Ca2+]i response associated with signaling of G protein-coupled receptors.

The influence of cooling on the intracellular concentration of Ca2+ ([Ca2+]i) was tested in cell lines expressing chemical receptors. First, when ATP was externally added to rat basophilic leukemia (RBL-2H3) cells, cooling from 37 degrees C to 27 degrees C induced a transient rapid increase in [Ca2+]i. In the absence of extracellular Ca2+, the [Ca2+]i response was induced whereas an inhibitor of microsomal Ca2+ ATPase, thapsigargin, largely abolished the [Ca2+]i response, suggesting that the internal Ca2+ store liberate the Ca2+. A purinergic receptor antagonist, suramin, completely inhibited the [Ca2+]i response to the cooling. Secondly, when serotonin (5-HT) was added to rat glioma C6BU-1 cells, the cooling induced a transient increase in [Ca2+]. This [Ca2+]i response was induced in the absence of external Ca2+, suggesting that the internal Ca2+ stores liberate the Ca2+. These results raise the possibility that some G protein-coupled receptors are sensitive to cooling in the presence of agonist for the receptor.

Chromosome analysis in human oocytes remaining unfertilized after in-vitro insemination: effect of maternal age and fertilization rate.

The incidence of chromosomal abnormalities was studied in 719 unfertilized human oocytes obtained from our in-vitro fertilization (IVF) programme. To make chromosome preparations, a gradual fixation/air-drying method was utilized. Of 388 oocytes successfully karyotyped, 70 (18.0%) were abnormal. The abnormalities included 33 aneuploidies (8.5%) (14 hyperhaploidies and 19 hypohaploidies), 25 diploidies (6.4%) and 15 structural abnormalities (3.9%), three of them being accompanied by aneuploidy. Of the 33 aneuploidies, 16 (48.5%) showed the loss or gain of dyads (so-called non-disjunction), while 17 (51.5%) showed the loss or gain of monads (so-called predivision). There was no maternal age-dependent increase in the incidence of aneuploidy. Unfertilized oocytes from patients with a high fertilization rate (>25%) had a significantly higher (11.4%, P < 0.05) incidence of diploidy compared with the oocytes from the remaining patients (4.3 and 4.0%), suggesting that diploid oocytes might have a lower fertilizing ability.