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.

A calcium-activated, large conductance and non-selective cation channel in Paramecium cell.

A non-selective cation channel was found in mutant Paramecium cells (K115). This cell had been selected as a resistant mutant in a high-K+ solution. In patch clamp studies of these cells in the inside-out configuration, this channel was activated by bath applications of elevated Ca2+ concentrations. The channels became very active when the Ca2+ concentration was above 3.2 microM. The channel was also activated by depolarization. The voltage dependency was steep upon depolarization, whereas upon hyperpolarization the channel activity barely changed. This channel had poor selectivity for monovalent alkali cations. Using the Goldman-Hodgkin-Katz equation for the reversal potential, the permeability ratios with respect to K+ for Na+, Rb+, Cs+ and Li+ were nearly 1. Although the permeability ratios were similar for each cation, the single channel conductances differed. The single channel conductances were 467 pS with K+ as the charge carrier, 406 pS with Na+, 397 pS with Rb+, 253 pS with Cs+ and 198 pS with Li+ upon depolarization in 100 mM cation solutions. A similar calcium-activated large conductance channel was observed in the wild-type (G3) Paramecium cells but was very rare.

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.

Cloning of a novel ubiquitin-conjugating enzyme (E2) gene from the ciliate Paramecium tetraurelia.

We isolated a 1.7 kb gene (UbcP1) for a ubiquitin-conjugating enzyme from a P. tetraurelia cDNA library and sequenced it. Its deduced polypeptide sequence consists of 425 amino acid residues (48 kDa). The UbcP1 protein contains novel N- and C-terminal extensions in addition to a UBC domain, and within the UBC domain it shares low identity with sequences of other known E2s. A constructed phylogenetic tree suggests that the UbcP1 protein may represent a member of a distinct subfamily of E2s. Southern blot analysis showed that the N-terminal extension of the UbcP1 is conserved in P. multimicronucleatum.

RECONSTITUTION OF METACHRONAL WAVES IN CILIATED CORTICAL SHEETS OF PARAMECIUM - WAVE STABILITIES

We reconstituted metachronal waves on ciliated cortical sheets prepared from detergent-extracted Paramecium multimicronucleatum cells. Ciliary movements of the cortical sheet, whose intracellular side adhered to a glass coverslip, were reactivated by perfusion of a basic reactivation medium containing ATP. In this condition, the ciliary field showed only unstable localised ripples. Addition of either cyclic AMP or cyclic GMP to the basic reactivation medium generated propagating metachronal waves characteristic of each nucleotide. In order to estimate the stability of the metachronal waves, autocorrelation coefficients were calculated from images of an 8 µm diameter region within the reactivated ciliary field. The decay time for the correlation coefficient to decrease to 0.5 was only 0.04 s in the basic reactivation medium, but was increased to 0.4 or 0.9 s by the addition of cyclic AMP or cyclic GMP, respectively. The decay time was dependent not only on the concentration of cyclic nucleotide but also on the wave frequency. In order to test whether cyclic-nucleotide-dependent phosphorylation affected the generation of waves, the ciliated cortical sheets were thiophosphorylated by incubation in ATP-gamma-S (adenosine-5'-o-3-thiotriphosphate) medium containing either cyclic AMP or cyclic GMP. Following this, perfusion with the basic reactivation medium generated metachronal waves only after cyclic GMP treatment. The effect of cyclic GMP is probably related to phosphorylation of ciliary proteins.

RECONSTITUTION OF METACHRONAL WAVES IN CILIATED CORTICAL SHEETS OF PARAMECIUM - ASYMMETRY OF THE CILIARY MOVEMENTS

In conditions in which ciliated cortical sheets prepared from detergent-extracted Paramecium multimicronucleatum cells adhered to glass coverslips on a microscope stage, perfusion of a reactivation medium containing ATP plus cyclic AMP or cyclic GMP generated metachronal waves. An analysis of the ciliary movements that generate these metachronal waves yielded the following results. During the generation of metachronal waves, there were phase differences in the ciliary orientation of adjacent cilia in the direction of wave propagation. Addition of cyclic AMP or cyclic GMP increased the rotational angular velocities during the effective stroke of ciliary beating, but did not increase the rotational angular velocity of the recovery stroke. When the ATP concentration in the cyclic GMP reactivation medium was increased, the rotational angular velocity during the effective stroke rose steeply and saturated at 0.8 mmol l-1 ATP, whereas that during the recovery stroke rose gradually. Addition of cyclic nucleotides caused a single cilium isolated from neighbouring cilia on the cortical sheet to incline almost parallel to the cortical surface during the recovery stroke. Addition of cyclic GMP increased the amplitude of bending of cilia detached from the cortical sheet. From these results, it was concluded that increases in the asymmetrical movement of individual cilia, caused by the addition of cyclic nucleotides, create the ciliary interaction that generates the metachronal waves.

Possible mediation of G-proteins in cold-sensory transduction in

The possible involvement of G-proteins in cold-sensory transduction was examined using voltage-clamped Paramecium multimicronucleatum into which non-hydrolyzable guanosine nucleotide analogues had been applied intracellularly. Guanosine-5'-O-3-thiotriphosphate, guanosine-5'-O-2-thiodiphosphate and aluminium fluoride all reduced the transient inward current in response to cooling, suggesting the possibility that G-proteins mediate cold-sensory transduction. Internal application of a Ca2+ chelator, EGTA, also reduced the current response. In addition to their effect on reducing the cold-sensory response, application of these chemicals modulated both the resting potential and the membrane conductance. Possible correlations between G-protein activity and the regulation of intracellular Ca2+ levels are discussed.

The photodynamic action of methylene blue on the ion channels of Paramecium causes cell damage.

The photodynamic effects of methylene blue (MB) on wild-type and mutant strains of Paramecium Were studied. From measurements of survival and cell motility under the continuous application of light in the presence of MB, the mutant strains remained alive for about three times longer than the wild-type strain. Although the resting potential of the mutant cells was similar to that of wild-type cells, the continuous photodynamic action shifted the membrane potentials of the mutant and wild-type cells to a depolarized level and a hyperpolarized level, respectively, from that before light application. Under voltage clamping, the mutant cells reduced not only the outward current elicited by the photodynamic action but also the outward tail current elicited by the preceding pulse of hyperpolarization. We conclude that the mutant strain is defective in the activation of Ca(2+)-dependent K+ channels. This defect might cause a reduction in the Ca2+ influx because of the suppression of the membrane hyperpolarization, which results in the elongation of survival time under the photodynamic action.

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.

Tuberculous spondylitis (Pott's disease) with bilateral pleural effusion.

We report a case of tuberculous spondylitis (Pott's disease) with bilateral pleural effusion in a 25-year-old male patient. Left-pleural effusion was observed on admission. The initial diagnosis was tuberculous pleuritis. However, during anti-tuberculosis chemotherapy, back pain and right-pleural effusion appeared. by further examination, we diagnosed an active tuberculous spondylitis of the 11th and 12th thoracic vertebrae possibly by the dissemination of Mycobacterium tuberculosis through blood circulation or lymphatic circulation. In cases in which anti-tuberculosis chemotherapy is not so effective, the possibility of the rarely-appearing extra-pulmonary manifestation of tuberculous spondylitis must be considered.

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.

Candidiasis caused by Candida glabrata in the forestomachs of a calf.

A female Japanese black calf suffering from diarrhoea was treated with six different antibiotics for 9 days. The diarrhoea worsened and the calf was killed when 17 days old. At necropsy, the mucosal surface of the forestomachs showed diffuse haemorrhagic necrosis and pseudomembrane formation. Candida glabrata infection was diagnosed histologically, immunohistochemically, and by isolation and examination of the yeast. The intensive antibiotic treatment was considered to have been a predisposing or exacerbating factor. This is the first report of C. glabrata infection of the bovine forestomachs.

Dual infection with attaching and effacing Escherichia coli and enterotoxigenic Escherichia coli in post-weaning pigs.

Post-weaning diarrhoea in pigs occurred on two farms in Hokkaido, Japan, in 1994. Four piglets aged 35 or 45 days were examined after death. At necropsy, ecchymotic haemorrhages were seen on the mucosal surface of the caecum and colon. Histopathologically, numerous Gram-negative bacilli adhered to the brush border of the small intestines, but the brush border itself was intact. Typical attaching and effacing (AE) lesions were seen in the caecum and colon. Immunohistochemically, the bacilli which adhered to the brush border gave positive results with antisera against serogroup O149 of Escherichia coli; the bacilli which caused the AE lesions, however, belonged to serogroup O45. It was concluded that the disease resulted from dual infection with attaching and effacing E. coli (AEEC) and enterotoxigenic E. coli (ETEC).

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.

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.

Exercise performance and limiting factors in patients with chronic lung diseases.

To demonstrate the clinical usefulness of the treadmill exercise test in patients with chronic lung diseases (CLD), 272 consecutive tests done from Jan. 1986 to Dec. 1988 were analyzed. The subjects mainly consisted of patients with chronic obstructive pulmonary disease (COPD) and interstitial lung disease (ILD). With the present incremental treadmill protocol the maximal stage attained by a patient or distance walked were closely related to the patient's maximal oxygen uptake. Comparing symptoms and cardio-pulmonary response, and exercise performance between patients with COPD and those with ILD, there was an evident difference in the exercise limiting factor (s): in the former ventilatory limitation was the most important whereas in the latter gas exchange and oxygen transport was critical. Assessment of breathlessness during the exercise test using the Borg scale was useful in evaluating various therapeutic modalities such as oxygen supplementation. In conclusion, the exercise performance in patients with CLD was properly assessed with the present treadmill protocol and the information derived thereby was of great clinical significance.

[A case of small cell carcinoma of the lung responding well to oral etoposide therapy].

A case of recurrent small cell carcinoma of the lung, accompanied by intraperitoneal metastasis, was orally treated with etoposide (50 mg/day) for 3 consecutive weeks. Subjective symptoms such as abdominal pain began to improve one week after the start of treatment. After three weeks of treatment, these symptoms disappeared completely, and CT findings also improved. The serum NSE level, which was as high as 89.5 ng/ml before treatment, fell to 8.7 ng/ml after 3 weeks of treatment. The patient is now being followed at the outpatient clinic of our hospital. As adverse reactions to the drug, alopecia and leukopenia were observed. The leukopenia appeared in the third week of treatment, but the white blood cell count returned to normal soon after the drug was discontinued.

[Indications for thoracotomy in pulmonary metastatic sarcoma and its prognosis].

Between 1960 and 1987, 46 patients underwent thoracotomy for pulmonary metastatic sarcoma. The histologic classification of the sarcoma was osteosarcoma in 33 patients, other malignant bone tumors in 6 and soft tissue sarcoma in 7. The cumulative five-year survival rate of the 33 patients with osteosarcoma was 23%. Ten patients survived more than 3 years and 8 of them are still alive without pulmonary metastasis. Prognosis was significantly better with intensive multidrug chemotherapy. In this series, prognosis was not significantly related to the interval from initial onset to initial treatment, that from initial treatment to pulmonary metastasis and that from pulmonary metastasis to initial thoracotomy. The five-year survival rate of the 6 patients with other malignant bone tumors was 35%, and that of the 7 patients with soft tissue sarcoma 26%. Good indications for thoracotomy in pulmonary metastatic osteosarcoma are: (1) the primary sarcoma is resected, (2) the interval from initial treatment to pulmonary metastases is more than 6 months, (3) the number of pulmonary metastases is less than 4 or 5 nodules, and (4) the number and diameter of pulmonary metastases is controlled with chemotherapy within 2 or 3 months after occurrence of metastasis.

[A case of superior vena cava syndrome treated with combination radiation and CRE (CBDCA and VP-16) therapy].

Carboplatin and etoposide were reported to be excellent radiation sensitizers. We encountered a patient with SVC syndrome due to lung cancer who was successfully treated by combination carboplatin, etoposide and hyperfractionation radiotherapy. A 72-year-old man was admitted to our hospital because of remarkable face edema. Computed tomography revealed a huge lung tumor and compressed SVC due to tumor growth. Acute tumor regression was essential for this case. We performed combination chemotherapy and radiation. The regime consisted of CBDCA 300 mg (day 1 1 hr drip infusion) and etoposide 50 mg/day for 21 days by oral administration. Two daily fractionations of 1.4 Gy were delivered 5 days-a-week, with a 4 h interval between fractions (total dose 49.8 Gy). Complete response of huge tumor was attained in this case. The major side effect associated with the therapy was myelosuppression. The patient's quality of life has been remarkably improved with this therapy.