Redox status of the oviduct and CDC2 activity in 2-cell stage embryos in heat-stressed mice

Mammalian preimplantation embryos are vulnerable to heat stress. However, the mechanisms by which maternal heat stress compromises embryonic development are unclear. We hypothesized that the loss of developmental competence in maternally heat-stressed embryos results from enhanced oxidative stress in the oviducts. In experiment 1, oviducts and zygotes were collected from mice that were heat-stressed at 35 degrees C and 60% relative humidity for 12 h on the day of pregnancy as well as from control mice. The zygotes were cultured for 84 h to assess their development, and the H(2)O(2) level, glutathione concentration, and free radical scavenging activity (FRSA) were measured in the oviduct. In experiment 2, zygotes were cultured for 22 h to reach the late G(2) phase in the 2-cell stage, and Cdc2 activity was assessed using immunoblotting. A high percentage (87.6%) of control embryos developed to morulae or blastocysts, whereas the majority (67.4%) of the heat-stressed group arrested at the 2-cell stage. Although heat stress did not alter the FRSA or glutathione concentration in the oviducts, the H(2)O(2) level (P < 0.01) and its ratio to the FRSA (P < 0.05) significantly increased in the heat-stressed group. The Cdc2 activation at the 2-cell stage, as shown by the ratio of the dephosphorylated form to the phosphorylated form, was evident in control embryos but absent in heat-stressed embryos, and the level was similar to that in embryos blocked at the 2-cell stage (positive control). These results indicate that maternal heat stress enhances oxidative stress in the oviducts and that loss of developmental competence in maternally heat-stressed embryos correlates with a defect in Cdc2 activity at the 2-cell stage.     

Vertical and temporal shifts in microbial communities in the water column and sediment of saline meromictic Lake Kaiike (Japan), as determined by a 16S rDNA-based analysis, and related to physicochemical gradients

The vertical and temporal changes in microbial communities were investigated throughout the water column and sediment of the saline meromictic Lake Kaiike by PCR-denaturing gradient gel electrophoresis (DGGE) of 16S rDNA. Marked depth-related changes in microbial communities were observed at the chemocline and the sediment-water interface. However, no major temporal changes in the microbial community below the chemocline were observed during the sampling period, suggesting that the ecosystem in the anoxic zone of Lake Kaiike was nearly stable. Although the sequence of the most conspicuous DGGE band throughout the anoxic water and in the top of the microbial mat was most similar to that of an anoxic, photosynthetic, green sulphur bacterium, Pelodyction luteolum DSM273 (97% similarity), it represented a new phylotype. A comparison of DGGE banding patterns of the water column and sediment samples demonstrated that specific bacteria accumulated on the bottom from the anoxic water layers, and that indigenous microbial populations were present in the sediment. The measurements of bicarbonate assimilation rates showed significant phototrophic assimilation in the chemocline and lithoautotrophic assimilation throughout the anoxic water, but were not clearly linked with net sulphide turnover rates, indicating that sulphur and carbon metabolisms were not directly correlated.     

Neuroprotection in ischemia: blocking calcium-permeable acid-sensing ion channels

Ca2+ toxicity remains the central focus of ischemic brain injury. The mechanism by which toxic Ca2+ loading of cells occurs in the ischemic brain has become less clear as multiple human trials of glutamate antagonists have failed to show effective neuroprotection in stroke. Acidosis is a common feature of ischemia and is assumed to play a critical role in brain injury; however, the mechanism(s) remain ill defined. Here, we show that acidosis activates Ca2+ -permeable acid-sensing ion channels (ASICs), inducing glutamate receptor-independent, Ca2+ -dependent, neuronal injury inhibited by ASIC blockers. Cells lacking endogenous ASICs are resistant to acid injury, while transfection of Ca2+ -permeable ASIC1a establishes sensitivity. In focal ischemia, intracerebroventricular injection of ASIC1a blockers or knockout of the ASIC1a gene protects the brain from ischemic injury and does so more potently than glutamate antagonism. Thus, acidosis injures the brain via membrane receptor-based mechanisms with resultant toxicity of [Ca2+]i, disclosing new potential therapeutic targets for stroke.     

Entomogenous fungus Nomuraea rileyi inhibits host insect molting by C22-oxidizing inactivation of hemolymph ecdysteroids

The entomogenous fungus Nomuraea rileyi reportedly secretes a proteinaceous substance inhibiting larval molt and metamorphosis in the silkworm Bombyx mori. We studied the possibility that N. rileyi controls B. mori development by inactivating hemolymph molting hormone, ecdysteroids. Incubation of ecdysone (E) and 20-hydroxyecdysone (20E) in fungal-conditioned medium resulted in their rapid modification into products with longer retention times in reverse-phase HPLC. Each modified product from E and 20E was purified by HPLC, and identified by NMR as 22-dehydroecdysone and 22-dehydro-20-hydroxyecdysone. Some other ecdysteroids with a hydroxyl group at position C22 were also modified. Injection of the fungal-conditioned medium into Bombyx mori larvae in the mid-4th instar inhibited larval molt but induced precocious pupal metamorphosis, and its injection into 5th instar larvae just after gut purge blocked pupal metamorphosis. In hemolymph of injected larvae, E and 20E disappeared and, in turn, 22-dehydroecdysone and 22-dehydro-20-hydroxyecdysone accumulated. These results indicate that N. rileyi secretes a specific enzyme that oxidizes the hydroxyl group at position C22 of hemolymph ecdysteroids and prevents molting in B. mori larvae.     

X-Linked inhibitor of apoptosis protein is involved in mutant SOD1-mediated neuronal degeneration

Mutations in the superoxide dismutase 1 (SOD1) gene cause the degeneration of motor neurons in familial amyotrophic lateral sclerosis (FALS). An apoptotic process including caspase-1 and -3 has been shown to participate in the pathogenesis of FALS transgenic (Tg) mouse model. Here we report that IAP proteins, potent inhibitors of apoptosis, are involved in the FALS Tg mouse pathologic process. The levels of X-linked inhibitor of apoptosis protein (XIAP) mRNA and protein were significantly decreased in the spinal cord of symptomatic G93A-SOD1 Tg mice compared with littermates. In contrast, the levels of cIAP-1 mRNA and protein were increased in symptomatic G93A-SOD1 Tg mice, whereas the levels of cIAP-2 mRNA and protein were unchanged. In situ hybridization showed that the expression of XIAP was remarkably reduced in the motor neurons of Tg mice, and the expression of cIAP-1 was strongly increased in the reactive astrocytes of Tg mice. Overexpression of XIAP markedly inhibited the cell death and caspase-3 activity in the neuro2a cells expressing mutant SOD1. Deletional mutant analysis revealed that the N-terminal domain of XIAP, the BIR1-2 domains, was essential for this inhibitory activity. These results suggest that XIAP plays a role in the apoptotic mechanism in the progression of disease in mutant SOD1 Tg mice and holds therapeutic possibilities for FALS.     

Comparative effects of a recombinant and a mutein type of granulocyte colony stimulating factor on the growth of Meth-A fibrosarcoma with 5-fluorouracil chemotherapy

The present study was designed to evaluate the effects of a recombinant human G-CSF (rhG-CSF) and a mutein G-CSF(KW-2228) on leucopenia and tumor growth in mice treated with 5-fluorouracil (5-FU). In normal mice, the number of leucocytes (white blood cell, WBC) reached the peak 12 hours after a single injection of either type of G-CSF and decreased to the normal level after 24 hours. Daily administration induced a continuous increase in the WBC count, however, administrations at intervals did not. Meth-A fibrosarcoma was subcutaneously inoculated into the backs of syngeneic BALB/c mice. The mice were treated with 5-FU alone or with G-CSFs. Chemotherapy with 5-FU alone resulted in leucopenia and an insignificant inhibition of tumor growth. The conjunctive administration of G-CSFs with 5-FU resulted in a significantly augmented inhibition of tumor growth, and leukopenia was not seen. This augmenting effect was more prominent with KW-2228.These results suggest that in 5-FU chemotherapy G-CSFs may be beneficial in restoring the number of leucocytes from leucopenic state and in augmenting the tumor inhibitory effect. Furthermore, KW-2228 may be more beneficial than the natural type rhG-CSF.     

High Efficiency Gene Transfer by Multiple Transfection Protocol

A high efficiency transfection protocol employing a common polycationic lipid is described. Using LipofectAMINE, a widely used transfection reagent, we transfected 293T cells with a plasmid harboring the -galactosidase (-gal) gene. The transfection efficiency was determined by direct staining for X-gal. The conventional transfection protocol achieved an efficiency of <40% while our protocol, which employs the repetition of transfection a few times, achieved an efficiency of approximately 80%. Thus, a dramatic increase in transfection efficiency can be obtained by simply repeating transfection with the use of a common polycationic lipid. This method will be useful in many molecular biological experiments.