Effect of polyethylene glycol and dimethyl sulfoxide on the fusion of bovine nuclear transfer using mammary gland epithelial cells

The effects of polyethylene glycol and dimethyl sulfoxide (PEG/DMSO) treatment of donor cells on the fusion and subsequent development of bovine nuclear transfer embryos using mammary gland epithelial (MGE) cells before electrofusion (fresh MGE cells) was studied. The same study was conducted on those cells that were frozen and stored in liquid nitrogen, and then thawed (frozen-thawed MGE cells). Experiment 1 showed that the exposure time and pH of PEG/DMSO solution affected the fusion of nuclear transfer, and that a higher fusion rate was obtained when fresh MGE cells were exposed to PEG/DMSO solution at pH 8.0 for 5 min. In Experiment 2, the proportion of fused oocytes with fresh PEG/DMSO-treated cells (70 +/- 6%) was significantly higher than that with non-treated cells (50 +/- 13%, p < 0.05). The same tendency was observed when frozen-thawed cells as donor nuclei were used (48 +/- 6% vs. 34 +/- 12%, p < 0.05). In addition, PEG/DMSO treatment has neither harmful nor beneficial effects on the cleavage and development of the blastocyst stage of reconstructed embryos (p > 0.05). The fusion and cleavage rates of frozen-thawed cells were significantly lower than those of fresh cells (p < 0.05). After 10 blastocysts, derived from fresh PEG/DMSO-treated cells, were transferred to five recipient heifers, one live female calf was obtained. Experiment 3 showed that PEG/DMSO treatment reduced the viability of both fresh and frozen-thawed MGE cells (p < 0.05). We conclude that the PEG/DMSO treatment of fresh MGE cells, as well as the frozen-thawed cells, before electrofusion has a positive effect on the fusion of nuclear transfer without decreasing the in vitro development of reconstructed embryos.     

Sorting specificity of spermatogenic cell specific region of mouse hexokinase-s (mHk1-s)

Hexokinase is the first enzyme involved in the glycolysis process that produces glucose phosphorylate. Our previous study reported on our cloning of mouse Hk1-s (mHk1-s) cDNA, which were expressed only in testis cells, and noted that this cDNA has a spermatogenic cell-specific region (SSR) that replaces the porin binding domain (PBD) in the Hk1of somatic cells. Although we know that PBD binds to the outer membrane of a mitochondrion, the role of the SSR is not yet understood. To investigate the intracellular localization of SSR, we constructed expression vectors with the epitope tag (GFP-, HA-), subcloned SSR, or PBD cDNA. We transfected these vectors in mouse fibroblast, NIH3T3 cells, after which we observed the localization of the SSR and PBD in the NIH3T3 cells. Our current study using the immunocytochemical method revealed that PBD is concentrated around the mitochondrion. However, the SSR could not be ascribed to the mitochondrion, ER, or nuclear colocalization. Moreover, subcellular fractionation analysis showed that PBD was detected in the mitochondrial fraction, and that SSR was detected in the cytosolic fraction. Our findings suggest that PBD of Hk1 targets mitochondrion, but the SSR of mHk1-s targets some specific organellae.     

A potential mechanism for the impairment of nitric oxide formation caused by prolonged oral exposure to arsenate in rabbits

We have recently found evidence for impairment of nitric oxide (NO) formation and induction of oxidative stress in residents of an endemic area of chronic arsenic poisoning in Inner Mongolia, China. To investigate the underlying mechanisms responsible for these phenomena, a subchronic animal experiment was conducted using male New Zealand White rabbits. After 18 weeks of continuous exposure of rabbits to 5 mg/l of arsenate in drinking water, a significant decrease in systemic NO production occurred, as shown by significantly reduced plasma NO metabolites levels (76% of control) and a tendency towards decreased serum cGMP levels (81.4% of control). On the other hand, increased oxidative stress, as shown by significantly increased urinary hydrogen peroxide (H(2)O(2)) (120% of control), was observed in arsenate-exposed rabbits. In additional experiments measuring aortic tension, the addition of either the calcium ionophore A23187 or acethylcholine (ACh) induced a transient vasoconstriction of aortic rings prepared from arsenate-exposed rabbits, but not in those prepared from control animals. This calcium-dependent contractility action observed in aorta rings from arsenate-exposed rabbits was markedly attenuated by the superoxide (O2(.-)) scavenging enzyme Cu, Zn-SOD, as well as diphenyleneiodonium (DPI) or N(G)-nitro-L-arginine methyl ester (L-NAME), which are inhibitors for nitric oxide synthase (NOS). However, the cyclooxygenase inhibitor indomethacin or the xanthine oxidase blocker allopurinol had no effect on this vasoconstriction. These results suggest that arsenate-mediated reduction of systemic NO may be associated with the enzymatic uncoupling reaction of NOS with a subsequent enhancement of reactive oxygen species such as O2(.-), an endothelium-derived vasoconstricting factor. Furthermore, hepatic levels of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH(4)), a cofactor for NOS, were markedly reduced in arsenate-exposed rabbits to 62% of control, while no significant change occurred in cardiac L-arginine levels. These results suggest that prolonged exposure of rabbits to oral arsenate may impair the bioavailability of BH(4) in endothelial cells and, as a consequence, disrupt the balance between NO and O2(.-) produced from endothelial NOS, such that enhanced free radicals are produced at the expense of NO.     

Calcium Antagonist Isradipine Reduces Metabolic Alterations in Acute Cerebral Ischemia in Spontaneously Hypertensive Rats

The present study was designed to examine the effect of a calcium antagonist isradipine (PN200-110: PN) on local cerebral blood flow and brain tissue metabolism after 1-hour supratentorial ischemia induced by bilateral carotid artery ligation (BCL) in spontaneously hypertensive rats (SHR). PN, dissolved in ethanol plus polyethylene glycol 400, diluted with saline to make the final concentration of 0.25mg/ml and 2.5mg/ml, was administered subcutaneously either 30 min prior to BCL or just after the induction of incomplete cerebral ischemia (n = 7 in each group). Vehicle injection was served as a control group (n = 7). Cerebral blood flow in the parietal cortex (CBF) and the cerebellar cortex (CeBF) was measured by hydrogen clearance technique, and the supra- and infratentorial metabolites of the brain frozen in situ were determined by the enzymatic method. Blood pressure was lowered, but CBF was increased by PN administration in pre-BCL treatment study. After 1 hour of BCL, CBF decreased to around 10% or less of the resting value, being insignificant among the groups. Brain adenosine triphosphate was better preserved in PN-administered groups. The increase in lactate level tended to reduce dose dependently by PN treatment. PN also reduced the metabolic alterations in brain tissue with significance, even when administered just after the induction of forebrain ischemia. It is considered that pre- as well as post-BCL administration of PN is beneficial to attenuate the metabolic alterations in incomplete forebrain ischemia in SHR.     

Adaptive Responses Limited by Intrinsic Noise

Sensory systems have mechanisms to respond to the external environment and adapt to them. Such adaptive responses are effective for a wide dynamic range of sensing and perception of temporal change in stimulus. However, noise generated by the adaptation system itself as well as extrinsic noise in sensory inputs may impose a limit on the ability of adaptation systems. The relation between response and noise is well understood for equilibrium systems in the form of fluctuation response relation. However, the relation for nonequilibrium systems, including adaptive systems, are poorly understood. Here, we systematically explore such a relation between response and fluctuation in adaptation systems. We study the two network motifs, incoherent feedforward loops (iFFL) and negative feedback loops (nFBL), that can achieve perfect adaptation. We find that the response magnitude in adaption systems is limited by its intrinsic noise, implying that higher response would have higher noise component as well. Comparing the relation of response and noise in iFFL and nFBL, we show that whereas iFFL exhibits adaptation over a wider parameter range, nFBL offers higher response to noise ratio than iFFL. We also identify the condition that yields the upper limit of response for both network motifs. These results may explain the reason of why nFBL seems to be more abundant in nature for the implementation of adaption systems.     

Quantitative Mass Density Image Reconstructed from the Complex X-Ray Refractive Index

We demonstrate a new analytical X-ray computed tomography technique for visualizing and quantifying the mass density of materials comprised of low atomic number elements with unknown atomic ratios. The mass density was obtained from the experimentally observed ratio of the imaginary and real parts of the complex X-ray refractive index. An empirical linear relationship between the X-ray mass attenuation coefficient of the materials and X-ray energy was found for X-ray energies between 8 keV and 30 keV. The mass density image of two polymer fibers was quantified using the proposed technique using a scanning-type X-ray microbeam computed tomography system equipped with a wedge absorber. The reconstructed mass density agrees well with the calculated one.     

Occurrence of a novel strain of Scirtothrips dorsalis (Thysanoptera: Thripidae) in Japan and development of its molecular diagnostics

Scirtothrips dorsalis Hood is a cosmopolitan and polyphagous thrips species. Recently, a novel strain of S. dorsalis attacking capsicum crops was found in Japan. A molecular phylogenetic analysis using mitochondrial cytochrome c oxidase subunit I sequences revealed that the capsicum-associated populations were genetically different from Japanese native strains and were closely related to Southeast Asian populations. We named the capsicum-associated populations “strain C” and the Japanese native ones “strain YT”. A total of 10 haplotypes were found in strain C and 26 in strain YT. To differentiate the two strains, we developed a multiplex-PCR method using the ribosomal ITS2 region.     

A conserved α helix of Bcs1, a mitochondrial AAA chaperone,is required for the Respiratory Complex III maturation

Bcs1 is a transmembrane chaperone in the mitochondrial inner membrane, and is required for the mitochondrial Respiratory Chain Complex III assembly. It has been shown that the highly-conserved C-terminal region of Bcs1 including the AAA ATPase domain in the matrix side is essential for the chaperone function. Here we describe the importance of the N-terminal short segment located in the intermembrane space in the Bcs1 function. Among the N-terminal 44 amino acid residues of yeast Bcs1, the first 37 residues are dispensable whereas a hydrophobic amino acid in the residue 38 is essential for integration of Rieske Iron-sulfur Protein into the premature Complex III from the mitochondrial matrix. Substitution of the residue 38 by a hydrophilic amino acid residue affects conformation of Bcs1 and interactions with other proteins. The evolutionarily-conserved short α helix of Bcs1 in the intermembrane space is an essential element for the chaperone function.     

Activation of activin type IB receptor signals in pancreatic β cells leads to defective insulin secretion through the attenuation of ATP-sensitive K channel activity

In studies of gene-ablated mice, activin signaling through activin type IIB receptors (ActRIIB) and Smad2 has been shown to regulate not only pancreatic β cell mass but also insulin secretion. However, it still remains unclear whether gain of function of activin signaling is involved in the modulation of pancreatic β cell mass and insulin secretion. To identify distinct roles of activin signaling in pancreatic β cells, the Cre-loxP system was used to activate signaling through activin type IB receptor (ActRIB) in pancreatic β cells. The resultant mice (pancreatic β cell-specific ActRIB transgenic (Tg) mice; ActRIBCAβTg) exhibited a defect in glucose-stimulated insulin secretion (GSIS) and a progressive impairment of glucose tolerance. Patch-clamp techniques revealed that the activity of ATP-sensitive K⁺ channels (K_ATP channels) was decreased in mutant β cells. These results indicate that an appropriate level of activin signaling may be required for GSIS in pancreatic β cells, and that activin signaling involves modulation of K_ATP channel activity.     

Effects of type III antifreeze protein on sperm and embryo cryopreservation in rabbit

We investigated the effects of antifreeze protein (AFP) III supplementation on the cryopreservation of rabbit sperm cells and embryos. Ejaculated semen was collected from male Japanese white (JW) rabbits and divided into four AFP-supplemented groups (0.1 μg/ml, 1 μg/ml, 10 μg/ml, 100 μg/ml) and one control group with no AFP-supplementation. The semen samples were treated with egg-yolk HEPES extender containing 6% acetamide before the sperm was cooled from room temperature to 5 °C, then packed into sperm straws. The straws were frozen in steam of liquid nitrogen (LN₂) and then preserved in the LN₂. The motility of the sperm after thawing in 37 °C water was analyzed. The percentage of rapidly motile sperm in the 1 μg/ml AFP group was significantly higher than in the control group. Morulae were collected from female JW rabbits and divided into three AFP-supplemented groups (100 ng/ml, 500 ng/ml, 1000 ng/ml) and one control group. The morulae, immersed in an embryo-freezing solution (M199-HEPES containing 20% ethylene glycol, 20% dimethylsulfoxide, 10% fetal bovine serum and 0.25 M sucrose), were packed into open pulled embryo straws and vitrified in LN₂. The frozen embryos were thawed in the embryo-freezing solution, and the rates of embryo survival and development to blastocyte stage were analyzed after incubation for 72 h. The development rate of the embryos in the 500 ng/ml AFP group was significantly higher than in the control group, but that in the 1000 ng/ml AFP group was significantly lower. In conclusion, the appropriate dose of AFP III increased the number of rapidly motile sperm and embryo survival following freezing and thawing. The results suggest that supplementation with AFP III can increase the efficiency of cryopreservation of rabbit sperm cells and embryos.