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.     

KRN5500, a novel antitumor agent, induces apoptosis or cell differentiation in HL-60 cells

BACKGROUND: KRN5500, a derivative of spicamycin, shows antitumor activity against a variety of tumor cell lines. However, the mechanism of cytotoxic action has remained unclear. METHODS: The viability of HL-60 human leukemic cells treated with KRN5500 was studied by the dye exclusion assay. Induction of apoptosis and effects on the cell cycle were investigated by flow cytometry: We measured cellular DNA content after extraction of fragmented DNA, and apoptosis-induced DNA strand breaks. Cell morphology was observed by light microscopy. DNA strand breaks at a nucleosomal unit were analyzed by electrophoresis. RESULTS: Our data demonstrated that KRN5500 caused inhibition of cell growth, and that apoptosis was the mode of cell death. G(1) phase cells were more susceptible to KRN5500 induced apoptosis. In addition, KRN5500 induced cell differentiation at lower concentration. CONCLUSIONS: It is anticipated that KRN5500 will be used clinically as an anti-leukemic agent. Its mechanism of antitumor action is to induce apoptosis or cell differentiation.     

Inotropic, chronotropic, and dromotropic effects mediated via parasympathetic ganglia in the dog heart

Some parasympathetic ganglionic cells are located in the epicardial fat pad between the medial superior vena cava and the aortic root (SVC-Ao fat pad) of the dog. We investigated whether the ganglionic cells in the SVC-Ao fat pad control the right atrial contractile force, sinus cycle length (SCL), and atrioventricular (AV) conduction in the autonomically decentralized heart of the anesthetized dog. Stimulation of both sides of the cervical vagal complexes (CVS) decreased right atrial contractile force, increased SCL, and prolonged AV interval. Stimulation of the rate-related parasympathetic nerves to the sinoatrial (SA) node (SAPS) increased SCL and decreased atrial contractile force. Stimulation of the AV conduction-related parasympathetic nerves to the AV node prolonged AV interval. Trimethaphan, a ganglionic nicotinic receptor blocker, injected into the SVC-Ao fat pad attenuated the negative inotropic, chronotropic, and dromotropic responses to CVS by 33 approximately 37%. On the other hand, lidocaine, a sodium channel blocker, injected into the SVC-Ao fat pad almost totally inhibited the inotropic and chronotropic responses to CVS and partly inhibited the dromotropic one. Lidocaine or trimethaphan injected into the SAPS locus abolished the inotropic responses to SAPS, but it partly attenuated those to CVS, although these treatments abolished the chronotropic responses to SAPS or CVS. These results suggest that parasympathetic ganglionic cells in the SVC-Ao fat pad, differing from those in SA and AV fat pads, nonselectively control the atrial contractile force, SCL, and AV conduction partially in the dog heart.     

NdWFamide: a novel excitatory peptide involved in cardiovascular regulation of Aplysia

Although diverse peptides are known to affect invertebrate cardiac activity, the peptidergic regulation of the cardiovascular system of Aplysia is still poorly understood. Asn-D-Trp-Phe-NH(2) (NdWFamide) is a recently purified cardioactive peptide in Aplysia. Pharmacological experiments showed that NdWFamide was one of the most potent cardioexcitatory peptides among the known endogenous cardioactive peptides in Aplysia. NdWFamide-immunopositive neuronal processes were abundant in the cardiovascular region of Aplysia, and many of them originated from neurosecretory cells in the abdominal ganglion (R3-R13 cells). The data suggest that NdWFamide is a cardioexcitatory peptide utilized by R3-R13 cells of Aplysia.     

Factors influencing lifespan dependency on agricultural crops by brown bears

Landscape and Ecological Engineering - Investigating factors underlying human-wildlife conflicts in agricultural landscapes is important for both preventing crop damage and wildlife conservation....     

Booster effect of canine distemper, canine parvovirus infection and infectious canine hepatitis combination vaccine in domesticated adult dogs

Domesticated adult dogs with antibody titer classified as below 'high' to one or more of canine distemper virus (CDV), canine parvovirus type-2 (CPV-2) and canine adenovirus type-1 (CAdV-1) were then given an additional inoculation, and the effectiveness of this booster evaluated 2 months later. Consequently, CDV and CAdV-1 antibody titer experienced a significant increase, but the same effect was not observed in the antibody titer of CPV-2. These findings suggest that with additional inoculation, a booster effect may be expected in increasing antibody titers for CDV and CAdV-1, but it is unlikely to give an increase in CPV-2 antibody titer.     

Sequence Analysis of Amplified DNA Fragments Containing the Region Encoding the Putative Lipase Substrate-Binding Domain and Genotyping of Aeromonas hydrophila

DNA fragments were amplified by PCR from all tested strains of Aeromonas hydrophila, A. caviae, and A. sobria with primers designed based on sequence alignment of all lipase, phospholipase C, and phospholipase A1 genes and the cytotonic enterotoxin gene, all of which have been reported to have the consensus region of the putative lipase substrate-binding domain. All strains showed lipase activity, and all amplified DNA fragments contained a nucleotide sequence corresponding to the substrate-binding domain. Thirty-five distinct nucleotide sequence patterns and 15 distinct deduced amino acid sequence patterns were found in the amplified DNA fragments from 59 A. hydrophila strains. The deduced amino acid sequences of the amplified DNA fragments from A. caviae and A. sobria strains had distinctive amino acids, suggesting a species-specific sequence in each organism. Furthermore, the amino acid sequence patterns appear to differ between clinical and environmental isolates among A. hydrophila strains. Some strains whose nucleotide sequences were identical to one another in the amplified region showed an identical DNA fingerprinting pattern by repetitive extragenic palindromic sequence-PCR genotyping. These results suggest that A. hydrophila, and also A. caviae and A. sobria strains, have a gene encoding a protein with lipase activity. Homologs of the gene appear to be widely distributed in Aeromonas strains, probably associating with the evolutionary genetic difference between clinical and environmental isolates of A. hydrophila. Additionally, the distinctive nucleotide sequences of the genes could be attributed to the genotype of each strain, suggesting that their analysis may be helpful in elucidating the genetic heterogeneity of Aeromonas.