Improvement of canine somatic cell nuclear transfer procedure

The purpose of the present study on canine somatic cell nuclear transfer (SCNT) was to evaluate the effects of fusion strength, type of activation, culture media and site of transfer on developmental potential of SCNT embryos. We also examined the potential of enucleated bovine oocytes to serve as cytoplast recipients of canine somatic cells. Firstly, we evaluated the morphological characteristics of in vivo-matured canine oocytes collected by retrograde flushing of the oviducts 72 h after ovulation. Secondly, the effectiveness of three electrical strengths (1.8, 2.3 and 3.3 kV/cm), used twice for 20 micros, on fusion of canine cytoplasts with somatic cells were compared. Then, we compared: (1) chemical versus electrical activation (a) after parthenogenetic activation or (b) after reconstruction of canine oocytes with somatic cells; (2) culture of resulting intergeneric (IG) embryos in either (a) mSOF or (b) TCM-199. The exposure time to 6-DMAP was standardized by using bovine oocytes reconstructed with canine somatic cells. Bovine oocytes were used for SCNT after a 22 h in vitro maturation interval. The fusion rate was significantly higher in the 3.3 kV/cm group than in the 1.8 and 2.3 kV/cm treatment groups. After parthenogenesis or SCNT with chemical activation, 3.4 and 5.8%, respectively, of the embryos developed to the morula stage, as compared to none of the embryos produced using electrical activation. Later developmental stages (8-16 cells) were transferred to the uterine horn of eight recipients, but no pregnancy was detected. However, IG cloned embryos (bovine cytoplast/canine somatic cell) were capable of in vitro blastocyst development. In vitro developmental competence of IG cloned embryos was improved after exposure to 6-DMAP for 4 h as compared to 0, 2 or 6h exposure, although the increase was not significantly different among culture media. In summary, for production of canine SCNT embryos, we recommend fusion at 3.3 kV/cm, chemical activation, culture in mSOF medium and transfer of presumptive zygotes to the oviduct of recipient animals. The feasibility of IG production of cloned canine embryos using bovine cytoplasts as recipient of canine somatic cells was demonstrated.     

Functional analysis of Streptococcus pyogenes nuclease A (SpnA), a novel group A streptococcal virulence factor

Streptococcus pyogenes nuclease A (SpnA) is a recently discovered DNase that plays a role in virulence as shown in a mouse infection model. SpnA is the only cell wall-anchored DNase found in S. pyogenes thus far and shows a unique protein architecture. The C-terminal nuclease domain contains highly conserved catalytic site and Mg(2+) binding site residues. However, expression of the SpnA nuclease domain alone resulted in a soluble, but enzymatically inactive protein. We found that at least two out of three oligonucleotide/oligosaccharide-binding fold motifs found in the N-terminal domain are required for SpnA activity, probably contributing to substrate binding. Using a combination of a spnA deletion mutant and a Lactococcus lactis'gain-of-function' mutant, we have shown that SpnA promotes survival in whole human blood and in neutrophil killing assays and this is, at least in part, achieved by the destruction of neutrophil extracellular traps (NETs). We observed higher frequencies for anti-SpnA antibodies in streptococcal disease patient sera (79%, n = 19) compared with sera from healthy donors (33%, n = 9) suggesting that SpnA is expressed during infection. Detection of anti-SpnA antibodies in patient serum might be useful for the diagnostic of post-streptococcal diseases, such as acute rheumatic fever or glomerulonephritis.     

The fermentation process for l-phenylalanine production using an auxotrophic regulatory mutant of Escherichia coli

Summary A process for l-phenylalanine production was studied using a tyrosine auxotrophic regulatory mutant of Escherichia coli, resistant to both -2-thienyl-dl-alanine and p-fluoro-dl-phenylalanine. Fermentations were carried out in a 30-1 fermentor with intermittent feeding of glucose plus phosphate. The mutant accumulated l-phenylalanine in the fermentation broth up to 15 g/l at pH 7.0 and 33°C. Column chromatography on a strong cation exchanger was employed as the most effective step in the purification of l-phenyl-alanine from the broth. This step brought about 4-fold concentration of the product with 96% recovery.     

Effects of NaCl stress on germination, antioxidant responses, and proline content in two rice cultivars

We investigated the physiological and biochemical bases for salt tolerance in two rice (Oryza sativa L.) cultivars — relatively salt-tolerant ‘Dongjin’ and salt-sensitive ‘Kumnam’. Salinized hydroponic cultures were studied at the germination and seedling stages. NaCI inhibited germination more severely in ‘Kumnam’ than in ‘Dongjin’. Increasing the salt concentration also deterred growth to a larger extent in the former. Moreover, the leaves of ‘Kumnam’ exhibited greater increases in lipid peroxidation and Na⁺ accumulation than those of ‘Dongjin’ under stress. The activities of constitutive and salt-induced superoxide dismutase (SOD, EC 1.15.1.1) and ascorbate peroxidase (AP, EC 1.11.1.11) were also higher in ‘Kumnam’, while only catalase (CAT, EC 1.11.1.6) activity was slightly higher in stressed plants of ‘Dongjin’. The positive correlation between leaf proline levels and NaCI concentration was more evident in ‘Kumnam’. However, ‘Dongjin’ seeds, which had higher germinability in the presence of NaCI, also contained more proline. These results suggest that the higher salt tolerance in ‘Dongjin’ seedlings could be ascribed to their lower NaCI accumulations in the leaves. This presumably is due to reductions in the uptake or transport rates of saline ions to the shoots from the roots. Finally, we believe that the higher germination rate by ‘Dongjin’ is caused by its higher seed proline content.     

Separation of protein and fatty acids from tuna viscera using supercritical carbon dioxide

Supercritical carbon dioxide extraction was investigated as a method for removing lipids and bad flavor from tuna viscera. To find the optimum conditions, different experimental variables, such as pressure, temperature, flow rate of solvent and sample size, were evaluated for the effective removal of lipids and the undesirable smell. Ethanol was used as the entrainer, with a 3% by vol CO₂ flow rate. By increasing the pressure at constant temperature, the efficiency of the lipid removal was improved and the protein was concentrated without denaturalization. The main fatty acids extracted from the tuna viscera were palmitic acid (16∶0), heptadecanoic acid (17∶1), oleic acid (18∶1) and docosahexaenoic acid (22∶6). The major amino acids in the tuna viscera treated by supercritical carbon dioxide were glutamic acid, leucine and lysine, and the free amino acids werel-proline, taurine andl-α-aminoadipic acid.     

The InR/Akt/TORC1 Growth-Promoting Signaling Negatively Regulates JAK/STAT Activity and Migratory Cell Fate during Morphogenesis

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Modeling Calcium Wave Based on Anomalous Subdiffusion of Calcium Sparks in Cardiac Myocytes

sparks and waves play important roles in calcium release and calcium propagation during the excitation-contraction (EC) coupling process in cardiac myocytes. Although the classical Fick’s law is widely used to model sparks and waves in cardiac myocytes, it fails to reasonably explain the full-width at half maximum(FWHM) paradox. However, the anomalous subdiffusion model successfully reproduces sparks of experimental results. In this paper, in the light of anomalous subdiffusion of sparks, we develop a mathematical model of calcium wave in cardiac myocytes by using stochastic release of release units (CRUs). Our model successfully reproduces calcium waves with physiological parameters. The results reveal how concentration waves propagate from an initial firing of one CRU at a corner or in the middle of considered region, answer how large in magnitude of an anomalous spark can induce a wave. With physiological currents (2pA) through CRUs, it is shown that an initial firing of four adjacent CRUs can form a wave. Furthermore, the phenomenon of calcium waves collision is also investigated.