Angiopoietin-1 protects heart against ischemia/reperfusion injury through VE-cadherin dephosphorylation and myocardiac integrin-β1/ERK/caspase-9 phosphorylation cascade

Early reperfusion after myocardial ischemia that is essential for tissue salvage also causes myocardial and vascular injury. Cardioprotection during reperfusion therapy is an essential aspect of treating myocardial infarction. Angiopoietin-1 is an endothelial-specific angiogenic factor. The potential effects of angiopoietin-1 on cardiomyocytes and vascular cells undergoing reperfusion have not been investigated. We propose a protective mechanism whereby angiopoietin-1 increases the integrity of the endothelial lining and exerts a direct survival effect on cardiomyocytes under myocardial ischemia followed by reperfusion. First, we found that angiopoietin-1 prevents vascular leakage through regulating vascular endothelial (VE)-cadherin phosphorylation. The membrane expression of VE-cadherin was markedly decreased on hypoxia/reoxygenation but was restored by angiopoietin-1 treatment. Interestingly, these effects were mediated by the facilitated binding between SH2 domain-containing tyrosine phosphatase (SHP2) or receptor protein tyrosine phosphatase μ (PTPμ) and VE-cadherin, leading to dephosphorylation of VE-cadherin. siRNA against SHP2 or PTPμ abolished the effect of angiopoietin-1 on VE-cadherin dephosphorylation and thereby decreased levels of membrane-localized VE-cadherin. Second, we found that angiopoietin-1 prevented cardiomyocyte death, although cardiomyocytes lack the angiopoietin-1 receptor Tie2. Angiopoietin-1 increased cardiomyocyte survival through integrin-β1-mediated extracellular signal-regulated kinase (ERK) phosphorylation, which inhibited caspase-9 through phosphorylation at Thr12? and subsequently reduced active caspase-3. Neutralizing antibody against integrin-β1 blocked these protective effects. In a mouse myocardial ischemia/reperfusion model, angiopoietin-1 enhanced cardiac function and reduction in left ventricular-end systolic dimension (LV-ESD) and left ventricular-end diastolic dimension (LV-EDD) with an increase in ejection fraction (EF) and fractional shortening (FS). Our findings suggest the novel cardioprotective mechanisms of angiopoietin-1 that are achieved by reducing both vascular leakage and cardiomyocyte death after ischemia/reperfusion injury.     

Rapid determination of perv copy number from porcine genomic DNA by real-time polymerase chain reaction

Here, we report the quantification of porcine endogenous retrovirus (PERV) copy numbers using real time PCR. After generating standard curves using plasmid DNA, copy numbers were determined for PERV pol and for a housekeeping gene, porcine estrogen receptor2 (ER2) with the same amount of genomic DNA. Using this method, we examined 6 pig breeds in Korea including two breeds of miniature pig, one domestic pig from Jeju, and imported pig breeds, Duroc, Landrace, and Yorkshire. All breeds showed PERV copy numbers ranging from 9 to 50. This method will be useful for monitoring of PERVs in a porcine xenograft.     

Enhancement of protein secretion by TatAC overexpression in <Emphasis Type="Italic">Streptomyces griseus</Emphasis>

Production of proteins in secretary form is one of the important factors affecting fermentation. The Tat (twin arginine translocation) protein secretion system, which includes the proteins TatA, TatB, and TatC, was identified in the genomic sequence of Streptomyces griseus IFO13350. The tatA and tatC genes were organized into a polycistronic operon, whereas tatB was located separately on the chromosome. Comparison of amino acid sequences suggested that TatC was a membrane-spanning protein, whereas TatA and TatB were found to be cytoplasmic proteins. Analysis of extracellular proteins and N-terminal amino acid sequencing revealed that secretion of SGR5556 was significantly enhanced by overexpression of TatAC in S. griseus HH1. Further, enzymatic study showed that SGR5556 encoded a glycerophosphoryl diester phosphodiesterase. In addition, other hydrolase activities, such as those of amylase, total protease, metalloprotease, trypsin, chymotrypsin, and Leuaminopeptidase, were also enhanced by 3, 3, 2.6, 2.3, 5.4, and 2.5 fold, respectively, in S. griseus upon TatAC overexpression. Overexpression of TatAC induced the production of a greenish-yellow pigment in S. griseus HH1 as well as more abundant sporulation at an earlier stage in Streptomyces coelicolor A3(2). In silico analysis by TatFIND, SignalP, and TMHMM identified 19 binding proteins, 28 enzymatic proteins, and 27 other proteins with unknown functions as putative TatAC-dependent secretary proteins. These results clearly indicate that TatA and TatC constitute a functional Tat system in S. griseus. Additionally, the S. griseus Tat system can be useful for the production of valuable proteins, including many hydrolytic enzymes and candidates of Tat-dependent secretary proteins, under industrial conditions.     

Biomass and Carbon Storage in an Age-Sequence of Korean Pine (<Emphasis Type="Italic">Pinus koraiensis</Emphasis>) Plantation Forests in Central Korea

This study examined the biomass and carbon pools of the main ecosystem components in an age sequence of five Korean pine plantation forest stands in central Korea. The C contents in the tree and ground vegetation biomass, coarse woody debris, forest floor, and mineral soil were estimated by analyzing the C concentration of each component. The aboveground and total tree biomass increased with increasing stand age. The highest C concentration across this chronosequence was found in the tree branch while the lowest C concentration was found in the ground vegetation. The observed C contents for tree components, ground vegetation, and coarse woody debris were generally lower than the predicted C contents estimated from a biomass C factor of 0.5. Forest floor C content was age-independent. Total mineral soil C content appeared to decline initially after establishing Korean pine plantations and recover by the stand age of 35 years. Although aboveground tree biomass C content showed considerable accumulation with increasing age, the relative contribution of below ground C to total ecosystem C content varied substantially. These results suggest that successional development as temporal factor has a key role in estimating the C storage in Korean pine plantation forests.     

The Protective Effect of ENA Actimineral Resource A on CCl4-Induced Liver Injury in Rats

ENA Actimineral Resource A (ENA-A) is alkaline water that is composed of refined edible cuttlefish bone and two different species of seaweed, Phymatolithon calcareum and Lithothamnion corallioides. In the present study, ENA-A was investigated as an antioxidant to protect against CCl(4)-induced oxidative stress and hepatotoxicity in rats. Liver injury was induced by either subacute or chronic CCl(4) administration, and the rats had free access to tap water mixed with 0% (control group) or 10% (v/v) ENA-A for 5 or 8?weeks. The results of histological examination and measurement of antioxidant activity showed that the reactive oxygen species production, lipid peroxidation, induction of CYP2E1 were decreased and the antioxidant activity, including glutathione and catalase production, was increased in the ENA-A groups as compared with the control group. On 2-DE gel analysis of the proteomes, 13 differentially expressed proteins were obtained in the ENA-A groups as compared with the control group. Antioxidant proteins, including glutathione S-transferase, kelch-like ECH-associated protein 1, and peroxiredoxin 1, were increased with hepatocyte nuclear factor 3-beta and serum albumin precursor, and kininogen precursor decreased more in the ENA-A groups than compared to the control group. In conclusion, our results suggest that ENA-A does indeed have some protective capabilities against CCl(4)-induced liver injury through its antioxidant function.     

The Effect of Ultrasonificated Extracts of <Emphasis Type="Italic">Spirulina maxima</Emphasis> on the Anticancer Activity

The effect of ultrasonic extraction on extraction yields, cytotoxicity, and anticancer activity of Spirulina maxima was investigated in this study. Optimal extraction conditions were determined as 60 kHz frequency at 60°C for 30 min with 120 W intensity, which resulted in 19.3% of extraction yields and 19.1% of cytotoxicity on normal human cells. Yields from conventional water and ethanol extraction were 15.8% at 100°C and 8.3% at 80°C, respectively. It was found that the extracts obtained by ultrasonic extraction process selectively inhibited the digestive-related cancer cell lines, such as human stomach cancer cells, having 89% of the highest inhibition ratio and 4.5 of the highest selectivity. In adding 0.5 mg/mL of the extract, human promyelocytic leukemia cells' cell differentiation was increased 1.72 times over that of the control. Expression level of B cell lymphoma-2 from Hep3B cell was also effectively suppressed by the extract obtained at 60 kHz and 60°C, leading to the inhibition of the early step of carcinogenesis. This work suggests that anticancer activity of the extracts is due to water-soluble polysaccharides rather than proteins and is further supported by the result that the ultrasonification extraction process can efficiently extract relatively intact polysaccharides rather than digesting the proteins in S. maxima by matrix assisted laser desorption ionization-time of flight and high performance size exclusion chromatography chromatogram analyses. Therefore, ultrasonic extraction increases both extraction yield and the biological activity of S. maxima extracts, which might be useful as an alternative natural anticancer agent in the medical and food industries.     

Characterization of <Emphasis Type="Italic">Bacillus</Emphasis> phage-K2 isolated from chungkookjang,a fermented soybean foodstuff

An investigation of a virulent Bacillus phage-K2 (named Bp-K2) isolated from chungkookjang (a fermented soybean foodstuff) was made. Bp-K2 differed in infectivity against a number of Bacillus subtilis strains including starter strains of chungkookjang and natto, being more infectious to Bacillus strains isolated from the chungkookjang, but much less active against a natto strain. Bp-K2 is a small DNA phage whose genome size is about 21 kb. Bp-K2 is a tailed bacteriophage with an isometric icosahedral head (50 nm long on the lateral side, 80 nm wide), a long contractile sheath (85–90 nm × 28 nm), a thin tail fiber (80–85 nm long, 10 nm wide), and a basal plate (29 nm long, 47 nm wide) with a number of spikes, but no collar. The details of the structures of Bp-K2 differ from natto phage ϕBN100 as well as other known Bacillus phages such as SPO1-like or ϕ 29-like viruses. These data suggest that Bp-K2 would be a new member of the Myoviridae family of Bacillus bacteriophages.