Kim, Ji-Hyun;Kim, Sul Ki;Ko, Eun Hye;Kim, Jin-Cheol;Kim, Jin-Seog
Weed & Turfgrass Science
/
v.2
no.2
/
pp.176-183
/
2013
To explore hydrolysis methods for the efficient manufacture of sugar solutions from the freshwater alga Water-net (Hydrodictyon reticulatum, HR), acid hydrolysis, enzymatic hydrolysis, and combined hydrolysis (acid followed by enzymatic hydrolysis) were investigated. In the one-step acid hydrolysis, the reaction of 8% solids content using 2% sulfuric acid at $120^{\circ}C$ for 1 hour was desirable. In this case, glucose 27.44 g 100 g $DM^{-1}$ could be obtained from the HR-d13 samples. In the two-step acid hydrolysis, the primary hydrolysis (HR powder : 72% sulfuric acid = 1 g : 1.5 mL) was carried out for 1 hour at $60^{\circ}C$, and then the secondary hydrolysis was done for 1 hour at $120^{\circ}C$ after addition of distilled water 23.5 mL. In this case, glucose 35.11 g/100 g DM could be obtained from the HR-d13 samples. In the combined hydrolysis, 25% solids content using 2% hydrochloric acid were reacted for 1 hour at $120^{\circ}C$, and then citrate buffer and hydrolysis enzyme complexes (E1 1.0 mL+E2 0.2 mL $g^{-1}$ dried matter) were added and reacted for 1 - 2 days at $50^{\circ}C$. In this case, glucose 33.5 g 100 g $DM^{-1}$ could be obtained from the HR-d23+26 samples. In conclusion, combined hydrolysis was likely to be more useful saccharification method of HR biomass at a practical level, considering the glucose productivity, generation of fermentation-inhibiting substances (hydroxyl methyl furfural, furfural), and limited use of strong acid.
Kim, Chang-Won;Park, Jin-Woo;Choi, Hyuk-Joon;Han, Bok-Kyung;Yoo, Seung-Seok;Kim, Byung-Yong;Baik, Moo-Yeol;Kim, Young-Rok
Journal of Life Science
/
v.21
no.2
/
pp.309-315
/
2011
Rice syrup meal (RSM) was enzymatically hydrolyzed using eight commercial proteases (Protamex, Neutrase, Flavourzyme, Alcalase, Protease M, Protease N, Protease A, Molsin F) for 4 hr at optimum pH and temperature. Proteolytic hydrolysates were examined in supernatant and precipitate using Lowry protein assay, semimicro Kjeldahl method and gravimetric method using weight difference before and after enzymatic hydrolysis. Although RSM contains a high amount of protein (71.2%), only a very small amount of protein was hydrolyzed. Two proteases (Protease M and Protease N) were found to be the most effective in the hydrolysis of RSM protein. In Lowry method, 57.5 and 59.0 mg protein/g RSM were hydrolyzed after Protease M and Protease N treatments, respectively. In gravimetric method, 80.0 and 85.4 mg protein/g RSM were hydrolyzed after Protease M and Protease N treatments. In Kjeldahl method, 67.43 and 70.43 mg protein/g RSM were hydrolyzed after Protamex and Protease N treatments, respectively. For synergistic effect, two or three effective commercial proteases (Protease M, Protease N and Protease A) were applied to RSM at one time. The highest hydrolysis of RSM protein was observed in both Lowry protein assay (80.3 mg protein/g RSM) and gravimetric methods (153.2 mg protein/g RSM) when three commercial proteases were applied at one time, suggesting the synergistic effect of those proteases.
Hwang, Jong Hee;Sung, Dong Kyung;Choi, Chang Won;Kang, Saem;Chang, Yun Sil;Park, Won Soon;Lee, Munhyang
Clinical and Experimental Pediatrics
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v.48
no.5
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pp.545-550
/
2005
Purpose : Newborn brain tissue has to be dissociated into a single cell suspension for flow cytometric analysis of cell death during hypoxia-ischemia. Thus the development of a method to dissociate cells from the brain tissue with least damage and maintenance of membrane and antigen integrity remains the challenge for the in vivo application of this technique. We evaluated the efficacy of mechanical or enzymatic (collagenase or tryspin) methods of brain tissue disaggregation. Methods : The extent of the damage to the plasma membrane and loss of the characteristics of the membrane induced with each dissociation method was determined by comparing the flow cytometric results labeled with both fluorescent annexin V and propidium iodide of the newborn rat pup brain tissue in the control group (n=10) and in the 48-hour after hypoxia-ischemia group (n=10). Results : In the control group, the cell percentage of damaged, apoptotic and necrotic cells of both hemispheres with the mechanical dissociation method was significantly increased compared to the trypsin or collagenase method. In the 48-hour after hypoxia-ischemia group, the cell percentage of apoptotic and necrotic cells of the right hemisphere with the collagenase method significantly increased, and live cells significantly decreased compared to the left hemisphere, control group. Although the same trend was observed, the extent of alterations made with the trypsin method was significantly less compared to the collagenase method. Conclusion : The dissociation of neonatal brain tissue for flow cytometric analysis with collagenase was most efficacious with the least cell damage and preservation of the plasma membrane characteristics.
Extrusion cooking treatment was compared with autoclaving/cooling treatment for formation of enzyme resistant starch of high amylose corn starch (HACS). Effects of barrel temperature $(100^{\circ}C,\;120^{\circ}C,\;140^{\circ}C)$ and feed moisture content (25%, 35%, 45%) on extrusion processing in a co-rotating twin-screw extruder under fixed screw speed (100 rpm) were investigated by measuring enzyme resistant starch (RS) yield. RS yield were estimated by in-vitro pancreatin digestion method and enzymatic-gravimetric method using termamyl. Barrel temperature and yield of RS were negatively correlated and feed moisture content and yield of RS was positively correlated as determined by in-vitro pancreatin method. The highest yield (38.4%) of RS was obtained from HACS extrudate processed at the barrel temperature of $100^{\circ}C$ and the feed moisture content of 45%, while the yield of RS by 5 times of autoclaving/cooling was 25%. The yield of RS by in vitro pancreatin digestion method was 20.7% with high amylose corn starch and 8.2% with ordinary corn starch (CS), respectively, under the same extrusion condition (barrel temperature $120^{\circ}C$, feed moisture content 35%). At the same condition, the yields of RS by enzyme-gravimetric method were 14.6% with HACS and 6.8% with CS, respectively. The yield of RS increased during the storage at $4^{\circ}C$ for 4 weeks and the highest yield (60%) was obtained by the storage of HACS extrudates extruded at $100^{\circ}C$ and 45% feed moisture content.
Objective: Hatching of the blastocyst from the zona pellucida (ZP) is a key event in mammalian implantation. In vivo, two factors have been identified as possible mediators of hatching: lysis of the ZP by substances elaborated either from the embryo or female reproductive tract and pressure exerted on the zona by expansion of the blastocyst. Two methods of zona manipulation were already in use to enhance the ability of embryos to hatch: mechanical PZD and chemical ZD by acidic Tyrode's solution. But several controversies of each method have been reported. The purpose of this study was to investigate the effect of pronase for mouse embryo hatching. Methods: Mouse embryos were obtained following ovulation induction of $F_1$ animals. Fresh and cryo-thawed morula embryos were exposed to 0.5, 1.0, 2.0, 5.0 ${\mu}g/ml$ pronase in Ham's F10 for 72 hrs. Main outcome measures were the rates of partial hatching and completely hatched blastocysts, and cell number of it. Results: In fresh and cryo-thawed group, the rates of completely hatched blastocyst were significantly higher in 5 ${\mu}g/ml$ pronase treatment group than control group. There was no difference in completely hatched blastocyst total cell number between pronase treatment group and control group. This suggest that pronase treatment did not harmful in mouse embryo development. In pronase treatment group, zona pellucida were thinner than control group. Conclusion: The addition of pronase to culture media may accelerate the hatching of embryo. So, enzymatic treatment of the zona may provide a valuable and effective assisted hatching technique for human in-vitro fertilization-embryo transfer.
Background: Various decellularization methods have been studied in order to develop tissue graft which is less immunogenic and more durable. This study was performed to investigate the physico-dynamic and histological effect of trypsin pretreatment on decellularization protocols. Material and Method: Two groups of bovine pericardium specimen each underwent decellularization process based on SDS and Triton X-100 or N-lauroylsarcosinate and Triton X-100. Two more groups additionally underwent pretreatment with 0.1% Trypsin/0.1% EDTA. After decellularization process, mechanical tensile strength was tested, then biomechanical test of permeability and compliance was tested before and after fatigue test. Light microscopy and electron microscopy was performed to observe histological findings. Result: There was no difference in mechanical tensile strength between groups, but permeability and compliance was decreased in trypsin pretreated groups. Light microscopic and electron microscopic findings revealed damage of the extracellular matrix in trypsin pretreated groups and in groups which underwent the fatigue test also. Conclusion: Trypsin pretreatment in decellularizing process of bovine pericardium damages extracellular matrix and increases permeability and compliance of the bovine pericardium, but did not decrease tensile strength. Further studies are needed to use enzymatic treatments in decellularization protocols.
Cancer is a complex disease and the genetic susceptibility to it could be an outcome of the inherited difference in the capacity of xenobiotic metabolizing enzymes. Glutathione-S-transferases (GSTs) are phase II metabolizing enzymes whose various genotypes have been associated with increased risk of different types of cancer. Null mutations caused by the deletion of the entire gene result in the absence of the enzymatic activity and increase in the risk of developing cancer including chronic myeloid leukaemia (CML). In the present case-control study we evaluated the effect of null mutations in GSTM1 and GSTT1 genes on the risk of developing CML. The study included 75 CML patients (43 males and 32 females; age (mean ${\pm}$ S.D) $42.3{\pm}13.4$ years) and unrelated non-malignant controls (76 male and 48 females; age (mean ${\pm}$ S.D) $41.5{\pm}12.9$). The distribution of GSTM1 and GSTT1 genotypes in CML patients and controls was assessed by multiplex-PCR method. Logistic regression was used to assess the relationship between GSTM1 and GSTT1 genotypes and risk of CML. Chi-square test was used to evaluate the trend in modulating the risk to CML by one or more potential high risk genotype. Although GSTM1 null genotype frequency was higher in CML patients (41%) than in the controls (35%), it did not reached a statistical significance (OD = 1.32, 95% CI: 0.73-2.40; P value = 0.4295). The frequency of GSTT1 null genotypes was higher in the CML patients (36%) than in the controls (21%) and the difference was found to be statistically significant (OD = 2.12, 95% CI: 1.12-4.02; P value = 0.0308). This suggests that the presence of GSTT1genotype may have protective role against the CML. We found a statistically significant (OD = 3.09, 95% CI: 1.122-8.528; P value = 0.0472) interaction between the GSTM1 and GSTT1 null genotypes and thus individuals carrying null genotypes of both GSTM1 and GSTT1 genes are at elevated risk of CML.
Yoon, Young Mi;An, Gi Hong;Kim, Jung Kon;Ahn, Seung-Hyun;Cha, Young-Lok;Yang, Jungwoo;Yu, Kyeong-Dan;Moon, Youn-Ho;Ahn, Jong-Woong;Koo, Bon-Cheol;Choi, In-Hoo
KSBB Journal
/
v.29
no.5
/
pp.316-322
/
2014
Xylanase have been used to convert the polymetric xylan into fermentable sugars from the production of ethanol and xylitol from plant biomass. The aim of this study was to isolate and identify xylanolytic bacterium from herbivore feces and was to used the xylanase for enzymatic hydrolysis of biomass. Xylanolytic strains were isolated from 59 different feces of herbivores from Seoul Grand Park located in Gwacheon Gyeonggi-do. The xylanolytic strains were selected by congo red staining and DNS method. Total 67 strains isolated from the herbivores feces were tested for xylanase activity. Among the strains, H10-1, which has the highest xylanase activity, was isolated from feces of Ceratotherium simum. The H10-1 strain was identified as Bacillus pumilus based on its morphological/biochemical characteristics and partial 16S rDNA gene sequences. Culture conditions of B. pumilus H10-1 such as initial medium pH, incubation temperature and incubation time were optimized for maximum xylanase production. And also xylanase produced by B. pumilus H10-1 was applied for the saccharification of Miscanthus sacchariflorus cv. 'Geodae 1', which was pretreated with 1.5M NaOH. The optimized culture conditions of B. pumilus H10-1 were pH 9, $30^{\circ}C$ incubation temperature, and 7 day incubation time, respectively. This xylanase activity under the optimized conditions was $20.4{\pm}3.3IU$. The crude xylanase produced by B. pumilus H10-1 was used for the saccharification of xylan derived from pretreated 'Geodae 1'. The saccharification conditions were $50^{\circ}C$, 200 rpm, and 5 days. Saccharification efficiency of pretreated 'Geodae 1' by B. pumilus H10-1 was 8.2%.
Background: We have previously reported that not only cGMP but also 8-Br-cGMP or 8-pCPT-cGMP, specific and potent stimulators of cGMP-dependent protein kinase (cGMP-PK), increased basal L-type calcium current $(I_{Ca})$ in rabbit ventricular myocytes. Our findings in rabbit ventricular myocytes were entirely different from the earlier findings in different species, suggesting that the activation of cGMP-PK is involved in the facilitation of $I_{Ca}}$ by cGMP. However, there is no direct evidence that cGMP-PK can stimulate $I_{Ca}}$ in rabbit ventricular myocytes. In this report, we focused on the direct effect of cGMP-PK on $I_{Ca}}$ in rabbit ventricular myocytes. Methods and Results: We isolated single ventricular myocytes of rabbit hearts by using enzymatic dissociation. Regulation of $I_{Ca}}$ by cGMP-PK was investigated in rabbit ventricular myocytes using whole-cell voltage clamp method. $I_{Ca}}$ was elicited by a depolarizing pulse to +10 mV from a holding potential of -40 mV. Extracellular 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP), potent stimulator of cGMP-dependent protein kinase (cGMP-PK), increased basal $I_{Ca}}$. cGMP-PK also increased basal $I_{Ca}}$. The stimulation of basal $I_{Ca}}$ by cGMP-PK required both 8-Br-cGMP in low concentration and intracellular ATP to be present. The stimulation of basal $I_{Ca}}$ by cGMP-PK was blocked by heat inactivation of the cGMP-PK and by bath application of 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate, Rp-isomer (Rp-pCPT-cGMP), a phosphodiesterase-resistant cGMP-PK inhibitor. When $I_{Ca}}$ was increased by internal application of cGMP-PK, IBMX resulted in an additional stimulation of $I_{Ca}}$. In the presence of cGMP-PK, already increased $I_{Ca}}$ was potentiated by bath application of isoprenaline or forskolin or intracellular application of cAMP. Conclusions: We present evidence that cGMP-PK stimulated basal $I_{Ca}}$ by a direct phosphorylation of L-type calcium channel or associated regulatory protein in rabbit ventricular myocytes.
The effect of thiol compounds on development and intracellular glutathione(GSH) concentrations of bovine embryos produced by in vitro maturation and in vitro fertilization(IVM/IVF) was examined in CRlaa medium with or without $\beta$-mercaptoethanol(0, 10, 25 and 50$\mu$MME) and cysteamine(0, 25, 50 and 75 $\mu$M). Numbers of cells comprising blastocysts were also counted using double fluorescence stain and the total glutathione levels(oxidized and reduced form) of morula and blastocyst embryos were than measured by an enzymatic method. Following routine IVM/IVF procedures oocytes and zygotes were cultured for 40 to 44h in CRlaa medium. Then 2 to 8-cell embyos had cumulus cell removed and were allotted randomly to the experimental medium. In Experiment 1, the proportion of embryos developing to and beyond morulae stages in 0, 10, 25 and 50 $\mu$M $\beta$-ME was 42.9%, 50.0%, 53.7% and 65.6%, respectively. Fifty $\mu$M $\beta$-ME group was significantly higher than those of any other groups (P<0.05). In Experiment 2, the percentages of embryos developed beyond morulae stages in 0, 25, 50 and 75 $\mu$M cysteamine was 42.9%, 40.4%, 60.0% and 59.2%, respectively. Fifty and 75$\mu$M cysteamine groups were significantly higher than in 0 and 25 $\mu$M cysteamine groups, but all of culture medium containing cysteamine(52.6%) was not significantly difference in control group(42.9%). In Experiment 3, the intracellular GSH concentrations of morulae and blastocyst embryos in 0 and 50 $\mu$M $\beta$-ME was 42.4 pM and 44.9 pM, 49.5 pM and 67.8 pM, respectively. Morulae embryos were not difference, but blastocyst embryos were significantly difference between treatments(P<0.05). In Experiment 4, the intracellular GSH concentrations of morulae in CRlaa with or without cysteamine were 39.8 pM and 45.6 pM, and blastocysts were 59.3 pM and 66.8 pM, respectively. Cell numbers of blastocysts were similar to in all experimental groups. These experiments indicate that thiol compounds can increase the proportion of embryos that developing to and beyond morulae stage and the intracellular GSH concentrations.
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