• Journal of Microbiology and Biotechnology
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• v.19 no.5
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• pp.511-519
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• 2009

A chimeric gene encoding enhanced green fluorescent protein (EGFP) and a S-layer protein from Lactobacillus brevis KCTC3102, and/or two copies of the Fe-binding Z-domain, a synthetic analog of the B-domain of protein A, was constructed and expressed in Escherichia coli BL21(DE3). The S-layer fusion proteins produced in a 500-1 fermentor were likely to be stable in the range of pH 5 to 8 and $0^{\circ}C$ to $40^{\circ}C$. Their adhesive property enabled an easy and rapid immobilization of enzymes or antibodies on solid materials such as plastics, glass, sol-gel films, and intestinal epithelial cells. Owing to their affinity towards intestinal cells and immunoglobulin G, the S-layer fusion proteins enabled the adhesion of antibodies to human epithelial cells. In addition, feeding a mixture of the S-layer fusion proteins and antibodies against neonatal calf diarrhea (coronavirus, rotavirus, Escherichia coli, and Salmonella typhimurium) to Hanwoo calves resulted in 100% prevention of neonatal calf diarrhea syndrome (p<0.01), whereas feeding antibodies only resulted in 56% prevention.

• Journal of Microbiology and Biotechnology
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• v.21 no.4
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• pp.421-429
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• 2011

For precise identification of a Lactobacillus K1 isolate, LC-MS/MS analysis of the putative surface layer protein was performed. The results obtained from LTQ-FT-ICR mass spectrometry confirmed that the analyzed protein spot is the surface layer protein originating from Lb. helveticus species. Moreover, the identified protein has the highest similarity with the surface layer protein from Lb. helveticus R0052. To evaluate the proteomic study, multilocus sequence analysis of selected housekeeping gene sequences was performed. Combination of 16S rRNA sequencing with partial sequences for the genes encoding the RNA polymerase alpha subunit (rpoA), phenylalanyl-tRNA synthase alpha subunit (pheS), translational elongation factor Tu (tuf), and Hsp60 chaperonins (groEL) also allowed to classify the analyzed isolate as Lb. helveticus. Further classification at the strain level was achieved by sequencing of the slp gene. This gene showed 99.8% identity with the corresponding slp gene of Lb. helveticus R0052, which is in good agreement with data obtained by nano-HPLC coupled to an LTQ-FT-ICR mass spectrometer. Finally, LC-MS/MS analysis of surface layer proteins extracted from three other Lactobacillus strains proved that the proposed method is the appropriate molecular tool for the identification of S-layer-possessing lactobacilli at the species and even strain levels.

• Journal of Microbiology and Biotechnology
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• v.22 no.9
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• pp.1271-1278
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• 2012

Genetic fusion of two proteins frequently induces beneficial effects to the proteins, such as increased solubility, besides the combination of two protein functions. Here, we study the effects of the bacterial surface layer protein SgsE from Geobacillus stearothermophilus NRS 2004/3a on the folding of a C-terminally fused enhanced green fluorescent protein (EGFP) moiety. Although GFPs are generally unable to adopt a functional confirmation in the bacterial periplasm of Escherichia coli cells, we observed periplasmic fluorescence from a chimera of a 150-amino-acid N-terminal truncation of SgsE and EGFP. Based on this finding, unfolding and refolding kinetics of different S-layer-EGFP chimeras, a maltose binding protein-EGFP chimera, and sole EGFP were monitored using green fluorescence as indicator for the folded protein state. Calculated apparent rate constants for unfolding and refolding indicated different folding pathways for EGFP depending on the fusion partner used, and a clearly stabilizing effect was observed for the SgsE_C fusion moiety. Thermal stability, as determined by differential scanning calorimetry, and unfolding equilibria were found to be independent of the fused partner. We conclude that the stabilizing effect SgsE_C exerts on EGFP is due to a reduction of degrees of freedom for folding of EGFP in the fused state.

• Journal of Microbiology and Biotechnology
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• v.21 no.8
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• pp.822-829
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• 2011

Evidence shows that probiotic bacteria can undergo substantial structural and morphological changes in response to environmental stresses, including antibiotics. Therefore, this study investigated the effects of penicillin G (0.015, 0.03, and 0.06 mg/l) on the morphology and adhesion of Lactobacillus acidophilus ATCC 4356, including the colony morphotype, biofilm production, hydrophobicity, $H_2_O2$ formation, S-layer structure, and slpA gene expression. Whereas only smooth colonies grew in the presence of penicillin, rough and smooth colony types were observed in the control group. L. acidophilus ATCC 4356 was found to be hydrophobic under normal conditions, yet its hydrophobicity decreased in the presence of the antibiotic. No biofilm was produced by the bacterium, despite testing a variety of different culture conditions; however, treatment with penicillin G (0.015-0.06 mg/l) significantly decreased its production of $H_2_O_2$ formation and altered the S-layer protein structure and slpA gene expression. The S-protein expression decreased with 0.015 mg/l penicillin G, yet increased with 0.03 and 0.06 mg/l penicillin G. In addition, the slpA gene expression decreased in the presence of 0.015 mg/l of the antibiotic. In conclusion, penicillin G was able to alter the S-layer protein production, slpA gene expression, and certain physicochemical properties of Lactobacillus acidophilus ATCC 4356.

• BMB Reports
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• v.44 no.5
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• pp.323-328
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• 2011

We screened four B. thuringiensis strains whose parasporal inclusions contained the S-layer protein (SLP), and cloned two slp genes from each strain. Phylogenetic analysis indicated these SLPs could be divided into two groups, SLP1s and SLP2s. To confirm whether SLPs were present in the S-layer or as a parasporal inclusion, strains CTC and BMB1152 were chosen for further study. Western blots with isolated S-layer proteins from strains CTC and BMB1152 in the vegetative phase showed that SLP1s and SLP2s were constituents of the S-layer. Immunofluorescence utilizing spore-inclusion mixtures of strains CTC and BMB1152 in the sporulation phase showed that SLP1s and SLP2s were also constituents of parasporal inclusions. When heterogeneously expressed in the crystal negative strain BMB171, four SLPs from strains CTC and BMB1152 could also form parasporal inclusions. This temporal and spatial expression is not an occasional phenomenon but ubiquitous in B. thuringiensis strains.

• Asian-Australasian Journal of Animal Sciences
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• v.13 no.11
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• pp.1548-1553
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• 2000

An experiment was conducted to study the nutritional quality of whole crop corn silage ensiled with cage layer manure in sheep. Treatments were designed as a $3{\times}3$ Latin square with 16-day periods. Sheep were allotted in one of three diet-treatments, which were whole crop corn silage (CS), whole crop corn+30% cage layer manure (CLM) silage (based on DM; MS) and rice straw+concentrate (SC) mixed at 8:2 ratio (on DM basis). Silage ensiled with CLM significantly increased (p<0.05) digestibilities of crude protein, NDF and ADF, TDN over the other treatments. Ruminal pH in sheep fed SC was significantly (p<0.05) higher than that of the other diets at 0.5, 1, 2, 4 and 8 h after feeding. Ruminal ammonia nitrogen concentration of the MS treatment was significantly (p<0.05) higher than that of the other treatments at 0, 1, 2 h after feeding. The MS treatment highly increased (p<0.05) feed intake, digestibility of organic matter and crude protein, nitrogen intake and retained nitrogen. The MS treatment highly increased (p<0.05) purine derivative (PD) excretion leading to higher microbial protein synthesis.

• Journal of KIISE:Databases
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• v.35 no.2
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• pp.132-142
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• 2008

In biological domain, there exist a variety of protein analysis databases which have their own meaning toward the same target of protein. If we integrate these scattered heterogeneous data efficiently, we can obtain useful information which otherwise cannot be found from each original source. Reflecting the characteristics of biological data, each data source has its own syntax and semantics. If we describe these data through RDF(S) models, one of the Semantic Web standards, we can achieve not only syntactic but also semantic integration. In this paper, we propose a new concept of integration layer based on the RDF unified schema. As a conceptual model, we construct a unified schema focusing on the protein information; as a representational model, we propose a technique for the wrappers to aggregate necessary information from the relevant sources and dynamically generate RDF instances. Two example queries show that our integration layer succeeds in processing the integrated requests from users and displaying the appropriate results.

• Journal of Microbiology and Biotechnology
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• v.20 no.4
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• pp.749-756
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• 2010

In many conditions, bacterial surface properties are changed as a result of variation in the growth medium and conditions. This study examined the influence of bile salt concentrations (0-0.1%) on colony morphotype, hydrophobicity, $H_2O_2$ concentration, S-layer protein production, and slpA gene expression in Lactobacillus acidophilus ATCC 4356. It was observed that two types of colonies (R and S) were in the control group and the stress condition. When the bile level increased in the medium, the amount of S type was more than the R type. A stepwise increment in the bile concentration resulted in a stepwise decline in the maximum growth rate. The results showed that hydrophobicity was increased in 0.01%-0.02% bile, but it was decreased in 0.1% bile. Treatment by bile (0.01%-0.1%) profoundly decreased $H_2O_2$ formation. S-Layer protein and slpA gene expression were also altered by the stress condition. S-Protein expression was increased in the stress condition. The slpA gene expression increased in 0.01%-0.05% bile and it decreased in 0.1% bile. However, we found that different bile salt concentrations influenced the morphology and some surface properties of L. acidophilus ATCC 4356. These changes were very different in the 0.1% bile. It appears that the bacteria respond abruptly to 0.1% bile.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.2
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• pp.254-265
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• 2002

Soybean meal (SBM) is a major protein source in poultry feeds and one of the best quality ingredients because of the relatively high protein content, good amino acid profile and bioavailability. But soybean meal quality is largely dependent on the processing technology and origins. In this experiment, effects of three different soybean meals were evaluated in layer (experiment 1) and broiler (experiment 2). Soybean meal sources used in the experiments were the US-originated dehulled soybean meal (USDHSBM), India-originated non-dehulled soybean meal (India SBM) and Brazil-originated non-dehulled soybean meal (Brazil SBM). Experiment 1 was conducted during growing and laying periods and evaluated the interactive effects of soybean meal sources according to feeding periods on growth performance and egg quality. Experiment 2 was conducted during growing period (day 1-35) and finishing period (day 35-42). The growth performance was measured for the same periods and any possible interaction between soybean meal origins and crude protein levels was also studied. In experiment 1, chicks fed India SBM utilized feed more efficiently (p<0.05) than those fed Brazil SBM from day 29 to day 42. The body weights of layers during the laying period had no relation to egg production. But egg weights were significantly heavier in all the USDHSBM fed groups than other groups (p<0.001) and depended on feed protein source during growing period (p<0.001). The average egg weight of the USDHSBM fed group scored the highest value (65.4 g), followed by the Brazil SBM fed group (62.1 g) and India SBM fed group (62.1 g). There was an effect of interaction between origins of soybean meal fed group in growing and laying period on eggshell color (p<0.01). Eggshell was significantly stronger in the USDHSBM fed (for growing period) groups than other groups (p<0.05) on $31^{st}$ week. Haugh's unit (HU), albumin index and yolk index of the USDHSBM fed group in growing stage were significantly superior (p<0.001) to other groups. In experiment 2, for the 7-week, chicks on the India SBM group gained less (p<0.001) weight than other groups. While daily gain of India SBM chicks was not affected by dietary crude protein level, those of the USDHSBM and Brazil SBM chicks were linearly increased as dietary crude protein level increased from 18% to 20%. The gain per feed ratio of the USDHSBM group was the highest (0.585), followed by the Brazil SBM group (0.568) and India SBM group (0.550) (p<0.01). Therefore, in this experiment, the use of USDHSBM with excellent protein quality and amino acid digestibility could be of advantage to the economic production of layer and broiler.

• The Korean Journal of Physiology and Pharmacology
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• v.4 no.4
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• pp.291-299
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• 2000

Morphine or butorphanol was continuously infused into cerebroventricle (i.c.v.) with the rate of $26\;nmol/{\mu}l/h$ for 3 days, and the withdrawal from opioid was rendered 7 hrs after the stopping of infusion. The expression of physical dependence produced by these opioids was evaluated by measuring the naloxone-precipitated withdrawal signs. The withdrawal signs produced in animals dependent on butorphanol (kappa opioid receptor agonist) were similar to those of morphine (mu opioid receptor agonist). Besides the behavioral modifications, opioid withdrawal affected G protein expression in the central nervous system. The G-protein ${\alpha}-subunit$ has been implicated in opioid tolerance and withdrawal. The effects of continuous infusion of morphine or butorphanol on the modulation of G protein ${\alpha}-subunit$ mRNA were investigated by using in situ hybridization study. In situ hybridization showed that the levels of $G\;{\alpha}s$ and $G\;{\alpha}i$ were changed during opioid withdrawal. Specifically, the level of $G\;{\alpha}s$ mRNA was decreased in the cortex and cerebellar granule layer during the morphine and butorphanol withdrawal. The level of $G\;{\alpha}i$ mRNA was decreased in the dentate gyrus and cerebellar granule layer during the morphine withdrawal. However, the level of $G\;{\alpha}i$ mRNA was significantly elevated during the butorphanol withdrawal. These results suggest that region-specific changes of G protein ${\alpha}-subunit$ mRNA were involved in the withdrawal from morphine and butorphanol.