• Journal of Microbiology and Biotechnology
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• v.29 no.2
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• pp.274-282
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• 2019

Mercury-resistant ($Hg^R$) bacteria were isolated from heavy metal polluted wastewater and soil collected near to tanneries of district Kasur, Pakistan. Bacterial isolates AZ-1, AZ-2 and AZ-3 showed resistance up to $40{\mu}g/ml$ against mercuric chloride ($HgCl_2$). 16S rDNA ribotyping and phylogenetic analysis were performed for the characterization of selected isolates as Bacillus sp. AZ-1 (KT270477), Bacillus cereus AZ-2 (KT270478) and Bacillus cereus AZ-3 (KT270479). Phylogenetic relationship on the basis of merA nucleotide sequence confirmed 51-100% homology with the corresponding region of the merA gene of already reported mercury-resistant Gram-positive bacteria. The merE gene involved in the transportation of elemental mercury ($Hg^0$) via cell membrane was cloned for the first time into pHLV vector and transformed in overexpressed C43(DE3) E. coli cells. The recombinant plasmid (pHLMerE) was expressed and the native MerE protein was obtained after thrombin cleavage by size exclusion chromatography (SEC). The purification of fusion/recombinant and native protein MerE by Ni-NTA column, dialysis and fast protein liquid chromatography (FPLC/SEC) involved unfolding/refolding techniques. A small-scale reservoir of wastewater containing $30{\mu}g/ml$ of $HgCl_2$ was designed to check the detoxification ability of selected strains. It resulted in 83% detoxification of mercury by B. cereus AZ-2 and B. cereus AZ-3, and 76% detoxification by Bacillus sp. AZ-1 respectively (p < 0.05).

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.8
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• pp.1135-1142
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• 2017

Objective: The objective of this study was to evaluate effects of heat treatment and soybean oil inclusion on protein oxidation of soy protein isolate (SPI) and of oxidized protein on redox status of broilers at an early age. Methods: SPI mixed with soybean oil (SPIO) heated at $100^{\circ}C$ for 8 h was used to evaluate protein oxidation of SPI. A total of two hundred and sixteen 1-day-old Arbor Acres chicks were divided into 3 groups with 6 replicates of 12 birds, receiving basal diet (CON), heat-oxidized SPI diet (HSPI) or mixture of SPI and 2% soybean oil diet (HSPIO) for 21 d, respectively. Results: Increased protein carbonyl, decreased protein sulfhydryl of SPI were observed as heating time increased in all treatments (p<0.05). Addition of 2% soybean oil increased protein carbonyl of SPI at 8 h heating (p<0.05). Dietary HSPI and HSPIO decreased the average daily gain of broilers as compared with the CON (p<0.05). Broilers fed HSPI and HSPIO exhibited decreased glutathione (GSH) in serum, catalase activity and total sulfhydryl in liver and increased malondialdehyde (MDA) and protein carbonyl in serum, advanced oxidation protein products (AOPPs) in liver and protein carbonyl in jejunal mucosa as compared with that of the CON (p<0.05). Additionally, broilers receiving HSPIO showed decreased glutathione peroxidase activity (GSH-Px) in serum, GSH and hydroxyl radical scavenging capacity in liver, GSH-Px activity in duodenal mucosa, GSH-Px activity and superoxide anion radical scavenging capacity in jejunal mucosa and increased AOPPs in serum, MDA and protein carbonyl in liver, MDA and AOPPs in jejunal mucosa (p<0.05). Conclusion: Protein oxidation of SPI can be induced by heat and soybean oil and oxidized protein resulted in redox imbalance in broilers at an early age.

• Journal of Animal Science and Technology
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• v.53 no.2
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• pp.119-126
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• 2011

The study was conducted to investigate the effects of dietary energy and protein levels on productivity and feed cost in crossbred chicks. Experiment was divided into starting (0~5 weeks), growing (6~10 weeks), and finishing (11~14 weeks) periods. Design of feeding trials was 3 ${\times}$ 3 factorial to feed different energy and protein levels for starting (ME 2,950, 3,000, and 3,050 kcal/kg CP 18, 19, and 20%), growing (ME 3,000, 3,050, and 3,100 kcal/kg CP 17, 18, and 19%) and finishing (ME 3,050, 3,100, and 3,150 kcal/kg CP 16, 17, and 18%) periods, respectively. In the starting period, weight gain and FCR was improved by dietary protein level (P<0.05). Interaction effect existed in feed intake and FCR (P<0.05). Weight gain was higher in 3,000 kcal/kg ME treatment than 3,100 kcal/kg ME treatment for growing period (P<0.05). In finishing period, feed intake was significantly decreased in ME 3,150 kcal/kg treatment than the other ME treatments (P<0.05). Feed cost/weight gain (FC/WG) was significantly decreased in chicks fed with 2,950 kcal/kg ME and 19% CP in starting period (P<0.05). For the growing period, FC/WG was notably increased in ME 3,000, 3,050 kcal/kg treatment than ME 3,100 kcal/kg treatment, and the FC/WG of CP 17, 18% treatment was significantly higher than CP 16% treatment (P<0.05). Thus, the optimum levels of ME and CP to improve the productivity and feed cost for starting, growing and finishing periods were 2,950 kcal/kg ME, 19% CP 3,000 kcal/kg ME, 18% CP and 3,100 kcal/kg ME, 17 or 16% CP, respectively.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.2
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• pp.741-746
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• 2015

Purpose: To investigate the influence of exogenous p53 upregulated modulator of apoptosis (PUMA) expression on cell proliferation and apoptosis in human non-small cell lung cancer A549 cells and transplanted tumor cell growth in nude mice. Materials and Methods: A549 cells were divided into the following groups: control, non-carrier (NC), PUMA (transfected with pCEP4-(HA) 2-PUMA plasmid), DDP ($10{\mu}g/mL$ cisplatin treatment) and PUMA+DDP (transfected with pCEP4-(HA)2-PUMA plasmid and $10{\mu}g/mL$ cisplatin treatment). The MTT method was used to detect the cell survival rate. Cell apoptosis rates were measured by flow cytometry, and PUMA, Bax and Bcl-2 protein expression levels were measured by Western blotting. Results: Compared to the control group, the PUMA, DDP and PUMA+DDP groups all had significantly decreased A549 cell proliferation (p<0.01), with the largest reduction in the PUMA+DDP group. Conversely, the apoptosis rates of the three groups were significantly increased (P<0.01), and the PUMA and DDP treatments were synergistic. Moreover, Bax protein levels significantly increased (p<0.01), while Bcl-2 protein levels significantly decreased (p<0.01). Finally, both the volume and the weights of transplanted tumors were significantly reduced (p<0.01), and the inhibition ratio of the PUMA+DDP group was significantly higher than in the single DDP or PUMA groups. Conclusions: Exogenous PUMA effectively inhibited lung cancer A549 cell proliferation and transplanted tumor growth by increasing Bax protein levels and reducing Bcl-2 protein levels.

• Journal of Pharmaceutical Investigation
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• v.35 no.2
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• pp.75-80
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• 2005

A promising application of Lactococcus lactis is its use as live vehicles for production and delivery of heterologous proteins of vaccines and therapeutic substances. Because L. lactis has GRAS ('generally regarded as safe') status, we tested whether L. lactis could function as the carrier of the Ll protein of human papillomavirus (HPV) type 16. The RNA level expression of Ll gene was detected in L. Lactis. The Ll protein was expressed in L. lactis with Ll gene. The growth of strains L. lactis with an empty plasmid (pAMJ328) and L. lactis with Ll-encoding plasmid (pAMJ328-Ll) was slightly decreased in comparison with the growth of strains L. lactis (wild type). However, all the three strains of L. lactis maintained the ability to ferment sugars primarily into lactic acid, indicating that Ll protein did not affect the biochemical property of L. lactis. These results suggest that L. lactis, capable of carrying Ll protein, might be further developed as a biocompatible oral protein delivery system.

• Korean Journal of Poultry Science
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• v.30 no.2
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• pp.73-81
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• 2003

This experiment was conducted to investigate the effects of dietary energy and protein levels on the growth rate of egg breeder pullets. A total of 360 Hy-Line Brown pullets aged 2 to 6 weeks (Phase I) were fed 5 rations differing in dietary protein (17, 19 and 20%) and energy (2,800, 2,950 and 3,050 kcal/kg, TMEn) leveIs for a period of 5 weeks and those aged 6 to 10 weeks (Phase II) were fed 5 rations differing in dietary protein (15, 16 and 17%) and energy (2,800, 2,900 and 3,000 kcal/kg, TMEn) levels in order to evaluate the optimum dietary energy and protein leveIs for egg breeder pullets reared in cages. Their body weight gains were significantly influenced by the dietary protein levels (P<0.05). The dietary energy levels did not greatly affect the growth performances throughout the experimental period. The low energy and protein regimen based on NRC requirement (control) was found to produce smaller pullets and lower tibia bone measurements as compared to the higher regimen groups. With the increase in dietary energy and protein levels, tibial bone strength and ash content also gradually increased (P<0.05). There were no significant differences in the flock uniformity among the treatments. These results indicate that increases in dietary energy and protein levels above the NRC requirements appeared to be more effective in obtaining the optimal growth and bone developments of egg breeder pullets reared in cage.

• Journal of Korean Neurosurgical Society
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• v.30 no.4
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• pp.443-450
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• 2001

Objective : The cyclin-dependent kinase inhibitor $p27^{kip1}$ protein is a negative regulator of the cell cycle, and its degradation is required for entry into the S phase. Loss of $p27^{kip1}$ expression has been reported to be associated with aggressive behavior in a variety of tumors of epithelial and lymphoid origin. However, its association with various astrocytic tumors has not been clearly demonstrated. We studied to investigate the relationship of $p27^{kip1}$ expression with the biological behavior of astrocytic tumors in addition to study on the role of $p27^{kip1}$ in the tumorigenesis of these tumors. Patients and Methods : From 1990 to 1998, a total of 29 astrocytic tumor of all grades obtained by operative resection were included for evaluation. We studied the expression of $p27^{kip1}$ protein immunohistochemical assay in astrocytic tumors and compared the findings with the clinicopathologic parameters. Immunohistochemical staining was performed on formalin-fixed paraffin-embedded sections by the avidin-biotin-peroxidase complex method. According to WHO classification, all cases were divided into astrocytomas(4 cases), anaplastic astrocytomas(9 cases), and glioblastomas(16 cases) by 3 pathologists. Clinical information was obtained from medical records, and others such as location and size of tumors from imaging studies. Results : Mean $p27^{kip1}$ protein labeling indexes(LI, mean${\pm}$standard deviation) of astrocytomas, anaplastic astrocytomas, and glioblastomas were $80.6{\pm}9.1$, $63.6{\pm}21.0$, and $28.9{\pm}18.7$, respectively, and were inversely correlated with grade of glial tumors(p<0.0001). Mean $p27^{kip1}$ protein LI in the recurrent group was lower than that in the nonrecurrent group, but there was no significant difference statistically(p=0.464). Additionally, $p27^{kip1}$ protein expression did not show any significant relationship to other prognostic factors such as age(p=0.1643), tumor size(p=0.8), or location(p=0.8). Conclusion : These results suggested that reduced expression of $p27^{kip1}$ protein may play a important role in the malignant transformation process of astrocytic tumor cells.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.5
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• pp.667-673
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• 2014

Eight multiparous Holstein cows ($632{\pm}12$ kg BW; $135{\pm}16$ DIM) were used in a replicated $4{\times}4$ Latin square design to evaluate the effects of forage sources on rumen fermentation characteristics, performance, and microbial protein (MCP) synthesis. The forage portion of the diets contained alfalfa hay (AH), oat hay (OH), Leymus chinensis (LC), or rice straw (RS) as the primary source of fiber. Diets were isonitrogenous and isocaloric, and cows were fed four corn silages based total mixed rations with equivalent nonfiber carbohydrate (NFC) and forage neutral detergent fiber (NDF). Dry matter intake was not affected by the source of dietary forages, ranging from 18.83 to 19.20 kg/d, consequently, milk yield was similar among diets. Because of the numerical differences in milk fat and milk protein concentrations, 4% FCM and ECM yields were unchanged (p>0.05). Mean rumen pH, NH3-N content, and concentrations of volatile fatty acids in the rumen fluid were not affected by the treatments (p>0.05). Dietary treatments did not affect the total tract apparent digestibility of dry matter, organic matter, and crude protein (p>0.05); however, digestibility of NDF and acid detergent fiber in RS diet was higher compared with AH, OH, and LC diets (p<0.05). Total purine derivative excretion was higher in cows fed AH, OH, and LC diets compared with those fed RS diet (p<0.05), consequently, estimated MCP synthesis was 124.35 g/d higher in cows fed AH diet compared with those fed RS diet (p<0.05). The results indicated that cows fed AH, OH, LC, and RS diets with an equivalent forage NDF and NFC have no unfavourable effect on the ruminal fermentation and productive parameters.

• Reproductive and Developmental Biology
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• v.29 no.1
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• pp.43-48
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• 2005

To search of a new porcine pheromonal odorants for biostimulation control system technologies to offer a potentially useful and practical way to improve reproductive efficiency in livestock species, the holographic quantitative structure activity relationship (HQSAR) model between odorants, 2-phenoxytetrahydrofurane (A), 2-cyclohexyl-oxytetrahydrofurane (B), derivatives and binding affinity constants (p[Od.]/sub 50/) for porcine odorant-binding protein (pOBP) as receptor of pig pheromones were derivated and disscused. The binding affinity constants of cyclohexyl substituents (A) for pOBP were higher (A>B) than that of phenyl substituents (B). It was revealed that the optimum HQSAR model XI using PLS analyses had a fragment length (5∼8) with chirality at 5 components and hologram length 97 bin, which had a cross-validated q²(predictablities) of 0.916, and a conventional correlation coefficient r² (fitness) of 0.988, respectively. From the atomic contribution, the C3 and C5 atom in 2-oxyfuryl group contributed to binding affinity constants, whereas the central carbon atom in tert-butyl group on the cyclohexyl ring and the C4 atom of furyl group parts showed no contribution.

• International Journal of Oral Biology
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• v.32 no.1
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• pp.35-43
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• 2007

Tooth loss in elderly is mainly caused by alveolar bone loss via severe periodontitis. Although the severity of periodontitis is known to be affected by age, the aging process or the genetic changes during the aging of periodontal tissue cells are not well characterized. In this study, we investigated the effect of in vitro aging on the change of gene expression pattern in periodontal fibroblasts. Gingival fibroblasts (GF) and periodontal ligament fibroblasts (PDL) were obtained from two young patients and replicative senescence was induced by sequential subcultivation. When more than 90% cells were positively stained with senescence-associated ${\beta},-galactosidase$, those cells were regarded as aged cells. In aged GF and PDL, the level of phosphorylated retinoblastoma (RB) and $p16^{INK4A}$ protein was significantly decreased and increased, respectively. However, the protein level of p53 and p21, well known senescence-inducing genes, did not increase in aged GF and PDL. Although $p27^{Kip1}$ and $p15^{INK4B}$, another cyclin-dependent kinase inhibitors, were reported to be involved in replicative senescence of human cells, they were decreased in aged GF and PDL. Because senescent cells showed flattened and enlarged cell shape and are known to have increased focal adhesion, we examined the protein level of several integrins. Aged GF and PDL showed increased protein level of integrin ${\alpha}2$, ${\alpha}v$, and ${\beta}1$. When the gene expression profiles of actively proliferating young cells and aged cells were compared by cDNA microarray of 3,063 genes and were confirmed by reverse transcription-polymerase chain reaction, 7 genes and 15 genes were significantly and commonly increased and decreased, respectively, in aged GF and PDL. Among them, included are the genes that were known to be involved in the regulation of cell cycle, gene transcription, or integrin signaling. The change of gene expression pattern in GF and PDL was minimally similar to that of oral keratinocyte. These results suggest that $p16^{INK4A}/RB$ might be involved in replicative senescence of periodontal fibroblasts and the change of gene expression profile during aging process is cell type specific.