• Journal of Microbiology and Biotechnology
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• v.30 no.4
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• pp.533-539
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• 2020

A quantitative real-time polymerase chain reaction (QPCR) was applied to estimate biokinetic coefficients of Clostridium cadaveris and Clostridium sporogenes, which utilize protein as carbon source. Experimental data on changes in peptone concentration and 16S rRNA gene copy numbers of C. cadaveris and C. sporogenes were fitted to model. The fourth-order Runge-Kutta approximation with non-linear least squares analysis was employed to solve the ordinary differential equations to estimate biokinetic coefficients. The maximum specific growth rate (μ_max), half-saturation concentration (K_s), growth yield (Y), and decay coefficient (K_d) of C. cadaveris and C.sporogenes were 0.73 ± 0.05 and 1.35 ± 0.32 h^-1, 6.07 ± 1.52 and 5.67 ± 1.53 g/l, 2.25 ± 0.75 × 10¹⁰ and 7.92 ± 3.71 × 10⁹ copies/g, 0.002 ± 0.003 and 0.002 ± 0.001 h^-1, respectively. The theoretical specific growth rate of C. sporogenes always exceeded that of C. cadaveris at peptone concentration higher than 3.62 g/l. When the influent peptone concentration was 5.0 g/l, the concentration of C.cadaveris gradually decreased to the steady value of 2.9 × 10¹⁰ copies/ml at 4 h Hydraulic retention time (HRT), which indicates a 67.1% reduction of the initial population, but the wash out occurred at HRTs of 1.9 and 3.2 h. The 16S rRNA gene copy numbers of C. sporogenes gradually decreased to steady values ranging from 1.1 × 10¹⁰ to 2.9 × 10¹⁰ copies/ml. C. sporogenes species was predicted to wash out at an HRT of 1.6 h.

• KSBB Journal
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• v.10 no.3
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• pp.264-270
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• 1995

Trigonopsis variabilis is a strong producer of D-amino acid oxidase that can transform cephalosporin C(ceph C) to ${\alpha}$-keto-adipyl-7-aminocephalosporanic acid(AKA-7ACA). Polymerase chain reaction (PCR) was applied to isolate the D-AAO gene from T. variabilis. To clone the PCR fragment, four different methods were examined using enzymatic reactions of Taq DNA polymerase, Klenow, T4 DNA polymerase I, Alkaline phosphatase Calf Intestinal, and T4 kinase. Ligation of phosphorylated blunt-end PCR fragment and dephosphorylated blunt-end of pUC18 plasmid yielded the best cloning efficiency One of recombinant E. coli transformants showed D-AAO activity against ceph C in both cell extracts and permeabilized cells.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.3
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• pp.401-409
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• 2010

FLICE inhibitory protein (FLIP) is one of the important anti-apoptotic proteins in the Fas/FasL apoptotic path which has death effect domains, mimicking the pro-domain of procaspase-8. To reveal the intracellular signal transduction molecules involved in the process of follicular development in the bovine ovary, we cloned the c-FLIP(L) gene in bovine ovary tissue with the reverse transcription polymerase chain reaction (RT-PCR), deleted the termination codon in its cDNA, and directionally cloned the amplified c-FLIP(L) gene into eukaryotic expression vector pAcGFP-Nl, including AcGFP, and successfully constructed the fusion protein recombinant plasmid. After identifying by restrictive enzyme BglII/EcoRI and sequencing, pAcGFP-bFLIP(L) was then transfected into follicular granulosa cells, mediated by Lipofectamine 2000, the expression of AcGFP observed and the transcription and expression of c-FLIP(L) detected by RT-PCR and Western blot. The results showed that the cattle c-FLIP(L) was successfully cloned; the pAcGFPbFLIP(L) fusion protein recombinant plasmid was successfuly constructed by introducing a BglII/EcoRI cloning site at the two ends of the c-FLIP(L) open reading frame and inserting a Kozak sequence before the start codon. AcGFP expression was detected as early as 24 h after transfection. The percentage of AcGFP positive cells reached about 65% after 24 h. A 1,483 bp transcription was amplified by RT-PCR, and a 83 kD target protein was detected by Western blot. Construction of the pAcGFP-bFLIP(L) recombinant plasmid should be helpful for further understanding the mechanism of regulation of c-FLIP(L) on bovine oocyte formation and development.

• Plant Resources
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• v.1 no.1
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• pp.13-21
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• 1998

A cDNA Fragment encoding iron storage protrin generated by polymerase chain reaction(PCR) using highly conserved regions of ferritin related genes were used to sereen a red pepper cDNA library. cDNA clone was designated as Fp1. Fp1 clone contatines a 5' nontranslated region of 51dp containing stop conds. Down stream from 5' UTP. an open reading frame of 750bp was observed. followed by a 3' UTR of 272bp. The deduces amino acid sequence of red pepper protein(Fp1) showed 84%, 48% and 36% identity with soybean(SolC). human(HuL H) and horse spleen(HoS-L) ferritin mRNA accumulation in response to iron. Ferritin mRNA accumulation was transient and particularly abundant in leaves. reaching a maxmum at 12h. The level of ferritin mRNA in roots was affected to a lesser extent than in leaves.

• Clinical and Experimental Pediatrics
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• v.53 no.2
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• pp.178-183
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• 2010

Purpose : The aim of this study was to identify mutations associated with macrolide resistance in Mycoplasma pneumoniae (MP) and to establish a cultural method to determine antimicrobial susceptibility. Methods : Nasopharyngeal aspirates (NPAs) were collected from 62 children diagnosed with MP pneumonia by a serologic method or polymerase chain reaction. The 23S rRNA and L4 ribosomal protein genes of MP were amplified and sequenced. To identify mutations in these 2 genes, their nucleotide sequences were compared to those of the reference strain M129. MP cultivation was carried out for 32 (28 frozen and 5 refrigerated) NPAs and M129 strain using Chanock's glucose broth and agar plate in a 5% $CO_2$ incubator at $37^{\circ}C$ and examined at 2-3 day intervals for 6 weeks. Results : Among the 62 specimens, 17 had M144V mutations in ribosomal protein L4. The A2064G mutation was observed in 1 specimen; its 23S rRNA gene was successfully sequenced. Culture for MP was successful from the M129 strain and 2 of the 5 NPAs that were refrigerated for no longer than 3 days. However, MP did not grow from the 28 NPAs that were kept frozen at $-80^{\circ}C$ since 2003. Conclusion : We found the M144V mutation of L4 protein to be common and that of domain V of 23S rRNA gene was relatively rare among MP. Studies on the prevalence of macrolide-resistant MP and the relationship between the mutations of 23S rRNA gene and ribosomal protein L4 will aid in understanding the mechanism of macrolide resistance in MP.

• Animal Bioscience
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• v.36 no.8
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• pp.1209-1220
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• 2023

Objective: The use of probiotics as an alternative to antibiotics in animal feed has received considerable attention in recent decades. Lactic acid bacteria (LAB) have remarkable functional properties promoting host health and are major microorganisms for probiotic purposes. The aim of this study was to characterize LAB strains of the chicken digestive tract and to determine their functional properties for further use as potential probiotics in poultry. Methods: A total of 2,000 colonies were isolated from the ileum and cecal contents of the chickens based on their phenotypic profiles and followed by a preliminary detection for acid and bile tolerance. The selected 200 LAB isolates with exhibited well-tolerance in acid and bile conditions were then identified by sequencing the 16S rDNA gene, followed by acid and bile tolerance, antimicrobial activity, adhesion to epithelial cells and additional characteristics on the removal of cholesterol. Then, the two probiotic strains (L. ingluviei and L. salivarious) which showed the greatest advantage in vitro testing were selected to assess their efficacy in broiler chickens. Results: It was found that 200 LAB isolates that complied with all measurement criteria belonged to five strains, including L. acidophilus (63 colonies), L. ingluviei (2 colonies), L. reuteri (58 colonies), L. salivarius (72 colonies), and L. saerimneri (5 colonies). We found that the L. ingluviei and L. salivarius can increase the population of LAB and Bifidobacterium spp. while reducing Enterobacteria spp. and Escherichia coli in the cecal content of chickens. Additionally, increased concentrations of valeric acid and short chain fatty acids were also observed. Conclusion: This study indicates that all five Lactobacillus strains isolated from gut contents of chickens are safe and possess probiotic properties, especially L. ingluviei and L. salivarius. Future studies should evaluate the potential for growth improvement in broilers.

• Journal of the Korean Society of Food Science and Nutrition
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• v.43 no.5
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• pp.749-755
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• 2014

Exopolysaccharides (EPSs) have been widely used in the food industry as viscofying, stabilizing, and emulsifying agents as well as in the pharmaceutical industry for their immunomodulatory, anti-tumor, and anti-inflammatory effects. A total of 458 lactic acid bacteria (LAB) strains isolated from several kinds of soybean pastes were screened for the production of homo-EPS (HoPS). LAB isolates were primarily screened using thin layer chromatography (TLC) and further screened polymerase chain reaction (PCR) targeting genes involved in HoPS production. Six LAB isolates producing high amounts of HoPS were identified by TLC. Among these isolates, glucansucrase gene was amplified in two strains (JSA57, JSB22), whereas the fructansucrase gene was detected in three strains (JSA57, JSB22, JSB66). After isolating the strains, their morphological characteristics and 16S rDNA sequences were determined. Six species were identified as L. alimentarius HSB15, L. plantarum JSA22, L. pentosus JSA57, L. brevis JSB22, L. alimentarius JSB66, and L. parabrevis JSB89. To evaluate the potential probiotic properties of these LAB, their survival rates against a simulated intestinal environment were determined. After 2 hr of incubation in artificial gastric juice, survival rates of JSA57, JSB90, JSB22, and JSB66 were all greater than 50%. After 2 hr of incubation in bile juice, viable cell count of JSB22 was similar with initial vial cell counts. Growth of the six LAB was screened in arabino-oligosaccharide (AOS)-containing MRS broth. Results showed that growth of the isolates selectively increased after culture in AOS-containing media. Strain JSB22 (6 hr), JSB66 (6 hr), HSB15 (20 hr), and JSA22 (29 hr) showed maximum growth rate. Especially, JSB22 showed the highest growth rate. These results suggest that EPS-producing LAB isolated from Deonjang could be applied as synbiotics.

• Nutrition Research and Practice
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• v.16 no.1
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• pp.14-32
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• 2022

BACKGROUND/OBJECTIVES: Peroxisome proliferator-activated receptor-gamma co-activator-1α (PGC-1α) has a central role in regulating muscle differentiation and mitochondrial metabolism. PGC-1α stimulates muscle growth and muscle fiber remodeling, concomitantly regulating lactate and lipid metabolism and promoting oxidative metabolism. Gynostemma pentaphyllum (Thumb.) has been widely employed as a traditional herbal medicine and possesses antioxidant, anti-obesity, anti-inflammatory, hypolipemic, hypoglycemic, and anticancer properties. We investigated whether G. pentaphyllum extract (GPE) and its active compound, gypenoside L (GL), affect muscle differentiation and mitochondrial metabolism via activation of the PGC-1α pathway in murine C2C12 myoblast cells. MATERIALS/METHODS: C2C12 cells were treated with GPE and GL, and quantitative reverse transcription polymerase chain reaction and western blot were used to analyze the mRNA and protein expression levels. Myh1 was determined using immunocytochemistry. Mitochondrial reactive oxygen species generation was measured using the 2'7'-dichlorofluorescein diacetate assay. RESULTS: GPE and GL promoted the differentiation of myoblasts into myotubes and elevated mRNA and protein expression levels of Myh1 (type IIx). GPE and GL also significantly increased the mRNA expression levels of the PGC-1α gene (Ppargc1a), lactate metabolism-regulatory genes (Esrra and Mct1), adipocyte-browning gene fibronectin type III domain-containing 5 gene (Fndc5), glycogen synthase gene (Gys), and lipid metabolism gene carnitine palmitoyltransferase 1b gene (Cpt1b). Moreover, GPE and GL induced the phosphorylation of AMP-activated protein kinase, p38, sirtuin1, and deacetylated PGC-1α. We also observed that treatment with GPE and GL significantly stimulated the expression of genes associated with the anti-oxidative stress response, such as Ucp2, Ucp3, Nrf2, and Sod2. CONCLUSIONS: The results indicated that GPE and GL enhance exercise performance by promoting myotube differentiation and mitochondrial metabolism through the upregulation of PGC-1α in C2C12 skeletal muscle.

• Korean Journal of Animal Reproduction
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• v.25 no.4
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• pp.389-397
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• 2001

Cocaine-amphetamine regulated transcript (CART), a satiety factor regulated by leptin, is associated with food intake and motor behavior. In knock out studies, Leu34Phe mutation of human CART gene resulted in obese phenotype but mice carrying a targeted deletion of the CART gene exhibited no dramatic increase of body weight on normal fat diet. To establish a new transgenic mouse model for determining the function of CART on feeding behavior in vivo, we constructed the fusion gene, CART gene under the control of neurofilament light chain promoter, which regulates gene expression at the stage of neuronal differentiation. Transgenic mice were generated by microinjection method and screened by PCR and Southern blot analyses. In these transgenic mice, overexpression of CART was detected by in situ hybridization in spinal cords and brains at 13.5 days post-coitum embryos. At six weeks of age, RT-PCR analysis showed that exogenous CART mRNA was expressed strongly in brains and spinal cords, but not much in other tissues. Our results suggest that these transgenic mice provide a new model to investigate the function of CART gene in neuronal network associated with feeding behavior.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.1
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• pp.7-11
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• 2007

The study was conducted on 162 adult male Haemonchus contortus of sheep collected from Avikanagar, Jaipur and Bikaner regions to diagnose the benzimidazole (BZ) resistance in H. contortus. The BZ resistance is primarily linked with the mutation in ${\beta}$-tubulin isotype 1 gene which substitute phenylalanine (Phe) into tyrosine (Tyr) at the 200 codon of the gene. An allele specific polymerase chain reaction (AS-PCR) technique was used for diagnosis of BZ resistance in H. contortus. In AS-PCR, one reverse primer (TGG 312) was used in two separate reactions with each of 2 forward primers (resistant TGG 331 and susceptible CAW 106 primer) that differed only at 3' nucleotide position. Therefore, the amplified products from resistant and susceptible parasites were produced 267 and 266 bp, respectively. A total of 162 parasites were genotyped, of which 130 parasites found homozygous resistant 'rr', 22 heterozygous 'rS' and 10 homozygous susceptible 'SS' type. The prevalence of 'rr' individuals was higher in Jaipur (98%) followed by Avikanagar (93%) and Bikaner (50%) regions. Overall, the prevalence of BZ resistant allele (r) was higher (87%) as compared to 13% of BZ susceptible allele (S).