• Microbiology and Biotechnology Letters
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• v.49 no.1
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• pp.75-87
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• 2021

Type I Interferons (IFNα) are known for their role as biological anticancer agents owing to their cell-apoptosis inducing properties. Development of an appropriate, cost-effective host expression system is crucial for meeting the increasing demand for proteins. Therefore, this study aims to develop codon-optimized IFNα-2b in L. lactis NZ3900. These cells express extracellular protein using the NICE system and Usp₄₅ signal peptide. To validate the mature form of the expressed protein, the recombinant IFNα-2b was screened in a human colorectal cancer cell line using the cytotoxicity assay. The IFNα-2b was successfully cloned into the pNZ8148 vector, thereby generating recombinant L. lactis pNZ8148-SP_Usp45-IFNα-2b. The computational analysis of codon-optimized IFNα-2b revealed no mutation and amino acid changes; additionally, the codon-optimized IFNα-2b showed 100% similarity with native human IFNα-2b, in the BLAST analysis. The partial size exclusion chromatography (SEC) of extracellular protein yielded a 19 kDa protein, which was further confirmed by its positive binding to anti-IFNα-2b in the western blot analysis. The crude protein and SEC-purified partial fraction showed IC₅₀ values of 33.22 ㎍/ml and 127.2 ㎍/ml, respectively, which indicated better activity than the metabolites of L. lactis NZ3900 (231.8 ㎍/ml). These values were also comparable with those of the regular anticancer drug tamoxifen (105.5 ㎍/ml). These results demonstrated L. lactis as a promising host system that functions by utilizing the pNZ8148 NICE system. Meanwhile, codon-optimized usage of the inserted gene increased the optimal protein expression levels, which could be beneficial for its large-scale production. Taken together, the recombinant L. lactis IFNα-2b is a potential alternative treatment for colorectal cancer. Furthermore, its activity was analyzed in the WiDr cell line, to assess its colorectal anticancer activities in vivo.

• Journal of fish pathology
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• v.6 no.2
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• pp.93-101
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• 1993

The RNAI mutation of Saccharomyces cerevisia is a recessive and temperature sensitive lethal mutation which interferes with the production of mRNA, rRNA, and tRNA. However, the precise role of RNAI gene have not been revealed until yet. We have cloned rna1-1 mutant gene from rna1-1 mutant yeast strain(R49 ; trpl, ura3-52, rna1-1). The 3.4kb BglII fragment of wild type RNAI clone(81-2-6) contains whole RNAI gene. The genomic southern blotting with BglII digested R49 genomic DNA as a probe shows the unique and identical band with wild type 3.4kb BglII fragment. Therefore, We prepared partial BglII genomic library(3~4kb BglII fragments) into BamH I site of pUC19. The rna 1-1 mutant clone was screened with Digoxigenin(DIG)-lableled probe by high density colony hybridization. The 5'-flanking region of rna1-1 gene was sequenced by dideoxy chain termination method. The 5'-flanking sequence of RNAI gene contains three TATA-like sequence ; TAATA, TATA and TTTTAA at position of -67, -45, and -36 from first ATG codon respectively. The 5'-flanking region of wild type RNA I gene from ATG codon to -103nt was deleted with Bal31 exonuclease digestion, generating $pUC{\Delta}$/RNA I. After constructing $pYEP{\Delta}RNA$ I (consists of -103nt deleting RNA I gene, URA3 gene, $2{\mu}m$ rep. origin), pYEPrna1-1(consists of Xba I fragment of pUCrna1-1. URA3 gene, $2{\mu}m$ rep. origin), and pYEPRNAI. each plasmid was transformed into host strain(trpl, ura3-52, rna1-1) by electroporation, respectively. Yeast transformant carrying $pYEP{\Delta}RNA$ I did not complement the thermal sensitivity of rna1-1 gene. It means that TATA-like sequences in 5'-flanking region is not TATA sequence for transcribing RNAI gene and there may be other essential sequence in upstream region for the transcription of RNAI gene.

• Clinical and Experimental Pediatrics
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• v.48 no.11
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• pp.1252-1255
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• 2005

Abnormalities in the levels of thyroxine-binding globulin (TBG) are not associated with clinical disease and they do not require treatment. Congenital TBG deficiency is inherited in an X-linked manner. To date, some complete and partial TBG variants and one polymorphism have been identified by analysis of the TBG gene. Two male neonates were referred to us because of their low $T_4$ levels that were noted on the neonatal screening test. They showed normal levels of free $T_4$ and TSH. Their serum TBG was not detectable and those values of their parents were within the normal ranges. The genomic DNA was extracted from their white blood cells and the four coding exons of the TBG gene were amplified by using polymerase chain reaction. Sequencing of the four coding regions and all the intron/exon junctions revealed a single nucleotide deletion of the first base of the codon 352 of the mature protein in both of the neonates. This mutation resulted in a frameshift and a premature stop codon (TGA) 374. Their mothers were shown to be heterozygotes. We detected a single nucleotide deletion resulting in a frameshift in two male Korean neonates who had complete TBG deficiency.

• Journal of the korean academy of Pediatric Dentistry
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• v.28 no.1
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• pp.175-179
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• 2001

Regional odontodysplasia is a relatively rare condition in which both enamel and dentin are hypoplastic and hypocalcified. The result is localized arrest in tooth development. The etiology of regional odontodysplasia is uncertain, but, disturbance in vascular supply, somatic mutation, latent virus infection, trauma, hyperpyrexia, irradiation, nutrition, metabolic disorders and hereditary transmission are supported to be etiologic factors. Females are more often affected than males. (1.4 : 1). The maxillary arch is more often affected than the mandibular arch with the maxillary left quadrant being the most commonly involved. Affected teeth are hypoplastic, typically discolored yellow or yellowish brown, smaller in size and display a variety of surface marking including pitting and grooving. Radiographically, the teeth affected have been described to have a "ghost like" appearance or "fuzzy" appearance. Pulp calcification and denticles may be present within the pulp chambers of the affected teeth. In it's case, a 2 years old male visited for a treatment of uneruption of lower right teeth. Partial eruption of lower right deciduous central incisor and unerupted deciduous lateral incisor, deciduous canine and deciduous first molar showed severly delayed eruption state. On radiographic appearance, "Ghost like appearance", shortened root and opened apexes on lower right region were observed. It was suspected regional odontodysplasia with clinical and radiographic condition.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.5
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• pp.2743-2746
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• 2013

Background: Dilantin sodium (phenytoin) is an antiepileptic drug, which is routinely used to control generalized tonic clonic seizure and partial seizure episodes. A few case reports of oral squamous cell carcinomas arising from regions of phenytoin induced gingival overgrowth (GO), and overexpression of mitogenic factors and p53 have presented this condition as a pathology with potential to transform into malignancy. We recently investigated the genetic status of p53 and H-ras, which are known to be frequently mutated in Indian oral carcinomas in GO tissues and found them to only contain wild type sequences, which suggested a non-neoplastic nature of phenytoin induced GO. However, besides p53 and H-ras, other oncogenes and tumor suppressors such as PIK3CA, p14ARF, p16INK4a and $p21^{Waf1/Cip1}$, are frequently altered in oral squamous cell carcinoma, and hence are required to be analyzed in phenytoin induced GO tissues to be affirmative of its non-neoplastic nature. Methods: 100ng of chromosomal DNA isolated from twenty gingival overgrowth tissues were amplified with primers for exons 9 and 20 of PIK3CA, exons $1{\alpha}$, $1{\beta}$ and 2 of p16INK4a and p14ARF, and exon 2 of $p21^{Waf1/Cip1}$, in independent reactions. PCR amplicons were subsequently gel purified and eluted products were sequenced. Results: Sequencing analysis of the twenty samples of phenytoin induced gingival growth showed no mutations in the analyzed exons of PIK3CA, p14ARF, p16INK4a and $p21^{Waf1/Cip1}$. Conclusion: The present data indicate that the mutational alterations of genes, PIK3CA, p14ARF, p16INK4a and $p21^{Waf1/Cip1}$ that are frequently mutated in oral squamous cell carcinomas are rare in phenytoin induced gingival growth. Thus the findings provide further evidence that phenytoin induced gingival overgrowth as a non-neoplastic lesion, which may be considered as clinically significant given the fact that the epileptic patients are routinely administered with phenytoin for the rest of their lives to control seizure episodes.

• The Korea Journal of Herbology
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• v.32 no.2
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• pp.87-95
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• 2017

Objectives : Infectious diseases by Methicillin-Resistant Staphylococcus aureus (MRSA) are a growing problem worldwide. Characteristic of MRSA is endlessly mutation to resist antibiotics. Daehwanggo (DHG) is one of the oriental medicine prescriptions contained in Principles and Practice of Eastern Medicine. Daehwanggo was mainly used for external preparation from old times. The purpose of this study is to confirm possibility as supplementary drug of DHG about antibiotics through observation of synergy effect between DHG and commercial antibiotics and to observe restriction on growth of MRSA on any pathway through observation of mechanism. Methods : The minimum inhibitory concentration (MIC) of DHG against MRSA is $500{\sim}2000{\mu}g/m{\ell}$ by broth dilution method. In the checkerboard method, the combinations of DHG with antibiotics has partial synergistic effect or synergy effect and DHG markedly reduced the MICs of the antibiotics oxacillin (OX), gentamicin (GT) against MRSA. In the inhibition of resistance mechanism of DHG against MRSA, the expression of resistance gene and protein about ${\beta}-lactam$ antibiotic was reduced. Also, we observed the effect of DHG about cell membrane permeability against MRSA, and confirmed that DHG suppressed growth of strains by increasing cell membrane permeability. Results : Basis on the result, we speculate that DHG increase antibacterial activity of antibiotics against MRSA by changing the structure of cell wall of MRSA. Conclusions : These data suggest that Daehwanggo possesses possibility as supplementary drug about antibiotics against MRSA.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.51 no.spc1
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• pp.283-291
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• 2006

To investigate seed non destructive and fast determination technique utilizing near infrared reflectance spectroscopy (NIRs) for screening ultra high oleic (C18:1) and linoleic (C18:2) fatty acid content sesame varieties among genetic resources and lines of pedigree generations of cross and mutation breeding were carried out in National Institute of Crop Science (NICS). 150 among 378 landraces and introduced cultivars were released to analyse fatty acids by NIRs and gas chromatography (GC). Average content of each fatty acid was 9.64% in palmitic acid (C16:0), 4.73% in stearic acid (C18:0), 42.26% in oleic acid and 43.38% in linoleic acid by GC. The content range of each fatty acid was from 7.29 to 12.27% in palmitic, 6.49% from 2.39 to 8.88% in stearic, 12.59% of wider range compared to that of stearic and palmitic from 37.36 to 49.95% in oleic and of the widest from 30.60 to 47.40% in linoleic acid. Spectrums analyzed by NIRs were distributed from 400 to 2,500 nm wavelengths and varietal distribution of fatty acids were appeared as regular distribution. Varietal differences of oleic acid content good for food processing and human health by NIRs was 14.08% of which 1.49% wider range than that of GC from 38.31 to 52.39%. Varietal differences of linoleic acid content by NIRs was 16.41% of which 0.39% narrower range than that of GC from 30.60 to 47.01%. Varietal differences of oleic and linoleic acid content in NIRs analysis were appeared relatively similar inclination compared with those of GC. Partial least square regression (PLSR) among multiple variant regression (MVR) in NIRs calibration statistics was carried out in spectrum characteristics on the wavelength from 700 to 2,500 nm with oleic and linoleic acids. Correlation coefficient of root square (RSQ) in oleic acid content was 0.724 of which 72.4 percent of sample varieties among all distributed in the range of 0.570 percent of standard error when calibrated (SEC) which were considerably acceptable in statistic confidence significantly for analysis between NIRs and GC. Standard error of cross validation (SECV) of oleic acid was 0.725 of which distributed in the range of 0.725 percent standard error among the samples of mother population between analyzed value by NIRs analysis and analyzed value by GC. RSQ of linoleic acid content was 0.735 of which 73.5 percent of sample varieties among all distributed in the range of 0.643 percent of SEC. SECV of linoleic acid was 0.711 of which distributed in the range of 0.711 percent standard error among the samples of mother population between NIRs analysis and GC analysis. Consequently, adoption NIR analysis for fatty acids of oleic and linoleic instead that of GC was recognized statistically significant between NIRs and GC analysis through not only majority of samples distributed in the range of negligible SEC but also SECV. For enlarging and increasing statistic significance of NIRs analysis, wider range of fatty acids contented sesame germplasm should be kept on releasing additionally for increasing correlation coefficient of RSQ and reducing SEC and SECV in the future.