• Asian-Australasian Journal of Animal Sciences
• /
• 제29권9호
• /
• pp.1363-1370
• /
• 2016

Rennet, a complex of enzymes found in the stomachs of ruminants, is an important component for cheese production. In our study, we described that yak chymosin gene recombinant Pichia pastoris strain could serve as a novel source for rennet production. Yaks total RNA was extracted from the abomasum of an unweaned yak. The yak preprochymosin, prochymosin, and chymosin genes from total RNA were isolated using gene specific primers based on cattle chymosin gene sequence respectively and analyzed their expression pattern byreal time-polymerase chain reaction. The result showed that the chymosin gene expression level of the sucking yaks was 11.45 times higher than one of adult yaks and yak chymosin belongs to Bovidae family in phylogenetic analysis. To express each, the preprochymosin, prochymosin, and chymosin genes were ligated into the expression vector $pPICZ{\alpha}A$, respectively, and were expressed in Pichia pastoris X33. The results showed that all the recombinant clones of P. pastoris containing the preprochymosin, prochymosin or chymosin genes could produce the active form of recombinant chymosin into the culture supernatant. Heterologous expressed prochymosin (14.55 Soxhlet unit/mL) had the highest enzyme activity of the three expressed chymosin enzymes. Therefore, we suggest that the yak chymosin gene recombinant Pichia pastoris strain could provide an alternative source of rennet production.

• Asian Pacific Journal of Cancer Prevention
• /
• 제15권7호
• /
• pp.3287-3291
• /
• 2014

Background: The interesting preponderance of Chinese with colorectal carcinoma (CRC) amongst the three major ethnic groups in Malaysia prompted a study to determine DNA mismatch repair (MMR) status in our CRC and attempt correlation with patient age, gender and ethnicity as well as location, grade, histological type and stage of tumour. Histologically re-confirmed CRC, diagnosed between $1^{st} $January 2005 and $31^{st}$ December 2007 at the Department of Pathology, University of Malaya Medical Centre, were immunohistochemically stained with monoclonal antibodies to MMR proteins, MLH1, MSH2, MSH6 and PMS2 on the Ventana Benchmark XT autostainer. Of the 142 CRC cases entered into the study, there were 82 males and 60 females (M:F=1.4:1). Ethnically, 81 (57.0%) were Chinese, 32 (22.5%) Malays and 29 (20.4%) Indians. The patient ages ranged between 15-87 years (mean=62.4 years) with 21 cases <50-years and 121 ${\geq}50$-years of age. 14 (9.9%) CRC showed deficient MMR (dMMR). Concurrent loss of MLH1 and PMS2 occurred in 10, MSH2 and MSH6 in 2 with isolated loss of MSH6 in 1 and PMS2 in 1. dMMR was noted less frequently amongst the Chinese (6.2%) in comparison with their combined Malay and Indian counterparts (14.8%), and was associated with right sided and poorly differentiated tumours (p<0.05). 3 of the 5 (60.0%) dMMR CRC cases amongst the Chinese and 1 of 9 cases (11.1%) amongst the combined Malay and Indian group were <50-years of age. No significant association of dMMR was noted with patient age and gender, tumour stage or mucinous type.

• BMB Reports
• /
• 제38권4호
• /
• pp.391-398
• /
• 2005

3-hydrogenwadaphnin (3-HK) is a new daphnane-type diterpene ester isolated from Dendrostellera lessertii with strong anti-tumoral activity in animal models and in cultures. Here, prolonged effects of this new agent on proliferation and viability of several different cancerous cell lines were evaluated. Using [$^3H$]thymidine incorporation, it was found that the drug inhibited cell proliferation and induced G1/S cell cycle arrest in leukemic cells 24 h after a single dose treatment. The cell viability of Jurkat cells was also decreased by almost 10%, 31% and 40% after a single dose treatment (7.5 nM) at 24, 48 and 72 h, respectively. The drug-treated cells were stained with acridine orange/ethidium bromide to document the chromatin condensation and DNA fragmentation. These observations were further confirmed by detection of DNA laddering pattern in the agarose gel electrophoresis of the extracted DNA from the treated cells. Treatment of K562 cells with the drug at 7.5, 15 and 30 nM caused apoptosis in 25%, 45% and 65% of the cells, respectively. Exogenous addition of $25-50\;{\mu}M$ guanosine and/or deoxyguanosine to the cell culture of the drug-treated cells restored DNA synthesis, released cell arrest at G1/S checkpoint and decreased the apoptotic cell death caused by the drug. These observations were not made using adenosine. However, the drug effects on K562 cells were potentiated by hypoxanthine. Based on these observations, perturbation of GTP metabolism is considered as one of the main reasons for apoptotic cell death by 3-HK.

• Asian-Australasian Journal of Animal Sciences
• /
• 제17권7호
• /
• pp.897-902
• /
• 2004

PSMC5 subunit, which belongs to the 26S proteasomal subunit family, plays an important role in the antigen presentation mediated by MHC class I molecular. Full-length cDNA of porcine PSMC5 was isolated using the in silico cloning and rapid amplification of cDNA ends (RACE). Amino acid was deduced and the primary structure was analyzed. Results revealed that the porcine PSMC5 gene shares the high degree of sequence similarity with its mammalian counterparts at both the nucleotide level and the amino acid level. The RT-PCR was performed to detect the porcine PSMC5 expression pattern in seven tissues and the result showed that high express level was observed in spleen, lung, marrow and liver while the low express level was in muscle. The full-length genomic DNA sequence of porcine PSMC5 gene was amplified by PCR and the genomic structure revealed that this gene was comprised by 12 exons and 11 introns. Best alignment of the cDNA and genomic exon DNA sequence presents 4 mismatches and this information potentially bears further study in gene polymorphisms.

• 대한관절경학회지
• /
• 제7권2호
• /
• pp.127-136
• /
• 2003

슬관절 후외측방 불안정성은 전방 및 후방 십자인대 손상과 흔히 동반되며 이 동반된 손상은 심각한 기능적 불안정성 및 관절 연골의 변성을 초래하게된다. 슬관절의 후외측 구조물 손상이 있는 경우 적절한 치료 없이 전방 및 후방 십자인대 재건술만 시행할 경우 십자인대 재건술이 실패하게된다. 이를 방지하기 위해 자세한 이학적 검사, 방사선 검사를 시행하여야하며 하지정열축 및 보행 형태를 평가하여야 한다. 급성 후외측방 구조물의 3등급 단독 손상이나 동반 손상에서는 3주이내에 일차 봉합을 하거나, 봉합이 어려울 경우 보강수술이나 재건수술을 시행하는 것이 좋다. 후방 및 후외측방 재건술에서 다양한 수술 방법 중 적절한 방법을 선택하여 동시에 시행하거나 2단계 재건수술을 가능한 빨리 시행하여야 한다. 만약 만성 후외측 불안정성 슬관절에서 내반 정열이 있으면서 varus thrust gait가 있는 경우 외측 연부조직 재건술로는 해결하기가 어려우므로 먼저 외반 절골술을 시행하여야 하고 약 6개월 뒤에 후외측 불안정성을 재평가하여 이후 연부조직 재건술을 시행할 수도 있다.

• 한국수산과학회지
• /
• 제42권4호
• /
• pp.322-328
• /
• 2009

Three-hundred and sixteen Escherichia coli strains from seawater, and a variety of farmed fishes, including oliver flounder (Paralichthys olivaceus), black rock fish (Sebastes schlegeli), red sea bream (Pagrus major) and sea bass (Lateolabrax japonicus) between May to October in 2004, were tested by agar dilution method to determine their susceptibility patterns to 17 antimicrobial agents. Overall, 92.1% of Escherichia coli isolates from samples showed antimicrobial resistance to at least one antimicrobial agent and the multiple resistance was seen in 173 isolates (54.7%). The resistance of E. coli isolates to tetracycline (74.1%) was highest, followed by cephalothin (69.9%), doxycycline (66.5%), streptomycin (47.2%), ampicillin (46.2%), cefazolin (31.6%), enrofloxacin (31.0%). norfloxacin (28.2%). The most frequent resistance pattern was TE-D-CF-CIP-ENO-NOR-AM-S-C-SXT-AmC-CZ (14.7%), followed by CF (6.2%), TE (5.1%), TE-CF (4.5%) in 177 isolates from fishes and TE-D-CF (7.2%) followed by TE-D-CF-S (5.8%), CF and TE-D-S (3.6%) in 139 isolates from seawater.

• Journal of Microbiology and Biotechnology
• /
• 제11권5호
• /
• pp.819-824
• /
• 2001

The principal DNA modification systems of the amino-acid-producing bacteria Corynebacterium glutamicum AS019, Brevibacterium flavum BF4, and B. lactofermentum BL1 was investigated using two approaches; digestion of plasmid DNA isolated from these species TseI and Fnu4HI, and sequence analysis of the putative methyltransferase target sites following the derivatization of DNA using metabisulfite treatment. The C. glutamicum and B. flavum strains showed similar digestion patterns to the two enzymes, indicating that the target for cytidine methyltransferase recognizes 5'-GCSGC-3'(where S is either G or C). Mapping the methylated cytidine sites by bisulfite derivatization, followed by PCR amplification and sequencing, was only possible when the protocol included an additional step eliminating any underivatized DNA after PCR amplification, thereby indicating that the derivatization was not $100\%$ efficient. This may have been due to the high G0C content of this genus. It was confirmed that C. glutamicum AS019 and B. flavum BF4 methylated the cytidine in the $Gm^5CCGC$ sequences, yet there were no similar patterns of methylation in B. lactofermentum, which was consistent with the distinctive degradation pattern seen for the above enzymes. These findings demonstrate the successful application of a modified bisulfite derivatization method with the Corynebacterium species for determining methylation patterns, and showed that different species in the geneus contain distinctive restriction and modification systems.

• Journal of Microbiology and Biotechnology
• /
• 제9권6호
• /
• pp.847-853
• /
• 1999

A Pseudomonas sp. strain that is capable of utilizing dicarboxylic acids as a sole carbon source was isolated from activated sludge by using the enrichment culture technique. This organism accumulated polyhydroxyalkanoates (PHAs) with an unusual pattern of monomer units that depends on the carbon sources used. Polyhydroxybutyrate (PHB) homopolyester was synthesized from glucose or small $C_{-even}$ alkanoic acids, such as butyric acid and hexanoic acid. Accumulation of PHB homopolyester was also observed in the cells grown on $C_{-odd}$ dicarboxylic acids, such as heptanedioic acid and nonanedioic acid as the sole carbon sources. In contrast, a copolyester consisting of 6 mol% 3-hydroxybutyrate (3HB) and 94 mol% 3-hydroxyvalerate (3HV) was produced with a PHA content of as much as 36% of the cellular dry matter. This strain produced PHAs consisting both of the short-chain-length (SCL) and the medium-chain-length (MCL) 3-hydroxyacid units when heptanoic acid to undecanoic acid were fed as the sole carbon sources. Most interestingly, polyester consisting of significant amount of relevant fractions, 3HB, 3HV, and 3-hydroxyheptanoate (3HHp), was accumulated from heptanoic acid. According to solvent fractionation experiments, the polymer produced from heptanoic acid was a blend of poly(3HHp) and of a copolyester of 3HB, 3HV, and 3HHp units. The hexane soluble fractions contained only 3HHp units while the hexane-insoluble fractions contained 3HB and 3HV units with a small amount of 3HHp unit. The copolyester was an elastomer with unusual mechanical properties. The maximum elongation ratio of the copolyester was 460% with an ultimate strength of 10 MPa, which was very different from those of poly(3HB-co-3HV) copolyesters having similar compositions produced from other microorganisms.

• Asian-Australasian Journal of Animal Sciences
• /
• 제13권9호
• /
• pp.1285-1289
• /
• 2000

This study investigated the lipogenesis capacity of hepatocytes and lipolysis rate of adipocytes of broilers as affected by different fatty acids (trial one) and different linoleic acid (C18:2) levels (trial two). Twenty 6-wk old broilers were used; their hepatocytes and adipocytes were isolated for the in vitro study. In trial one, four treatments were tested. The control group in which no fatty acid was added, and the test groups to which were added $300{\mu}M$ of C16:0, C18:1 and C18:2, respectively. For trial two, different levels (0, $300{\mu}M$ and 1 mM) of C18:2 combined to fatty acid-free bovine serum albumin (BSA) were added to the medium. According to results of trial one, added fatty acids significantly reduced the incorporation by hepatocytes of [U,$^{14}C$]glucose into total lipid (p<0.05); the lipogenesis capacity in C18:2 group was the lowest. Although a similar pattern was found with [l,$^{14}C$]acetate, the groups only slightly differed in terms of lipogenesis capacity (p=0.11). In addition, the C18:2 group had a significantly (p<0.05) greater lipolysis rate than the C16:0 and control groups. Results of trial two indicated that C18:2 significantly (p<0.05) reduced lipogenesis capacity both for [U,$^{14}C$]glucose and [l,$^{14}C$]acetate, and markedly stimulated the lipolysis rate (p<0.05), displaying a dose response. Results presented herein demonstrate that C18:2 can reduce lipogenesis capacity and stimulate the lipolysis rate in broilers.

• Asian Pacific Journal of Cancer Prevention
• /
• 제14권2호
• /
• pp.1147-1150
• /
• 2013

Background: Cancer diagnostic biomarkers have a wide range of applications that include early detection of oral precancerous lesions and oral squamous cell carcinomas, and assessing the metastatic status of lesions. The interferon stimulated ISG15 gene encodes an ubiquitin-like protein, which conjugates to stabilize activation status of associated proteins. Hence a deregulated expression of ISG15 may promote carcinogenesis. Indeed overexpression of ISG15 has been observed in several cancers and hence it has been proposed as a strong candidate cancer diagnostic biomarker. Given the emerging relationship between malignant transformation and ISG15, we sought to examine the expression pattern of this gene in tumor biopsies of oral squamous cell carcinoma (OSCC) tissues collected from Indian patients. Materials and Methods: Total RNA isolated from thirty oral squamous cell carcinoma tissue biopsy samples were subjected to semi-quantitative RT-PCR with ISG15 specific primers to elucidate the expression level. Results: Of the thirty oral squamous cell carcinomas that were analyzed, ISG15 expression was found in twenty four samples (80%). Twelve samples expressed low level of ISG15, six of them expressed moderately, while the rest of them expressed very high level of ISG15. Conclusions: To the best of our knowledge, the results show for the first time an overexpression of ISG15 in up to 80% of oral squamous cell carcinoma tissues collected from Indian patients. Hence ISG15 may be explored for the possibility of use as a high confidence diagnostic biomarker in oral cancers.