• BMB Reports
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• v.48 no.3
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• pp.178-183
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• 2015

Dendritic cells play an important role in determining whether na${\ddot{i}}$ve T cells mature into either Th1 or Th2 cells. We determined whether heat-shock protein X (HspX) purified from Mycobacterium tuberculosis regulates the Th1/Th2 immune response in an ovalbumin (OVA)-induced murine model of asthma. HspX increased interferon-gamma, IL-17A, -12 and transforming growth factor (TGF)-${\beta}$ production and T-bet gene expression but reduced IL-13 production and GATA-3 gene expression. HspX also inhibited asthmatic reactions as demonstrated by an increase in the number of eosinophils in bronchoalveolar lavage fluid, inflammatory cell infiltration in lung tissues, airway luminal narrowing, and airway hyper-responsiveness. Furthermore, HspX enhanced OVA-induced decrease of regulatory T cells in the mediastinal lymph nodes. This study provides evidence that HspX plays critical roles in the amelioration of asthmatic inflammation in mice. These findings provide new insights into the immunotherapeutic role of HspX with respect to its effects on a murine model of asthma.

• Journal of Acupuncture Research
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• v.19 no.6
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• pp.111-124
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• 2002

A line of study reported that electroacupuncture(EA) modulate natural killer cell(NK Cell) activities. One report suggested that EA enhanced splenic interferon-gamma($IFN-{\gamma}$), interleukin-2(IL-2), and NK cell activity in Sprague-Dawley rats. Another study suggested that $IFN-{\gamma}$ mediates the up-regulation of NK cell activity, and endogenous ${\beta}$-endorphin secretion also play a role in the up-regulation of NK cell activity induced by EA stimulation. In order to better understand the molecular regulation underlying the activation of NK cell induced by EA, we have utilized cDNA microarray to elucidate how EA alters program of gene expression of spleen in rats. First, we divided three groups, group I was EA group treated with EA in restriction holder, group II was sham group with only holder stress, and last group III was control group with no treatment. We measured NK cell activity after EA stimulation three times for 2 days using $^{51}Cr$ release assay. Second, Biotin-labeled cDNA probes synthesized from EA group and sham group, were competitively hybridized to the microarray that contained variable genes. Such high-throughput screening has identified a number of EA-responsive gene candidates. Of these, we found that EA induced a subset of genes of genes that functionally could modulatory effects on NK cell activity. Genes(vascular cell adhesion molecule-1, protein-tyrosine kinase, CD94 mRNA) related to boost NK cell activity, were increased by EA And, genes(protein-tyrosine-phospatase mRNA, protein-tyrosine phosphatase(SHP-1) mRNA) related to inhibit NK cell activity, were decreased by EA. These EA-responsive genes may provide key insights from which to understand mechanisms of activation of NK cell induced by EA.

• BMB Reports
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• v.38 no.4
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• pp.500-506
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• 2005

HOX11 encodes a homeodomain-containing transcription factor which directs the development of the spleen during embryogenesis. While HOX11 expression is normally silenced through an unknown mechanism in all tissues by adulthood, the deregulation of HOX11 expression is associated with leukemia, such as T-cell acute lymphoblastic leukemia. The elucidation of regulatory elements contributing to the molecular mechanism underlying the regulation of HOX11 gene expression is of great importance. Previous reports of HOX11 regulatory elements mainly focused on the 5'-flanking region of HOX11 on the chromosome related to transcriptional control. To expand the search of putative cis-elements involved in HOX11 regulation at the post-transcriptional level, we analyzed HOX11 mRNA 3'-untranslated region (3'UTR) and found an AU-rich region. To characterize this AU-rich region, in vitro analysis of HOX11 mRNA 3'UTR was performed with human RNA-binding protein HuR, which interacts with AU-rich element (ARE) existing in the 3'UTR of many growth factors' and cytokines' mRNAs. Our results showed that the HOX11 mRNA 3'UTR can specifically bind with human HuR protein in vitro. This specific binding could be competed effectively by typical ARE containing RNA. After the deletion of the AU-rich region present in the HOX11 mRNA 3'UTR, the interaction of HOX11 mRNA 3'UTR with HuR protein was abolished. These findings suggest that HOX11 mRNA 3'UTR contains cis-acting element which shares similarity in the action pattern with RE-HuR interactions and may involve in the post-transcriptional regulation of the HOX11 gene.

• Proceedings of the Microbiological Society of Korea Conference
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• 2007.05a
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• pp.120-122
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• 2007

All living organisms use numerous signal-transduction pathways to sense and respond to their environments and thereby survive and proliferate in a range of biological niches. Molecular dissection of these signalling networks has increased our understanding of these communication processes and provides a platform for therapeutic intervention when these pathways malfunction in disease states, including infection. Owing to the expanding availability of sequenced genomes, a wealth of genetic and molecular tools and the conservation of signalling networks, members of the fungal kingdom serve as excellent model systems for more complex, multicellular organisms. Here, we employed Cryptococcus neoformans as a model system to understand how fungal-signalling circuits operate at the molecular level to sense and respond to a plethora of environmental stresses, including osmoticshock, UV, high temperature, oxidative stress and toxic drugs/metabolites. The stress-activated p38/Hog1 MAPK pathway is structurally conserved in many organisms as diverse as yeast and mammals, but its regulation is uniquely specialized in a majority of clinical Cryptococcus neoformans serotype A and D strains to control differentiation and virulence factor regulation. C. neoformans Hog1 MAPK is controlled by Pbs2 MAPK kinase (MAPKK). The Pbs2-Hog1 MAPK cascade is controlled by the fungal "two-component" system that is composed of a response regulator, Ssk1, and multiple sensor kinases, including two-component.like (Tco) 1 and Tco2. Tco1 and Tco2 play shared and distinct roles in stress responses and drug sensitivity through the Hog1 MAPK system. Furthermore, each sensor kinase mediates unique cellular functions for virulence and morphological differentiation. We also identified and characterized the Ssk2 MAPKKK upstream of the MAPKK Pbs2 and the MAPK Hog1 in C. neoformans. The SSK2 gene was identified as a potential component responsible for differential Hog1 regulation between the serotype D sibling f1 strains B3501 and B3502 through comparative analysis of their meiotic map with the meiotic segregation of Hog1-dependent sensitivity to the fungicide fludioxonil. Ssk2 is the only polymorphic component in the Hog1 MAPK module, including two coding sequence changes between the SSK2 alleles in B3501 and B3502 strains. To further support this finding, the SSK2 allele exchange completely swapped Hog1-related phenotypes between B3501 and B3502 strains. In the serotype A strain H99, disruption of the SSK2 gene dramatically enhanced capsule biosynthesis and mating efficiency, similar to pbs2 and hog1 mutations. Furthermore, ssk2, pbs2, and hog1 mutants are all hypersensitive to a variety of stresses and completely resistant to fludioxonil. Taken together, these findings indicate that Ssk2 is the critical interface protein connecting the two-component system and the Pbs2-Hog1 pathway in C. neoformans.

• Molecules and Cells
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• v.44 no.3
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• pp.146-159
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• 2021

DNA methylation, and consequent down-regulation, of tumour suppressor genes occurs in response to epigenetic stimuli during cancer development. Similarly, human oncoviruses, including human papillomavirus (HPV), up-regulate and augment DNA methyltransferase (DNMT) and histone deacetylase (HDAC) activities, thereby decreasing tumour suppressor genes (TSGs) expression. Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1), an epigenetic regulator of DNA methylation, is overexpressed in HPV-induced cervical cancers. Here, we investigated the role of UHRF1 in cervical cancer by knocking down its expression in HeLa cells using lentiviral-encoded short hairpin (sh)RNA and performing cDNA microarrays. We detected significantly elevated expression of thioredoxin-interacting protein (TXNIP), a known TSG, in UHRF1-knockdown cells, and this gene is hypermethylated in cervical cancer tissue and cell lines, as indicated by whole-genome methylation analysis. Up-regulation of UHRF1 and decreased TXNIP were further detected in cervical cancer by western blot and immunohistochemistry and confirmed by Oncomine database analysis. Using chromatin immunoprecipitation, we identified the inverted CCAAT domain-containing UHRF1-binding site in the TXNIP promoter and demonstrated UHRF1 knockdown decreases UHRF1 promoter binding and enhances TXNIP expression through demethylation of this region. TXNIP promoter CpG methylation was further confirmed in cervical cancer tissue by pyrosequencing and methylation-specific polymerase chain reaction. Critically, down-regulation of UHRF1 by siRNA or UHRF1 antagonist (thymoquinone) induces cell cycle arrest and apoptosis, and ubiquitin-specific protease 7 (USP7), which stabilises and promotes UHRF1 function, is increased by HPV viral protein E6/E7 overexpression. These results indicate HPV might induce carcinogenesis through UHRF1-mediated TXNIP promoter methylation, thus suggesting a possible link between CpG methylation and cervical cancer.

• Development and Reproduction
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• v.4 no.1
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• pp.95-100
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• 2000

A large number of man-made chemicals that have been released into the environment have the potential to disrupt the endocrine system of animals and humans. There is a critical developmental period during which sexual brain differentiation proceeds irreversibly under the influence of gonadal hormone. Recently we identified KAP3 gene expressed during the critical period of rat brain sexual differentiation. KAP3 functions as a microtubule-based motor that transports membranous organelles anterogradely in cells, including neurons. In the present study, we aimed to investigate the effect of endocrine disrupter, Dichlorodiphenyl trichloroethane (DDT), on the KAP3 gene expression during critical period of rat brain development. Maternal exposure to DDT increased the level of KAP3 mRNA in male and female fetus brains when examined on the gestational day 17 (GDl7). In postnatal day 6, DDT suppressed the expression of KAP3 gene in male and female rat brain. Also, the body weight and fertilization rate were decreased in the DDT exposured rats. These results showed that endocrine disrupter, DDT, can affect the transcriptional level of brain sexual differentiation related gene, KAP3, in the prenatal and the neonatal rat brain and that maternal exposure to endocrine disruptors may lead to a toxic response in embryonic differentiation of brain. And so KAP3 gene may be used a gene maker to analyse the molecular mechanism for toxic response in animal nerve tissues exposed to endocrine disruptors.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.7
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• pp.1023-1029
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• 2007

Inhibins participate in the regulation of pituitary follicle-stimulating hormone synthesis and secretion, follicular maturation and steroidogenesis in the female. Inhibin ${\beta}_A$ gene (INHBA) was studied as a candidate gene for the prolificacy of sheep. Single nucleotide polymorphisms of the entire coding region and partial 3' untranslated region of INHBA were detected by PCR-SSCP in two high fecundity breeds (Small Tail Han and Hu sheep) and six low fecundity breeds (Dorset, Texel, German Mutton Merino, South African Mutton Merino, Chinese Merino and Corriedale sheep). Only the PCR products amplified by primers 3, 4 and 5 displayed polymorphisms. For primer 3, genotype CC was only detected in Chinese Merino sheep, genotype AA was detected in the other seven sheep breeds. Genotype BB was only detected in Hu sheep. Only Hu sheep displayed polymorphism. Eight or four nucleotide mutations were revealed between BB or CC and AA, respectively, and these mutations did not result in any amino acid change. For primer 4, genotypes EE, EG and GG were detected in Dorset and German Mutton Merino sheep, genotypes EE, EF and FF were detected in Chinese Merino sheep, only genotype EE was detected in the other five sheep breeds. Only Dorset, German Mutton Merino and Chinese Merino sheep displayed polymorphism. Sequencing revealed one nucleotide mutation ($114G{\rightarrow}A$) of exon 2 of INHBA gene between genotype FF and genotype EE, and this mutation did not cause any amino acid change. Another nucleotide change ($143C{\rightarrow}T$) was identified between genotype GG and genotype EE, and this mutation resulted in an amino acid change of $serine{\rightarrow}leucine$. For primer 5, genotypes KK and KL were detected in German Mutton Merino and Corriedale sheep, genotypes KK, LL and KL were detected in the other six sheep breeds. Genotype MM was only detected in Hu sheep. All of these eight sheep breeds displayed polymorphism. Sequencing revealed one nucleotide mutation ($218A{\rightarrow}G$) of exon 2 of the INHBA gene between genotype LL and genotype KK, and nine nucleotide mutations between genotype MM and genotype KK. These mutations did not alter amino acid sequence. The partial sequence (395 bp for exon 1 and 933 bp for exon 2) of the INHBA gene in Small Tail Han sheep (with genotype KK for primer 5) was submitted into GenBank (accession number EF192431). Small Tail Han sheep displayed polymorphisms only in the fragment amplified by primer 5. The Small Tail Han ewes with genotype LL had 0.53 (p<0.05) or 0.63 (p<0.05) more lambs than those with genotype KL or KK, respectively. The Small Tail Han ewes with genotype KL had 0.10 (p>0.05) more lambs than those with genotype KK.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.8
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• pp.1069-1077
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• 2009

Castration of male pig produces significant negative effects on skeletal muscle development. The androgen receptor (AR), two splice variants of insulin-like growth factor-I (IGF-I Ea and MGF) and the myostatin gene may play important roles in this process. In the present study, the expression of AR, IGF-I Ea, MGF and myostatin genes in three skeletal muscles, the brachialis, longissimus and semitendinosus, were studied using real-time quantitative RT-PCR. Our experimental design used 14 pairs of male Landrace sire${\times}$Yorkshire dam piglets. The two piglets in each pair were full sibs, one of which was castrated at 21 d of age; the other remained intact. The study group was divided into subgroups of equal size. Animals in the first subgroup were slaughtered at 147 d and those of the second at 210 d of age. Carcass weight and lean meat yield were similar between boars and barrows at 147 d of age (p>0.05), whereas barrows had lower carcass weight and less lean meat yield at 210 d of age (p<0.05). Castration caused down-regulation of AR gene expression at both 147 and 210 d of age (p<0.05). The two splice variants of the IGF-I gene from porcine skeletal muscle were cloned using RT-PCR, and it was found that MGF differs from IGF-I Ea in having a 52-base insert in the last coding exon of the mRNA. Both splice variants were down-regulated by castration only at 210 d of age (p<0.05). No differences in expression of the myostatin gene were observed between boars and barrows at either 147 or 210 d of age (p>0.05). These results suggest that the downregulation of AR, IGF-I Ea and MGF gene expression following castration helps to explain the negative effect of castration on skeletal muscle development.

• Journal of fish pathology
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• v.26 no.1
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• pp.19-30
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• 2013

Megalocytivirus is a major fish pathogen in marine aquaculture of Asian countries including Korea. Despite of many species affected by this pathogen, little is known interaction between megalocytivirus and the fish immune system. One of the cyclooxygenase isoforms, named COX-2, is playing an important role in immune regulation, and distinct from COX-1 isoform of constitutive activity. COX-2 enzyme is induced by various inflammatory signals, including injection of lipopolysaccharide or infection by pathogenic agents. We cloned COX-2 gene in rock bream using degenerated primers designed from reported sequences of other fish species in PCR followed with 5'- and 3'-end RACE-PCR. The full length of cDNA of rbCOX2 (rock bream COX-2) gene are 2655 bp and that translates into 609 amino acids. The rbCOX-2 genomic organization are found to span 10 exons separated by 9 introns. We also studied if the experimental infection of rock bream with megalocytivirus could affect the expression of COX-2 gene. When injected with LPS, expression of the COX-2 gene was reached peak level at 1 day post injection and showed 13.10 fold increased level compared with that of control. While, when injected with megalocytivirus, we were not able to find significantly increased COX-2 gene expression different from that of control. Cloned and analyzed COX-2 gene in rock bream will help to understand defence mechanisms in fish after viral infection and will also support the development of the measures for treatment and prevention of viral infection.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.14
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• pp.5635-5641
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• 2015

Background: Cirrhosis is regarded as a possible end stage of many liver diseases, including viral infection. It occurs when healthy liver tissue becomes damaged and is replaced by scar tissue and finally may lead to hepatocellular carcinoma. Interferons (IFNs)are two general categories, type I and II. Type I includes one beta interferon and over 20 different alpha interferons. Alpha interferons are very similar in how they work, interacting with other proteins on cells like receptors. The main objective of this study was to compare Mx gene productivity post different cell line treatment with imported and Egyptian biosimilar locally produced IFNs, as well as the efficacy of those tested IFNs. Also, an assessment was made of sensitivity of different cell lines as alternatives to that recommended for evaluation of antiviral activity. Materials and Methods: Different cell lines (Vero, MDBK and Wish) were employed to evaluate cytotoxicity using the MTT assay. Antiviral activity was evaluated compared with standard IFN against VSV, Indiana strain -156, on tested rh-IFNs (imported; innovated and Egyptian biosimilar locally produced IFNs) in the pre-treated cell lines previously mentioned. The virus was propagated in the Wish cell line as recommended. Finally we estimated up-regulation of the Mx gene as a biomarker. Results: Data recorded revealed that test IFNs were safe in test cell lines. Viability was around 100%. Locally tested interferon did not realize the international potency limits, while the imported one was accepted compared with the standard IFN. These results were the same either using infectivity titer reduction assay or crystal violet staining of residual non- infected cells. Mx protein production was cell type related and confirmed by the detected Mx gene expressed in imported and locally produced IFN pre-treated cell lines. The expression of the gene was arranged in the order of Vero> wish > MDBK for the imported IFN, while for the Egyptian biosimillar locally produced one it was MDBK> Vero> wish. With regard to the antiviral activity there was a significant difference of imported IFN potency compared with the locally produced IFN (P<0.05), the IFN potential (antiviral activity) was not cell line related and showed non-significant difference for each separate product. Conclusions: Vero cells can be used as an alternative cell line for evaluation of IFN potency in case of unavailable USP recommended cell lines. Alternative potency evaluation assay could be used and proved significant difference in IFN potency in case of local and imported agents. Evaluation of antiviral activity could be used in parallel to viral infectivity reduction assay for better accuracy. Mx gene can be used as a marker for IFN potential.