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Closely correlated expression patterns between ubiquitin specific peptidase 9X-linked (USP9X) and adherens junction formation factor (Afadin) in mouse testis development suggests that Usp9x regulates the deubiquitination of Af-6 (also known as Afadin, AFDN), and subsequently, the cell adhesion dynamics during gametogenesis. However, this relationship has not yet been tested in other domestic animals. The study was examined the temporal and spatial expression patterns of porcine USP9X and AFDN from the pre-pubertal to adult stages using real time-PCR and immunohistochemistry. Furthermore, we detected the transcripts of USP9X and AFDN in the testis of 1-, 6- and 12-months old boar, respectively. USP9X and AFDN were found to have similar expressions patterns, with basal expression after 1 month followed by a significant up-regulation from 6 months (puberty) onwards. In addition, neither the AFDN or USP9X proteins were detected in spermatogenic cells but they were expressed in the leydig cells and sertoli cells. USP9X was detected around the basal lamina during pre-puberty, and predominantly expressed in the leydig cells at puberty. Finally, in adult testis, USP9X was increased at the sertoli cell-cell interface and the sertoli cell-spermatid interface. In summary, closely correlated expression patterns between USP9X and AFDN in boar testis supports the previous findings in mice. Furthermore, the junction connections between the sertoli cells may be regulated by the ubiquitination process mediated via USP9X.

This study aimed to explore gene expression changes in the inferior colliculus (IC) after single-sided deafness (SSD). Forty 8-week-old female Sprague–Dawley rats were used. Twenty rats underwent right-side cochlear ablation, and IC tissues were harvested after 2 weeks (SSD 2-week group). Twenty rats underwent a sham operation and were sacrificed after 2 weeks (control group). Both sides of the IC were analyzed using a gene expression array. Pathway analyses were performed on genes that were differentially expressed compared with their levels in the control group. The expression levels of genes involved in the candidate pathways were confirmed using reverse transcription polymerase chain reaction (RT-PCR). Among the genes with ≥ 1.5-fold changes in expression levels and P

Studies have shown that particulate matter (PM) induces the expression of the aryl hydrocarbon receptor (Ahr) leading to the activation of the oxidative stress response. This study is aimed at characterizing the specific impact of fine PM on the expression profile of the Ahr and oxidative stress response in the primary auditory cortex. PM2.5 ( n = 14 per group), respectively. One week after intracranial injection, the temporal cortex was harvested. Transmission electron microscopy was performed to evaluate the distribution of PM2.5 within the temporal cortex. Additionally, the mRNA and protein expression levels of cytochrome P450 1A1 (CYP1A1), CYP1B1, inducible nitric oxide synthase (iNOS), Ahr, and brevican mRNA and protein were measured using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) or western blotting, respectively. Finally, the protein expression levels of the receptor for advanced glycation end products (RAGE) were estimated using enzyme-linked immunosorbent assay (ELISA). PM2.5 was observed in intracellular vesicles within the temporal cortex following intracranial injection. Levels of oxidative stress molecules (i.e., CYP1A1, CYP1B1, and iNOS), Ahr, Brevican, and RAGE were higher in the PM2.5 group compared with the control group. Intracranial administration of PM2.5 led to increased levels of Ahr and markers of an oxidative stress response in the temporal cortex. The oxidative stress response-mediated increases in the levels of brevican and RAGE.

Deafferentation and compensatory neural plastic changes in the inferior colliculus (IC) have been suggested following single-sided deafness (SSD). We explored related miRNA changes in the IC of SSD rats using miRNA microarray analyses.Eight-week-old rats were divided into control and SSD rats (n = 8 for each group). SSD rats underwent right-side cochlear ablation surgery, with the IC harvested two weeks post-surgery. miRNA microarray analysis was performed using GeneChip miRNA 4.0, microarray (Affymetrix Inc.). miRNAs whose expression levels differed between SSD and control rats with a fold-change ≥ 1.5 and P 0.05 were examined using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Target genes of differentially expressed miRNAs were predicted using TargetScan software. The pathways related to predicted target genes were analyzed. mRNA levels of predicted target genes were estimated using qRT-PCR.The expression of miR-15b-5p, miR-202-5p, and miR-212-3p was lower in the contralateral (left) IC of SSD rats than that of control rats. In SSD rats, miRNA expression levels in the contralateral IC were 0.45-, 0.25-, and 0.50-fold lower for miR-15b-5p, miR-202-5p, and miR-212-3p, respectively (P 0.05). The expression of predicted target genes (Spred1, Rasa1, Lsm11, and Srsf1) was higher in the contralateral IC of SSD rats than in control rats. The targets were predicted to be related with cleavage of growing transcripts in the termination region, mitogen-activated protein kinase family signaling cascades, RAF/AMP kinase cascade, regulation of RAS by GTPase activating proteins (GAPs), and RNA polymerase II transcription termination. For ipsilateral ICs, miR-425-3p, miR-199a-5p, and miR-134-3p showed lower expressions in SSD rats than in control rats, which were 0.55-, 0.61-, and 0.69-fold lower, respectively (P 0.05). The expression of predicted target genes (Atp2b2, Grin2b, Foxp1, Ztbt20, Zfp91, and Strn) was higher in the ipsilateral IC of SSD rats; the regulation of synaptic plasticity, cAMP signaling pathway, metal ion binding, and calcium ion transport can be associated with these target genes.Adult rats with unilateral auditory deprivation showed miRNA changes in the IC. The contralateral IC showed decreased miRNA expression predicted to be related to MAPK and RAS signaling, whereas the ipsilateral IC revealed decreased miRNA expression predicted to be associated with synaptic plasticity and calcium ion transport.

Nanoparticulate matter activates the aryl hydrocarbon receptor (AhR) pathway in the respiratory system in a process involving the AhR nuclear translocator (ARNT) and cytochrome P450 family 1, member A1 (CYP1A1). We examined changes in AhR-related pathways following intranasal instillation of nanoparticulate matter in the olfactory bulb and cerebral cortex. Twice a day for 5 days per week for 1 week or 2 weeks, 8-week-old Sprague–Dawley rats were intranasally instilled with 10 µL nanoparticulate matter (nano group; n = 36). An equal volume of saline was intranasally instilled in control rats (n = 36). One week after intranasal instillation, olfactory function and Y-maze tests were performed. The expression levels of AhR in the olfactory bulb and temporal cortex were analyzed using western blotting and immunofluorescence assays. The expression levels of AhR, CYP1A1, inducible nitric oxide synthase (iNOS), and five genes encoding cation transporters (ARNT, ATP7B, ATPB1, OCT1, and OCT2) in the olfactory bulb were analyzed using quantitative reverse transcription. The olfactory discrimination capability was reduced in the nano group compared with the control group. Proportional changes in the Y-maze test were not significantly different between the nano and control groups. AhR mRNA and protein expression in the olfactory bulb increased 1.71-fold (P P = 0.008), respectively. However, no significant changes were observed in the temporal cortex. In the olfactory bulb, the expression of ARNT, ATP7B, ATPB1, and OCT2 was downregulated. CYP1A1 and iNOS expression in the olfactory bulb was upregulated compared with that in the temporal cortex. The intranasal instillation of nanoparticulate matter decreased the olfactory discrimination ability, which was accompanied by upregulation of AhR expression and downregulation of cation transporters in the olfactory bulb.

The 3D8 single-chain variable fragment (scFv) is a mini-antibody sequence with independent nuclease activity that shows antiviral effects against all types of viruses in chickens and mice. In this study, chickens were treated daily with an oral dose of 109 CFU Lactobacillus paracasei (L. paracasei) expressing either a secreted or anchored 3D8 scFv for three weeks. After L. paracasei administration, the chickens were challenged with avian influenza virus (AIV). From each experimental group, three chickens were directly infected with 100 µL of 107.5 EID50/mL H9N2 AIV and seven chickens were indirectly challenged through contact transmission. oropharyngeal and cloacal swab samples were collected at 3, 5, 7, and 9 days post-inoculation (dpi) from AIV-challenged chickens, AIV Shedding titres were measured by quantitative real-time PCR. Contact transmission in the chickens that were fed 3D8 scFv-secreting L. paracasei showed a significant reduction in viral shedding when compared with other groups. These results suggest that L. paracasei secreting 3D8 provides a basis for the development of ingestible antiviral probiotics with activity against AIV.

Infectious bronchitis (IB) generated by the infectious bronchitis virus (IBV) causes economic difficulties for livestock farmers. The 3D8 single chain variable fragment (scFv) protein is a recombinant antibody with nuclease activity that shows antiviral effects against various DNA and RNA viruses in mice and chickens. In this experiment, 3D8 scFv G2 transgenic chickens produced by crossing 3D8 scFv G1 transgenic rooster and wild type hens were screened by genomic PCR and immunohistochemistry analysis. 3D8 scFv transgenic chickens, wild type sibling chickens, and SPF chickens were directly infected with IBV (5 chickens per group) and indirectly infected by airborne propagation (15 chickens per group). The relative IBV shedding titers were measured by quantitative real-time PCR using oropharyngeal and cloacal swabs on days 3 and 5 after intraocular infection. The viral load was significantly decreased in the 3D8 scFv transgenic chickens from the contact transmission group. Additionally, blood was collected from each group on day 17 post-infection. The ELISA results showed a marked reduction of the antibody titer against IBV in the 3D8 scFv transgenic chickens from the contact transmission group. These results suggest that the 3D8 scFv protein potentially inhibits infectious bronchitis virus transmission in chickens., Highlights • Produced G2 3D8 single chain variable fragment (scFv) transgenic chickens. • 3D8 scFv transgenic chickens showed reduced infectious bronchitis viral shedding level in the contact transmission group. • 3D8 scFv transgenic chickens were 40% lower than the response in the control groups in IBV serum antibody titer.

Previous preclinical studies have demonstrated the otoprotective effects of resveratrol (RV) at low doses. This study aimed to investigate the dose-dependent effects of RV in rats with cisplatin (CXP)-induced hearing loss. Sprague-Dawley rats (8-weeks old) were divided into six treatment groups (n = 12/group) and treated as follows: control, 0.5 mg/kg RV, 50 mg/kg RV, CXP, 0.5 mg/kg RV + CXP), and 50 mg/kg RV + CXP groups. CXP (3 mg/kg) was intraperitoneally injected for 5 days. RV (0.5 or 50 mg/kg) was intraperitoneally injected for 10 days from the first day of CXP administration. Auditory brainstem response (ABR) thresholds were measured before and within 3 days at the end of the drug administration. Cochlear tissues were harvested, and the outer hair cells were examined using cochlear whole mounts. The mRNA expression of NF&kappa, B, IL6, IL1&beta, and CYP1A1, and protein levels of aryl hydrocarbon receptor (AhR) and cytosolic and nuclear receptor for advanced glycation endproducts (RAGE) were evaluated. The ABR threshold increased in the 50 mg/kg RV and CXP groups at 4, 8, 16, and 32 kHz. The 0.5 mg/kg RV + CXP group demonstrated decreased hearing thresholds at 4 and 32 kHz compared to the CXP group. Cochlear whole-mount analysis revealed loss of outer hair cells in the 50 mg/kg RV and CXP groups and partial prevention of these cells in the 0.5 mg/kg RV + CXP group. The mRNA expressions of NF&kappa, B, IL6, and IL1&beta, were increased in the 50 mg/kg RV and CXP groups compared to the control group. In contrast, these levels were decreased in the 0.5 mg/kg RV + CXP group compared to the CXP group. The mRNA expression of CYP1A1 was increased in the CXP group, while it was decreased in the 0.5 mg/kg RV + CXP group compared to the control group. The protein levels of AhR and cytosolic RAGE decreased in the 0.5 mg/kg RV group. Low-dose RV had partial otoprotective effects on CXP ototoxicity. The otoprotective effects of RV may be mediated through anti-oxidative (CYP1A1 and RAGE) and anti-inflammatory (NF&kappa, ) responses. High-dose RV exerted an inflammatory response and did not ameliorate CXP-induced ototoxicity.

Background/Aims: Renal ischemia followed by reperfusion (I/R) is a leading cause of acute kidney injury (AKI), which is closely associated with high morbidity and mortality. Studies have shown that induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (iMSCs) exert powerful therapeutic effects in renal ischemia. However, the efficacy of iMSC-derived exosomes (iExo) on I/R injuries remains largely unknown.Methods: Human iPSCs were differentiated into iMSCs using a modified one-step method. Ultrafiltration, combined with purification, was used to isolate iExo from iMSCs. iExo was administered following I/R injury in a mouse model. The effect of iExo on I/R injury was assessed through changes in renal function, histology, and expression of oxidative stress, inflammation, and apoptosis markers. Further, we evaluated its association with the extracellular signal-regulated kinase (ERK) 1/2 signaling pathway.Results: Mice subjected to I/R injury exhibited typical AKI patterns; serum creatinine level, tubular necrosis, apoptosis, inflammatory cytokine production, and oxidative stress were markedly increased compared to sham mice. However, treatment with iExo attenuated these changes, significantly improving renal function and tissue damage, similar to the renoprotective effects of iMSCs on I/R injury. Significant induction of activated ERK 1/2 signaling molecules was observed in mice treated with iExo compared to those in the I/R injury group.Conclusions: The present study demonstrates that iExo administration ameliorated renal damage following I/R, suggesting that iMSC-derived exosomes may provide a novel therapeutic approach for AKI treatment.

A synthetic progestin altrenogest (ALT) is used to synchronize the estrus cycle of swine for fixed-time artificial insemination (AI) and has been shown to improve follicular development and reproductive performances in post-weaning sows. However, the effects of ALT treatment on reproductive tracts, including the ovaries, oviducts and uterus have not been yet clarified. In this study, we examined the expression of genes involved in endometrial responses in ALT-treated sows. ALT did not significantly alter luteinizing hormone (LH), follicle-stimulating hormone (FSH) and estradiol profiles in blood compared to untreated control. Quantitative RT-polymerase chain reaction (qRT-PCR) analysis showed that the expression of genes encoding galectin-3 (LGALS3) and fibroblast growth factor 9 (FGF9) was upregulated in the reproductive tracts of ALT-treated sows, including the ovaries, oviducts and uteri. Moreover, ALT treatment induced the expression of FGF9 and galectin-3 proteins, and promoted their localization to the luminal epithelium of the oviducts and uterus. Our findings suggest that the enhancement of reproductive performance shown by ALT-treated sows is associated with the upregulation of galectin-3 and FGF9, which are essential for endometrial receptivity, successful implantation, and pregnancy.

Tissue plasminogen activator (tPA) is a protein involved in the breakdown of blood clots. We have previously produced a human tPA (htPA)-overexpressing transgenic pig using a mammary gland-specific promoter. In this study, we have established a transgenic pig mammary gland cell line that produces recombinant htPA. The mammary gland cells grew well and retained their character over long periods of culture. There was no difference in the extent of apoptosis in transgenic cells compared to wild-type mammary gland cells. In addition, the transgenic mammary gland cells expressed and secreted htPA into the conditioned media at a concentration similar to that in milk. This transgenic cell line represents a simple and ethical method for recombinant htPA production.

Previously, Escherichia coli harboring the codon-optimized 3D8scFv gene (E. coli 3D8scFv) was developed as a feed additive for use in preventing norovirus infection. Here, we evaluated whether the 3D8scFv gene affects the colonization of E coli when E. coli 3D8scFv passes through the mouse gastrointestinal tract. To determine the colonization ability of E. coli 3D8scFv, E. coli cells with or without the 3D8scFv gene were fed to mice. Total DNA was extracted from the animals’ stools, stomach, small intestine and colon. All samples were amplified using 3D8scFv gene-specific primer sets. E. coli 3D8scFv begins to be excreted 1 h after feeding and that all E. coli 3D8scFv cells were excreted between 12 and 24 h after the last feeding of the cells. The previously measured gastrointestinal transit time of the mice was between 8 h and 22 h. The results of this study therefore show that E. coli 3D8scFv cannot colonize the gastrointestinal tracts of mice. In addition, if the purified 3D8 scFv protein is used as a feed additive, any associated E. coli 3D8scFv bacteria will not colonize the gastrointestinal tracts of the livestock. Thus, this feed additive meets the safety assessment criteria for the commercial use of bacteria., Highlights • It is evaluated whether E. coli 3D8scFv colonizes in the gastrointestinal tracts of mice. • Orally ingested E. coli 3D8scFv is excreted from mice without colonization of the gastrointestinal tract. • Purified 3D8 scFv is suitable for a feed additive according to the concept of substantial equivalence.

Transplantation is considered to be a very useful approach to improve human welfare and to prolong life-span. Heterologous organ transplantation using pig organs which are similar to human beings and easy to make mass-production has known as one of the alternatives. To ensure potential usage of the pig organ for transplantation application, it is essentially required to generate transgenic pig modifying immuno-related genes. Previously, we reported production of heterozygous α 1,3-galactosyltransferase (GalT) knock-out and human membrane cofactor protein (MCP) expressing pig (GalT-MCP/+), which is enforced for suppression of hyperacute and acute immunological rejection. In this study, we reported generation of homozygous pig (GalT-MCP/-MCP) by crossbreeding GalT-MCP/+ pigs. Two female founders gave birth to six of GalT-MCP/-MCP, and seven GalT-MCP/+ pigs. We performed quantitative real-time PCR, western blot, and flow cytometry analyses to confirm GalT and MCP expression. We showed that fibroblasts of the GalT-MCP/-MCP pig do not express GalT and its product Gal antigen, while efficiently express MCP. We also showed no expression of GalT, otherwise expression of MCP at heart, kidney, liver and pancreas of transgenic pig. Taken together, we suggest that the GalT-MCP/-MCP pig is a useful candidate to apply xenotransplantation study.

The aim of this study was to determine the typical incidence of micronuclei (MNi) in the peripheral blood lymphocytes of healthy middle-aged Koreans using the cytokinesis-block micronucleus cytome (CBMN-Cyt) assay. Non-smoking, low-risk alcohol-drinking healthy Korean men and women aged 30 to 59 years were recruited. Participants were divided into three groups according to age, i.e., 30 to 39, 40 to 49, and 50 to 59 years. Fifty participants were included in each age group, for a total of 300 participants. DNA damage was measured based on the number of binucleated (BN) cells with MNi, nucleoplasmic bridges (NPBs), and nuclear buds (NBUDs) using the CBMN-Cyt assay. The frequencies of BN cells with MNi in men were 14.0±4.9 (mean±SD) in 30-39year olds, 20.0±6.1 in 40-49year olds, and 21.7±7.6 in 50-59year olds. In women, they were 19.7±7.1 in 30-39year olds, 28.7±11.2 in 40-49year olds, and 31.9±12.9 in 50-59year olds. MNi and NPBs scores were higher in females than in males. The elder groups showed higher MNi frequencies for both genders, and the NPB frequency was higher in elder groups than younger groups, but only for males. Based on a regression analysis of the CBMN-Cyt parameters, MNi frequencies showed a positive relationship with age for both genders. BMI and blood vitamin B concentration were not significantly associated with CBMN-Cyt parameters, except vitamin B6 levels, which were positively associated with MNi scores in males. These results provide the standard frequencies of MNi, NPBs, and NBUDs in peripheral blood lymphocytes in middle-aged Korean individuals with healthy lifestyles. In this group, CBMN-Cyt assay parameters varied according to gender and age; however, BMI and micronutrient levels were not significantly associated with assay parameters.

The disialoganglioside GD3 has been considered to be involved in tumor progression or suppression in various tumor cells. However, the significance of the biological functions of GD3 in breast cancer cells is still controversial. This prompted us to study the possible relationship(s) between GD3 expression and the metastatic potential of a breast cancer MDA-MB231 cells as an estrogen receptor negative (ER-) type. The human GD3 synthase cDNA was transfected into MDA-MB231 cells, and G-418 bulk selection was used to select cells stably overexpressing the GD3 synthase. In vitro invasion potentials of the GD3 synthase over-expressing cells (pc3-GD3s) were significantly suppressed when compared with control cells. Expression of intercellular adhesion molecule-1 (ICAM-1; CD54) was down-regulated in the pc3-GD3s cells and the decrease in ICAM-I expression is directly related to the decrease in invasiveness of the pc3-GD3s cells. Another type of ER negative SK-BR3 cells exhibited the similar level of ICAM-1 expression as MDA-MB231 cells, while the ER positive MCF-7 cells (ER+) showed the increased expression level of ICAM-1. Then, we investigated signaling pathways known to control ICAM-1 expression. No difference was observed in the phosphorylation of ERK and p38 between the pc3-GD3s and control cells (pc3), but the activation of AKT was inhibited in pc3-GD3s, and not in the control (pc3). In addition, the composition of total gangliosides was changed between control (pc3) and pc3-GD3s cells, as confirmed by HPTLC. The pc3-GD3s cells had an accumulation of the GD2 instead of the GD3. RT-PCR results showed that not only GD3 synthase, but also GM2/GD2 synthase (β4-GalNc T) expression was increased in pc3-GD3s cells. Overexpression of GD3 synthase suppresses the invasive potential of human breast cancer MDA-MB-231 cells through down-regulation of ICAM-1 and the crucial pathway to allow the apoptotic effect has been attributed to accumulation of the GD2 ganglioside. ER has been linked to the ICAM-1 expression with GD3 to GD2 conversion in human breast cancer cells. This is the first finding of the endogenous sialyltransferase functions in tumor cells.

The 3D8 single-chain variable fragment (scFv) is a mini-antibody sequence with independent nuclease activity that shows antiviral effects against all types of viruses in chickens and mice. In this study, chickens were treated daily with an oral dose of 10

Transmembrane B cell lymphoma 2-associated X protein inhibitor motif-containing (TMBIM) 6, a Ca2+ channel-like protein, is highly up-regulated in several cancer types. Here, we show that TMBIM6 is closely associated with survival in patients with cervical, breast, lung, and prostate cancer. TMBIM6 deletion or knockdown suppresses primary tumor growth. Further, mTORC2 activation is up-regulated by TMBIM6 and stimulates glycolysis, protein synthesis, and the expression of lipid synthesis genes and glycosylated proteins. Moreover, ER-leaky Ca2+ from TMBIM6, a unique characteristic, is shown to affect mTORC2 assembly and its association with ribosomes. In addition, we identify that the BIA compound, a potentialTMBIM6 antagonist, prevents TMBIM6 binding to mTORC2, decreases mTORC2 activity, and also regulates TMBIM6-leaky Ca2+, further suppressing tumor formation and progression in cancer xenograft models. This previously unknown signaling cascade in which mTORC2 activity is enhanced via the interaction with TMBIM6 provides potential therapeutic targets for various malignancies., TMBIM6, a member of the transmembrane BI-1 motif-containing family of proteins, is overexpressed in many cancer types. Here, the authors show that TMBIM6 regulates AKT activation through mTORC2 assembly and ribosome association and identify an antagonist of TMBIM6 with anti-tumor properties.

The present study aimed to investigate the characteristics of mPEA15 expressing transgenic pig (TG pig) as a potential model for diabetes. Expression analysis confirmed the ubiquitous expression of mPEA15 in TG pigs at F4. Oral glucose tolerance test results showed that restoration of normal glucose levels was significantly delayed in the TG pigs when compared with that in the wild-type pigs (WT pigs). Primary skeletal muscle cells isolated from TG pigs demonstrated reduced glucose uptake and reduced GLUT4 translocation to the plasma membrane in response to insulin treatment. Combined, these results suggest that mPEA15 expressing pigs has a glucose intolerance and insulin resistance which are known to mediate the pathophysiology of type 2 diabetes mellitus. Thus, mPEA15 transgenic pigs would serve as a promising model for diabetes translational research. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-019-2021-0) contains supplementary material, which is available to authorized users.

Chicken serum has been suggested as a supplement to promote chicken cell proliferation and development. However, the molecular mechanisms by which chicken serum stimulates chicken cell proliferation remain unknown. Here, we evaluated the effects of chicken serum supplementation on chicken embryo fibroblast (CEF) and DF-1 cell proliferation. We also sought to elucidate the molecular pathways involved in mediating the effects of chicken serum on fibroblasts and DF-1 cells by overexpression of chicken 78 kDa glucose-regulated protein (chGRP78), which is important for cell growth and the prevention of apoptosis. Our data demonstrated that the addition of 5% chicken serum significantly enhanced fibroblast proliferation. Moreover, knockdown of chGRP78 using siRNA decreased fibroblast proliferation and increased apoptosis. Based on these results, we suggest that the chGRP78-mediated signaling pathway plays a critical role in chicken serum-stimulated fibroblast survival and anti-apoptosis. Therefore, our findings have important implications for the maintenance of chicken fibroblast cells through the inhibition of apoptosis and may lead to the development of new treatments for avian disease.

Tissue plasminogen activator (tPA) is a protein involved in the breakdown of blood clots. We have previously produced a human tPA (htPA)-overexpressing transgenic pig using a mammary gland-specific promoter. In this study, we have established a transgenic pig mammary gland cell line that produces recombinant htPA. The mammary gland cells grew well and retained their character over long periods of culture. There was no difference in the extent of apoptosis in transgenic cells compared to wild-type mammary gland cells. In addition, the transgenic mammary gland cells expressed and secreted htPA into the conditioned media at a concentration similar to that in milk. This transgenic cell line represents a simple and ethical method for recombinant htPA production.

Recently, diabetes mellitus (DM) has shown rapid global increases with about five million deaths annually. Animal models are imperative to understand disease mechanisms and develop diagnostic, preventive, and therapeutic interventions in translational research. Rodent and mini-pig models have been established and widely used for DM research. However, domestic pig models are limited in spite of advantages such as pharmacokinetic and physiopathological availability. This study examines the potential use of domestic pigs expressing recombinant human erythropoietin (rhEPO) as disease and therapeutic response models for DM. We previously generated transgenic pigs (n = 16, EPO Tg) in which rhEPO was expressed and circulated in all organs. Thirty-two pigs, including 16 controls, were fed high fat (HF) diets for 42 weeks. Subsequently, blood samples for chemical and metabolic analysis were collected after fasting for 24 h and glucose loading for oral glucose tolerance tests (OGTTs). We found increased activation of the PI3 K/Akt signaling pathway under hypoxic conditions after rhEPO treatment, and HF diet-inducible-obesity in the EPO Tg and control pigs. OGTTs showed lower fasting glucose levels in the EPO Tg pigs than in controls before and after the HF diet, suggesting that rhEPO may affect glucose concentrations. Insulin and C-peptide concentrations responded slowly to glucose administration and returned to initial levels after 2 h. The blood test results suggest that EPO might affect metabolic and chemical components such as glucose, high-density lipoprotein, glucagon, triglyceride, and free fatty acid. Our findings support the use of rhEPO transgenic domestic pigs as model animals for translational DM research.

The mammary gland serves as a valuable bioreactor system for the production of recombinant proteins in lactating animals. Pharmaceutical-grade recombinant protein can be harvested from the milk of transgenic animals that carry a protein of interest under the control of promoter regions genes encoding milk proteins. Whey acidic protein (WAP), for example, is predominantly expressed in the mammary gland and is regulated by lactating hormones during pregnancy. We cloned the 5'-flanking region of the porcine WAP gene (pWAP) to confirm the sequence elements in its promoter that are required for gene-expression activity. In the present study, we investigated how lactogenic hormones--including prolactin, hydrocortisone, and insulin--contribute to the transcriptional activation of the pWAP promoter region in mammalian cells, finding that these hormones activate STAT5 signaling, which in turn induce gene expression via STAT5 binding sites in its 5'-flanking region. To confirm the expression and hormonal regulation of the 5'-flanking region of pWAP in vivo, we generated transgenic mice expressing human recombinant granulocyte colony stimulating factor (hCSF2) in the mammary gland under the control of the pWAP promoter. These mice secreted hCSF2 protein in their milk at levels ranging from 242 to 1,274.8 ng/ml. Collectively, our findings show that the pWAP promoter may be useful for confining the expression of foreign proteins to the mammary gland, where they can be secreted along with milk.

Understanding the behavior of transgenes introduced into oocyte or embryos is essential for evaluating the methodologies for transgenic animal production. To date, many studies have reported the production of transgenic pig embryos with, however, low efficiency in environment of blastocyst production. The aim of present study was to determine the expression and duration of transgene transferred by intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT). Embryos obtained from the ICSI-MGT procedure were analysed for the expression of GFP and then for the transmission of the transgene. Briefly, fresh spermatozoa were bound to exogenous DNA after treatment by Triton X-100 and Lipofectin. When ICSI-MGT was performed using sperm heads with tails removed, the yield of blastocyst (25.3%), treated with Lipofectin (18.8%) and Triton X-100 (19.2%) were observed. Treatments of Lipofectin or Triton X-100 did not further improve the rates of blastocysts. Moreover, the apoptosis rates of embryos were obtained from the control and LIpofectin groups (8.7%, 9.7%, respectively), but were significantly higher in the Triton X-100 group (13.0%). Our results demonstrated that ICSI-MGT caused minimal damage to oocytes that could develop to full term. Moreover, the embryos derived by ICSI-MGT have shown prolonged exogenous DNA expression during preimplantation stage in vivo. However, more efforts will be required to improve the procedures of both sperm treatments cause of high frequency of mosaicisms.

Transmembrane Bax Inhibitor Motif-containing 6 (TMBIM6) is upregulated in several cancer types and involved in the metastasis. Specific downregulation of TMBIM6 results in cancer cell death. However, the TMBIM6 gene transcriptional regulation in normal and cancer cells is least studied. Here, we identified the core promoter region (&minus, 133/+30 bp) sufficient for promoter activity of TMBIM6 gene. Reporter gene expression with mutations at transcription factor binding sites, EMSA, supershift, and ChIP assays demonstrated that Sp1 is an essential transcription factor for basal promoter activity of TMBIM6. The TMBIM6 mRNA expression was increased with Sp1 levels in a concentration dependent manner. Ablation of Sp1 through siRNA or inhibition with mithramycin-A reduced the TMBIM6 mRNA expression. We also found that the protein kinase-C activation stimulates promoter activity and endogenous TMBIM6 mRNA by 2- to 2.5-fold. Additionally, overexpression of active mutants of PKC&iota, PKC&epsilon, and PKC&delta, increased TMBIM6 expression by enhancing nuclear translocation of Sp1. Immunohistochemistry analyses confirmed that the expression levels of PKC&iota, Sp1, and TMBIM6 were correlated with one another in samples from human breast, prostate, and liver cancer patients. Altogether, this study suggests the involvement of Sp1 in basal transcription and PKC in the enhanced expression of TMBIM6 in cancer.

To profile the proteome in porcine plasma, blood samples were collected from adult male barrows and those plasma were retrieved. For the depletion or pre-fractionation of high-abundance proteins, plasma samples were treated with commercial kits. Then, protein profiling was initiated using one and two-dimensional electrophoresis. Proteins were spotted and then identified by MALDI-TOF-TOF and LC-MS-MS. In the results, more than forty six proteins were identified and the reference map was constructed. The pre-treatment for the removal of high-abundance proteins caused the changes in 2-DE images and some of the proteins were newly uncovered after the most of high abundant proteins were removed. However, it is expected for further steps necessary to identify more low-abundance proteins that may contain potential bio-markers.

Satellite cells were derived from muscular tissue in postnatal pig. Satellite cell is an important to growth and development in animal tissues or organs. However, the progress underlying induced differentiation is not clear. The aim of this study was to evaluate the morphologic and the transcriptome changes in porcine satellite cell (PSC) treated with insulin, rosiglitazone, or dexamethasone respectively. PSC was obtained from postnatal muscle tissue. In study 1, for study the effect of insulin and FBS on the differentiated satellite cells, cells were cultured at absence or presence of insulin treated with FBS. Total RNA was extracted for determining the expression levels of myogenic PAX3, PAX7, Myf5, MyoD, and myogenin genes by real-time PCR. Myogenic genes decreased expression levels of mRNA in treated with insulin. In study 2, in order to clarify the relationship between rosiglitazone and lipid in differentiated satellite cells, we further examined the effect of FBS on lipid accumulation in the presence or absence of the rosiglitazone and lipid. Significant differences were observed between rosiglitazone and lipid by FBS. The mRNA of FABP4 and PPARγ increased in rosiglitazone treatment. In study 3, we examined the effect of dexamethasone on osteogenic differentiation in PSC. The mRNA was increased osteoblasotgenic ALP and ON genes treated with dexamethasone in 2% FBS. Dexamethasone induces osteoblastogenesis in differentiated PSC. Taken together, in differentiated PSCs, FABP4 and PPARγ increased to rosiglitazone. Whereas, no differences to FBS and lipid. These results were not comparable with previous reports. Our results suggest that adipogenic, myogenic, and osteoblastogenic could be isolated from porcine skeletal muscle, and identify culture conditions which optimize proliferation and differentiation formation of PSC.

Muscular satellite cell (SC), which is stem cell of postnatal pig, is an important for study of differentiation into adipogenesis, myogenesis, and osteoblastogenesis. In this study, we isolated and examined from pig muscle tissue to determine capacity in proliferate, differentiate, and expression of various genes. Porcine satellite cells (PSC) were isolated from semimembranosus (SM) muscles of 90∼100 days old pigs according to standard conditions. The cell proliferation increased in multi-potent cell by Masson’s, oil red O, and Alizarin red staining respectively. We per-formed the expression levels of differentiation related genes using real-time PCR. We found that the differentiation into adipocyte increased expression levels of both fatty acid binding protein 4 (FABP4) and peroxisome proliferator- acti-vated receptor gamma (PPARγ) genes (p

The use of chlorella as an immune stimulant to enhance nonspecific host defense mechanisms or as an antimicrobial to inhibit bacterial growth has been reported. Thus, the aim of the present study was to clarify the effect of recombinant chlorella supplementation on growth performance, meat quality, and the blood profile, excreta microflora, and nutrient digestibility in broilers. A total of 375 one-day-old ROSS 308 broilers (male and female) were allotted to 5 dietary treatments using 5 cages with 15 chicks per cage. Treatments were: 1) NC, basal diet supplemented with 1.0% E. coli fermented liquor (EFL); 2) PC1, 0.2% EFL with chlorella; 3) PC2, 1.0% EFL with chlorella; 4) T1, 0.2% EFL with chlorella (anti-viral); and 5) T2, 1.0% EFL with chlorella (anti-viral). The broilers in the T2 treatment groups showed higher body weight gain (BGW) by 2.55% (P 0.01) and lower feed conversion ratio (FCR) by 2.75% (P 0.05) compared with those fed the control NC treatment group. Moreover, the blood contents of blood urea nitrogen (BUN), creatinine, and IgA in the broilers of the T2 treatment group were significantly increased by 28.12, 23.07, and 29.72%, respectively -more than those found in the broilers of the NC treatment group (P 0.01). In contrast, the LDL/C in the blood from the animals in the T2 treatment group was significantly decreased by 23.23% - more than that in the blood from the NC broilers (P 0.05). Based on these results, we suggest that the dietary supplementation of broilers with recombinant chlorella could improve their growth performance, increase the concentration of IgA and apparently metabolizable nitrogen in the blood, and decrease ammonia emissions. Therefore, our findings have important implications for the effect of recombinant chlorella supplementation through increasing the concentration of IgA and the level of metabolizable nitrogen.

Accurate detection of oestrus is important for artificial insemination. The aim of this study was to identify oestrous-specific bovine cervical mucus proteins that could be used to determine the optimal time for artificial insemination. Non-oestrous and controlled internal drug release (CIDR)-induced oestrous-stage mucus proteins were purified and subjected to surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, sodium dodecyl sulphate polyacrylamide gel electrophoresis and MALDI-TOF/TOF. Among differentially expressed proteins, lactoferrin (LF) and glutamate receptor-interacting protein 1 (GRIP1) showed a twofold increase during the CIDR-induced oestrous stage compared to the levels in non-oestrous stage in bovine cervical mucus. The RT-PCR, Western blotting and immunohistochemistry results showed that LF and GRIP1 expression was significantly increased during the oestrous stage in the uterus. This study demonstrated that bovine LF and GRIP1 exist during the oestrous stage, but not during the non-oestrous stage, suggesting that cervical mucus LF and GRIP1 are useful oestrous detection markers in cattle.

Granulocyte colony-stimulating factor (G-CSF) is used for heart failure therapy and promotes myocardial regeneration by inducing mobilization of bone marrow stem cells to the injured heart after myocardial infarction; however, this treatment has one weakness in that its biological effect is transient. In our previous report, we generated 5 mutants harboring N-linked glycosylation to improve its antiapoptotic activities. Among them, one mutant (Phe140Asn) had higher cell viability than wild-type hG-CSF in rat cardiomyocytes, even after treatment with an apoptotic agent (H2O2). Cells treated with this mutant significantly upregulated the antiapoptotic proteins, and experienced reductions in caspase 3 activity and PARP cleavage. Moreover, the total number of apoptotic cells was dramatically lower in cultures treated with mutant hG-CSF. Taken together, these results suggest that the addition of an N-linked glycosylation was successful in improving the antiapoptotic activity of hG-CSF, and that this mutated product will be a feasible therapy for patients who have experienced heart failure. [BMB Reports 2012; 45(12): 742-747]

Steroidogenesis requires coordination of the anabolic and catabolic pathways of lipid metabolism, but the profile of proteins associated with progesterone synthesis in cyclic and pregnant corpus luteum (CL) is not well-known in cattle. In Experiment 1, plasma progesterone level was monitored in cyclic cows (n = 5) and pregnant cows (n = 6; until d-90). A significant decline in the plasma progesterone level occurred at d-19 of cyclic cows. Progesterone level in abbatoir-derived luteal tissues was also determined at d 1 to 5, 6 to 13 and 14 to 20 of cyclic cows, and d-60 and -90 of pregnant cows (n = 5 each). Progesterone level in d-60 CL was not different from those in d 6 to 13 CL and d-90 CL, although the difference between d 6 to 13 and d-90 was significant. In Experiment 2, protein expression pattern in CL at d-90 (n = 4) was compared with that in CL of cyclic cows at d 6 to 13 (n = 5). Significant changes in the level of protein expression were detected in 32 protein spots by two-dimensional polyacrylamide gel electrophoresis (2-DE), and 23 of them were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Six proteins were found only in pregnant CL, while the other 17 proteins were found only in cyclic CL. Among the above 6 proteins, vimentin which is involved in the regulation of post-implantation development was included. Thus, the protein expression pattern in CL was disorientated from cyclic luteal phase to mid pregnancy, and alterations in specific CL protein expression may contribute to the maintenance of pregnancy in Korean native cows.

To investigate reproductive disorder in human erythropoietin (EPO)-expressing pig, we performed comparative proteomic analyses of testicular tissues from human erythropoietin (hEPO) gene-harboring transgenic pigs and wild type pigs born from natural conception. In hEPO TG pigs, we found relatively low sperm motility and higher death rate indicating impaired sperm development. Consistently, plasma concentration of testosterone was significantly lower in the transgenic post-pubertal boars compared with wild type boars. Normalized protein spots showing higher than 2-fold differential expression intensity in two-dimensional polyacrylamide gel electrophoresis were selected for matrix associated laser desorption/ionization time-to-flight mass spectrometry analysis. Specific proteins were identified by searching the NCBI protein sequence databases. Among 55 proteins selected, 12 proteins were identified as those differentially expressed between transgenic and wild type pigs. Three downregulated proteins (β-globin, carbonyl reductase 1, and peroxiredoxin 6) and nine upregulated proteins (cytoskeletal β-actin, α 2,3-sialyltransferase, apolipoprotein A-I, tubulin α-1A chain, tropomodulin 3, thioredoxin, heat shock Protein 70.2, ch4/domains of swine IgM, and albumin), all of which are closely related to apoptosis and cytoskeletal development, were found in the transgenic boar testes. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay confirmed the increased occurrence of apoptosis in the transgenic boar testes compared with the wild type boar testes. Reproductive defects of the hEPO-expressing transgenic pigs may be caused by the abnormal expression of the genes identified in this study.

Xenotransplantation involves the transplantation of organs, tissues and cells from one species to another. A major barrier to successful xenotransplantation is the rejection of the donor tissue by the recipient immune system. Swine leukocyte antigens (SLA) are important molecules within the immune system and play an essential role in fighting infectious diseases and viruses. The present study investigated three SLA class I (SLA-1, SLA-3 and SLA-2) and three SLA class II (DRB1, DQB1 and DQA) alleles in 60 NIH miniature pigs using PCR with sequence-specific primers (PCR-SSP). As the results, nine combinations of SLA class I and II haplotypes, comprising of three homozygous and six heterozygous haplotypes, were examined. The SLA homozygous haplotype Lr-2.4/2.4 was the most prevalent, with an overall frequency of 28.3% (17/60) and heterozygous haplotype Lr-2.2/4.4 was the second most common (20.0%; 12/60), followed by haplotype Lr-4.2/4.2 (16.7%; 10/60), Lr-2.2/2.4 (15.0%; 9/60), Lr-2.2/2.2 (5.0%; 3/60), Lr-2.2/4.2 (5.0%; 3/60), Lr-2.4/4.4 (5.0%; 3/60) and Lr-2.2/3.3 (3.3%; 2/60), Lr-4.2/4.4 (1.7%; 1/60), respectively. These results provide useful information that can be used to establish highly inbred pig lines with fixed SLA homozygous alleles and haplotypes.

Maintaining an appropriate balance between subsets of CD4(+) helper T cells and T regulatory cells (Tregs) is a critical process in immune homeostasis and a protective mechanism against autoimmunity and inflammation. To identify the role of vitamin A-related compounds, we investigated the regulation of interleukin (IL)-17-producing helper T cells (Th17 cells) and Tregs treated with all-trans-retinal (retinal). CD4(+)T cells or total cells from the spleens of C57BL/6 mice were stimulated under Treg-polarizing (anti-CD3/CD28 and TGF-β) or Th17-polarizing (anti-CD3/CD28, TGF-β, and IL-6) conditions in the presence or absence of retinal. To analyze their suppressive abilities, retinal-induced Tregs or TGF-β-induced Tregs were co-cultured with responder T cells. Collagen-induced arthritis (CIA) was established in interferon (IFN)-γ knockout mice. On day 13, retinal-induced Tregs were adoptively transferred to mice with established CIA after second immunizations. Compared with TGF-β-induced Treg cells, retinal-induced Tregs showed increased Foxp3 expression and mediated stronger suppressive activity. Under Th17-polarizing conditions, retinal inhibited the production of IL-17 and increased the expression of Foxp3.Retinal-induced Tregs showed therapeutic effects in IFN-γ knockout CIA mice. Thus, we demonstrated that retinal reciprocally regulates Foxp3(+) Tregs and Th17 cells. These findings suggest that retinal, a vitamin A metabolite, can regulate the balance between pro- and anti-inflammatory immunity. A better understanding of the manipulation of Foxp3 and Tregs may enable the application of this tremendous therapeutic potential in various autoimmune diseases.

BAX Inhibitor-1 (BI-1), a transmembrane protein on the endoplasmic reticulum, has been studied previously in various physio/pathological conditions, but not in bone cells. In this study, using the MG63 osteoblast cell line and osteoblasts differentiated from stem cells, the role of BI-1 was studied. First, expression of BI-1 was confirmed in osteoblasts, as well as osteoclasts, in mouse tibiae bone immunohistochemistry. For evaluation of a recently published property of BI-1, an acidic pH-dependent Ca²⁺ channel-like effect in osteoblasts, acidic pH-associated cell death, and pro-inflammatory cytokine release were examined. In MG63 osteoblasts, acidic pH induced a pH-dependent increase in cell death and ER stress, as determined by elevated expression of GRP78, CHOP, phospho-eIF2α, IRE-1α, spliced XBP-1, and phospho-JNK. In osteoblasts, mitochondrial Ca²⁺ also showed a strong pH-dependent increase. BI-1 knock-down using siRNA protected cells against acidic pH, regulating mitochondrial Ca²⁺ accumulation, possibly via the acidic pH-dependent Ca²⁺ channel-like effect of BI-1. BI-1 knock-down also resulted in inhibition of acidic pH-induced release of pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α. In addition, bone marrow stem cells were differentiated into human osteoblasts, which showed increased expression of BI-1 mRNA and protein. In differentiated primary human osteoblasts, acidic pH-associated cell death, mitochondrial Ca²⁺ accumulation, and pro-inflammatory cytokine release were more significant than in non-differentiated stem cells. In summary, endogenous expression of BI-1 is associated with acidic pH-induced Ca²⁺ release, cell death, and pro-inflammatory cytokine release in human osteoblasts.

Combinatory responses of proinflamamtory cytokines have been examined on the nitric oxide-mediated function in cultured mouse calvarial osteoblasts. Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) induced iNOS gene expression and NO production, although these actions were inhibited by L-NG-monomethylarginine (L-NMMA) and decreased alkaline phosphatase (ALPase) activity. Furthermore, NO donors, sodium nitroprusside (SNP) and NONOate dose-dependently elevated ALPase activity. In contrast, transforming-growth factor-beta (TGF-beta) decreased NO production stimulated by IL-1beta, TNF-alpha and interferon-gamma (IFN-gamma). iNOS was expressed by mouse calvarial osteoblast cells after stimulation with IL-1beta, TNF-alpha, and IFN-gamma. Incubation of mouse calvarial osteoblast cells with the cytokines inhibited growth and ALPase activity. However, TGF-beta-treatment abolished these effects of IL-1beta, TNF-alpha and IFN-gamma on growth inhibition and stimulation of ALPase in mouse calvarial osteoblast cells. In contrast, IL-1beta, TNF-alpha, and IFN-gamma exerted growth-inhibiting effects on mouse calvarial osteoblast cells which were partly NO-dependent. The results suggest that NO may act predominantly as a modulator of cytokine-induced effects on mouse calvarial osteoblast cells and TGF-beta is a negative regulator of the NO production stimulated by IL-1beta, TNF-alpha and IFN-gamma.

STUDY DESIGN Retrospective study. OBJECTIVE To determine whether magnetic resonance myelography (MRM) improves the evaluation of the severity of stenosis in patients with multilevel lumbar stenosis. SUMMARY OF BACKGROUND DATA MRM shows a similar image as myelography in a noninvasive manner. METHODS One hundred patients over 50 years of age with multiple lumbar stenosis who were prospectively referred for MR imaging (MRI) with MRM were enrolled in the study. The most severe stenotic segment and the degree of stenosis of that segment, as assessed by the extent of remaining subarachnoidal space (1: normal to 50%; 2: over 50% but not a total blockage; 3: total blockage) were evaluated in a blinded manner by 2 observers. Conventional MRI (class A), MRM (class B), and MRI combined with MRM (class C) were evaluated independently and interobserver and intraobserver reliability were assessed. RESULTS In the selection of the most severe segment and degree of stenosis, for both observers, the consensus between class (B) and class (C), was higher than that of class (A) and class (C). The average kappa values for interobserver agreement in the selection of the most severe segment/assessing the degree of stenosis for classes (A), (B), and (C) were 0.649/0.727, 0.782/0.771, and 0.832/0.784, respectively. Intraobserver kappa values were also highest for class (B), followed by class (C), and then class (A). Observations were within the range of "almost perfect" (0.81< or =kappa< or =1), with the exception of the selection of the most severe segment in class (A) by one of the observers. CONCLUSIONS When employed in routine practice, MRM could be of value for improving observer reliability in the assessment of severity of stenosis in multiple lumbar stenosis.

Scutellaria barbata D. Don (Lamiaceae; SB) inhibited the growth of leiomyomal cells (LM). A time-dependent antiproliferative effect was noted when 10−5m buserelin, gonadotrophin-releasing hormone (GnRH) agonist or 20–40 µg/mL SB was added. The inhibition of cell growth decreased with the addition of the PKC activator (12-O-tetradecanoylphorbor-13-acetate; TPA) much as it did with the addition of SB, and the decreases in the viable cells caused by the addition of SB were reversed completely by pretreatment with a protein kinase C (PKC) inhibitor (calphostin C). The findings suggest that SB inhibits cell proliferation in cultured human uterine leiomyoma cells accompanied by PKC activation. Next, the study investigated the effect of SB on fetal development for toxicity. Pregnant Sprague-Dawley rats, from gestation day 6–15, were administered 20 g/L or 50 g/L SB in the drinking water and then killed on day 20. No maternal toxicity was observed, however, embryonic loss in the treatment groups was double that of the controls (p < 0.05). No gross morphologic malformations were seen in the treated fetuses. Fetuses exposed to SB were found to be significantly heavier than the controls, an effect that was greater in female fetuses and was not correlated with increased placental size. The results suggest that the SB had no toxicity and that in utero exposure to SB resulted in increased early embryo loss with increased growth in surviving fetuses. On the other hand, Western blot analyses revealed that Bcl-2 protein of a 26 kDa was abundant in leiomyomal cells, but not in normal myometrial cells. The addition of progesterone (100 ng/mL) resulted in a striking increase in Bcl-2 protein expression in the cultured leiomyoma cells. However, the addition of SB (20 µg/mL) resulted in a significant reduction in Bcl-2 protein expression in the cells. The results indicated that human uterine leiomyomal cells express Bcl-2 protein and progesterone enhances its expression, however, SB reduces the expression of Bcl-2 protein in human uterine leiomyoma cells. Copyright © 2008 John Wiley & Sons, Ltd.

Anti-bone resorption properties of the Korean herbal formulation, Gami-Honghwain (HJ), which comprises Carthamus tinctorius L. seed and hominis placenta, were investigated. We demonstrate that the production of PGE2 is inhibited by 20-100 microg/ml HJ in nontransformed osteoblastic cells (MC3T3-E1 cells), indicating that HJ inhibits PGE2 production. The effect of HJ on the proliferation and osteoblastic differentiation in MC3T3-E1 was also studied. HJ dose-dependently increased DNA synthesis (significant at 20-100 microg/ml), and increased alkaline phosphatase (ALP) and prolyl hydroxylase activities of MC3T3-E1 cells (20-100 microg/ml), while anti-estrogen tamoxifen eliminated the stimulation of proliferation and ALP activity of MC3T3-E1 which was induced by HJ. These results indicate that HJ directly stimulates cell proliferation and differentiation of osteoblasts. Also, when we assessed the effects of HJ on osteoblastic differentiation in MC3T3-E1, HJ enhanced ALP activity and mineralization in a dose- and time-dependent fashion. This stimulatory effect of the HJ was observed at relatively low doses (significant at 20-100 microg/ml and maximal at 100 microg/ml). Northern blot analysis showed that the HJ (60 microg/ml) increased in bone morphogenetic protein-2 as well as ALP mRNA concentrations in MC3T3-E1 cells. HJ (100 microg/ml) slightly increased in type I collagen mRNA abundance throughout the culture period, whereas it markedly inhibited the gene expression of collagenase-1 between days 15 and 20 of culture. These results indicate that HJ has anabolic effect on bone through the promotion of osteoblastic differentiation, suggesting that it could be used for the treatment of common metabolic bone diseases.

A rapid and simple immunochromatography (IC) strip test, for specific detection of porcine rotavirus (PRV) in stool specimen, was developed. Monoclonal antibodies (mAbs) to the OSU strain of PRV have been produced in mice. Among them, two hybridoma clones that generate mAb-1 and mAb-2, respectively, specific for VP6 protein of PRV, have been selected. In the IC configuration, mAb-1, one of the selected mAbs was used to the designed coat microparticles (MP), while another mAb-2 was used to fix it on the nitrocellulose membrane strip to form a result line. The control line was formed on the same membrane strip past the result line by fixing anti-mouse IgG antibody. The IC test was capable of detecting 1000 plaque-forming units of PRV/ml in less than 5min, and the binding capacity was demonstrated by specific recognition of PRV only, but not other porcine diarrhea viruses, transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV). The IC test produced positive results with all the nine PRV-positive stool specimens and negative results with five different non-PRV specimens, which were identified previously by the polymerase chain reaction (PCR) test, respectively. The results indicate an excellent concordance between the two methods, suggesting a potential application of the three combinated IC tests (PRV, TGEV and PEDV) for the on-site, rapid screening of porcine diarrhea cases.

It is well known that the pro-inflammatory mediators such as nitric oxide (NO) and prostaglandin (PG)E(2) are involved in several inflammatory diseases and lipopolysaccharide (LPS) can stimulate these inflammatory responses. Oleanolic acid 3-O-beta-d-glucopyranosyl(1-->3)-alpha-l-rhamnopyranosyl(1-->2)-alpha-l-arabinopyranoside (OA) was purified from edible plant Aralia elata. OA inhibited LPS-induced NO and PGE(2) production in raw 264.7 murine macrophages in a dose-dependent manner and RT-PCR analysis indicated OA inhibited mRNA transcriptions of iNOS and COX-2 genes in LPS-induced cells. EMSA and Western blot analysis revealed that OA drastically reduced NF-kappaB translocation by the inhibition effects of LPS-induced phosphorylation of IkappaBalpha. In addition, it was found that OA inhibited the phosphorylation of ERK1/2, p38 and JNK MAPK, and the treatment of U0126 in LPS-induced raw 264.7 cells showed significant inhibition activity on the NO production and the phosphorylation of IkappaBalpha. Taken together, it is suggested that OA from A. elata has an anti-inflammatory activity via down-regulation of NF-kappaB.