Somatic cell nuclear transfer (SCNT) is a well-known laboratory technique. The principle of the SCNT involves the reprogramming a somatic nucleus by injecting a somatic cell into a recipient oocyte whose nucleus has been removed. Therefore, the nucleus donor cells are considered as a crucial factor in SCNT. Cell cycle synchronization of nucleus donor cells at G0/G1 stage can be induced by contact inhibition or serum starvation. In this study, acteoside, a phenylpropanoid glycoside compound, was investigated to determine whether it is applicable for inducing cell cycle synchronization, cytoprotection, and improving SCNT efficiency in canine fetal fibroblasts. Primary canine fetal fibroblasts were treated with acteoside (10, 30, 50 μM) for various time periods (24, 48 and 72 hours). Cell cycle synchronization at G0/G1 stage did not differ significantly with the method of induction: acteoside treatment, contact inhibition or serum starvation. However, of these three treatments, serum starvation resulted in significantly increased level of reactive oxygen species (ROS) (99.5 ± 0.3%) and apoptosis. The results also revealed that acteoside reduced ROS and apoptosis processes including necrosis in canine fetal fibroblasts, and improved the cell survival. Canine fetal fibroblasts treated with acteoside were successfully arrested at the G0/G1 stage. Moreover, the reconstructed embryos using nucleus donor cells treated with acteoside produced a healthy cloned dog, but not the embryos produced using nucleus donor cells subjected to contact inhibition. In conclusion, acteoside induced cell cycle synchronization of nucleus donor cells would be an alternative method to improve the efficiency of canine SCNT because of its cytoprotective effects.
Female adnexal tumor of probable Wolffian origin (FATWO) is a rare disease entity that arises from the mesonephric duct system. FATWO is different than other gynecological cancers in terms of embryology. Here, we describe the case of a 52-year-old woman with malignant FATWO. The patient underwent explorative laparotomy and surgical staging after a frozen section revealed malignancy. Detailed examination of the pathologic findings were consistent with FATWO. Counseling and further testing were provided to the patient to assess the risk of germline mutation and epigenetic change. An O-6-methylguanine-DNA methyltransferase gene methylation test was positive, and all other tests were normal. This is the first study to report a case of O-6-methylguanine-DNA methyltransferase methylation with FATWO in Korea.
Epigenesis, Genetic; O(6)-methylguanine-DNA methyltransferase methylation; Wolffian tumor
Previously, we showed that B-cell receptor associated protein 31 (BAP31), an endoplasmic reticulum (ER) membrane chaperone, is also expressed on the cell surface by two monoclonal antibodies (MAbs) 297-D4 and 144-A8. Both MAbs recognize the same linear epitope on the C-terminal domain of BAP31, although they were independently established. Here, flow cytometric analysis showed that 144-A8 had additional binding properties to some cells, as compared to 297-D4. Quantitative antigen binding assays also showed that 144-A8 had higher antigen binding capacity than 297-D4. Affinity measurement revealed that 144-A8 had 1.54-fold higher binding affinity than 297-D4. Analysis of the heavy- and light-chain variable region sequences of two MAbs revealed that both MAbs belonged to the same heavy chain (Igh-V3660 VH3) and light chain subgroup (IGKV21) with just two amino acid differences in each framework region, indicating that both MAbs arise from the same germline origin. Seven amino acid differences were found between the complementarity determining regions (CDRs) of the two MAbs. Molecular modeling of the epitope-paratope complexes revealed that the epitope appeared to reside in closer proximity to the CDRs of 144-A8 than to those of 297-D4 with the stronger hydrogen bond interactions with the former than the latter. More interestingly, an additional hydrophobic interaction appeared to be established between the leucine residue of epitope and the paratope of 144-A8, due to the substitution of H-Tyr101 for H-Phe101 in 144-A8. Thus, the different binding specificity and affinity of 144-A8 appeared to be due to the different hydrogen bonds and hydrophobic interaction induced by the alterations of amino acids in CDRs of 144-A8. The results provide molecular insights into how the binding specificities and affinities of antibodies evolve with the same epitope in different microenvironments.
The basidiomycetous fungus Cryptococcus neoformans has been known to be highly radiation resistant and has been found in fatal radioactive environments such as the damaged nuclear reactor at Chernobyl. To elucidate the mechanisms underlying the radiation resistance phenotype of C. neoformans, we identified genes affected by gamma radiation through genome-wide transcriptome analysis and characterized their functions. We found that genes involved in DNA damage repair systems were upregulated in response to gamma radiation. Particularly, deletion of recombinase RAD51 and two DNA-dependent ATPase genes, RAD54 and RDH54, increased cellular susceptibility to both gamma radiation and DNA-damaging agents. A variety of oxidative stress response genes were also upregulated. Among them, sulfiredoxin contributed to gamma radiation resistance in a peroxiredoxin/thioredoxin-independent manner. Furthermore, we found that genes involved in molecular chaperone expression, ubiquitination systems, and autophagy were induced, whereas genes involved in the biosynthesis of proteins and fatty acids/sterols were downregulated. Most importantly, we discovered a number of novel C. neoformans genes, the expression of which was modulated by gamma radiation exposure, and their deletion rendered cells susceptible to gamma radiation exposure, as well as DNA damage insults. Among these genes, we found that a unique transcription factor containing the basic leucine zipper domain, named Bdr1, served as a regulator of the gamma radiation resistance of C. neoformans by controlling expression of DNA repair genes, and its expression was regulated by the evolutionarily conserved DNA damage response protein kinase Rad53. Taken together, the current transcriptome and functional analyses contribute to the understanding of the unique molecular mechanism of the radiation-resistant fungus C. neoformans.
Although there are no natural environments under intense radiation, some living organisms have been found to show high radiation resistance. Organisms harboring the ability of radiation resistance have unique regulatory networks to overcome this stress. Cryptococcus neoformans is one of the radiation-resistant fungi and is found in highly radioactive environments. However, it remains elusive how radiation-resistant eukaryotic microorganisms work differentially from radiation-sensitive ones. Here, we performed transcriptome analysis of C. neoformans to explore gene expression profiles after gamma radiation exposure and functionally characterized some of identified radiation resistance genes. Notably, we identified a novel regulator of radiation resistance, named Bdr1 (a bZIP TF for DNA damage response 1), which is a transcription factor (TF) that is not closely homologous to any known TF and is transcriptionally controlled by the Rad53 kinase. Therefore, our work could shed light on understanding not only the radiation response but also the radiation resistance mechanism of C. neoformans.
Oct4 is a crucial germ line-specific transcription factor expressed in different pluripotent cells and downregulated in the process of differentiation. There are two conserved enhancers, called the distal enhancer (DE) and proximal enhancer (PE), in the 5′ upstream regulatory sequences (URSs) of the mouse Oct4 gene, which were demonstrated to control Oct4 expression independently in embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs). We analyzed the URSs of the pig Oct4 and identified two similar enhancers that were highly consistent with the mouse DE and PE. A dual-fluorescence reporter was later constructed by combining a DE-free-Oct4-promoter-driven EGFP reporter cassette with a PE-free-Oct4-promoter-driven mCherry reporter cassette. Then, it was tested in a mouse ESC-like cell line (F9) and a mouse EpiSC-like cell line (P19) before it is formally used for pig. As a result, a higher red fluorescence was observed in F9 cells, while green fluorescence was primarily detected in P19 cells. This fluorescence expression pattern in the two cell lines was consistent with that in the early naïve pluripotent state and late primed pluripotent state during differentiation of mouse ESCs. Hence, this reporter system will be a convenient tool for screening out ESC-like naïve pluripotent stem cells from other metastable state cells in a heterogenous population.
The normal cells derived from human embryonic stem cells (hESCs) are regarded as substitutes for damaged or dysfunctional adult cells. However, tumorigenicity of hESCs remains a major challenge in clinical application of hESC-derived cell transplantation. Previously, we generated monoclonal antibody (MAb) 57-C11 specific to the surface molecule on undifferentiated hESCs. The aim of this study is to prove whether 57-C11-positive hESCs are pluripotent and tumorigenic in immunodeficient mice.
Undifferentiated hESCs were mixed with retinoic acid (RA)-differentiated hESCs at different ratios prior to 57-C11-mediated separation. To isolate 57-C11-positive hESCs from the mixture, biotinylated 57-C11 and streptavidin-coated magnetic beads were added to the mixture. Unbound 57-C11-negative hESCs were first isolated after applying magnet to the cell mixture, and 57-C11-bound hESCs were then released from the magnetic beads. In order to measure the efficiency of separation, 57-C11-positive or -negative hESCs were counted after isolation. To evaluate the efficiency of teratoma formation in vivo, 57-C11-positive or negative cells were further injected into left and right, respectively, testes of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice.
Approximately 77~100% of undifferentiated hESCs were isolated after applying 57-C11-coated magnetic beads to the mixed cell populations. Importantly, teratomas were not observed in NOD/SCID mice after the injection of isolated 57-C11-negative hESCs, whereas teratomas were observed with 57-C11-positive hESCs.
57-C11-positive hESCs are pluripotent and tumorigenic. The combination of 57-C11 and magnetic beads will be useful to eliminate remaining undifferentiated hESCs for the safe cell transplantation.
Human embryonic stem cells; Surface marker; 57-C11; Magnetic beads; Teratoma
Women with Lynch syndrome have an increased risk of developing colorectal and gynecologic malignancies such as endometrial cancer. Complex hyperplasia has about a 30% risk of developing into endometrial cancer. The aim of this study was to determine the genetic risk for developing endometrial cancer by immunohistochemical staining of premalignant lesions for mutL homolog 1, mutS homolog 2, mutS homolog 6, and postmeiotic segregation increased 2.
Twenty cases (n=20) were selected from among patients with available sample blocks for analysis. Clinical information was obtained from medical chart review. Immunohistochemical staining was performed for all of the tumor blocks. Staining was scored based on the intensity (intensity score 0-3) .
Among the 20 cases of complex endometrial hyperplasia, 11 (55%) patients showed loss of expression of at least one of the following proteins: mutL homolog 1, mutS homolog 2, mutS homolog 6, or postmeiotic segregation increased 2. Seven (35%) patients were negative for the expression of two or more proteins, and one patient (5%) was negative for the expression of all four proteins.
More than half of the patients showed loss of expression of at least one mismatch repair protein in our study population. Genetic risk counseling and further tests are recommended for these patients.
Endometrial hyperplasia; Endometrial premalignancy; Mismatch repair gene
We recently experienced a case of endometrial cancer 5 years after the diagnosis of breast cancer in a patient with a mutation in the BRCA2 gene. A 55-year-old Korean woman who had a past history of breast cancer in her 50s underwent an operation for endometrial cancer. Final pathology confirmed stage Ia, and no adjuvant treatment was performed. After surgery, considering her history of sequential cancer occurrence, genetic counseling was offered. The result showed the BRCA2 variation of unknown significance mutation. This is the first case report of sequential cancers (endometrial and breast) in a patient with a BRCA2 mutation among a Korean population.
BRCA; Breast neoplasms; Endometrial neoplasms
Borderline ovarian tumors (BOT) are premalignant lesions. Approximately 10% of all epithelial ovarian cancers are known to be hereditary with hereditary breast and ovarian cancer (HBOC) accounting for approximately 90% of cases; the remaining 10% are attributable to Lynch syndrome, also known as hereditary nonpolyposis colorectal cancer (HNPCC). The aim of our study is to estimate this risk based on screening immunohistochemistry (IHC).
Thirty-four patients diagnosed with BOT were identified. Family history, clinical characteristics, and IHC data (breast cancer 1, early onset [BRCA1], breast cancer 2, early onset [BRCA2], mutS homolog 2 [MSH2], mutL homolog 1 [MLH1]) were collected for all cases from the patients' medical charts. Nuclear staining of the tumor was scored as negative and positive.
Among 32 patients, 14 (44%) had serous type and 18 (56%) had mucinous type. The mean patient age was 44 years (range 19-86).The number of patients with weak IHC staining for MSH2 and BRCA2 was 1 (3%) and 6 (19%) respectively. The median follow up was 21.8 months.
According to the results, we discovered that 3% and 19% of patients with BOT had a risk of hereditary cancer based on IHC analysis respectively. This pilot study may help clinician to counsel effectively for confirmative tests.
Lynch syndrome; Ovarian neoplasmsms
Ovarian cancer is the seventh most common cancer in the world and the fifth most common cause of death from cancer; it is responsible for over half of all deaths related to gynecological cancers. The presence of lymphatic metastasis is an important prognostic factor in ovarian cancer. Nodal metastases to the pelvic and the para-aortic lymph nodes are common, particularly in an advanced of the disease (stages III-IV). The finding of distant nodal metastasis, especially subclavian lymph node metastasis, from ovarian carcinoma is very uncommon. 18F-fluorodeoxyglucose-positron emission tomography (FDG-PET) or FDG-PET/computed tomography (CT) provides an improved imaging for detecting metastatic lymph nodes in patients with ovarian cancer. Immunohistochemically, ovarian carcinoma cells are positive for estrogen receptor, progesterone receptor, cancer antigen 125, Wilms' tumor 1 protein, and p53; they are negative for thyroid transcription factor (TTF-1) and caudal-related homeobox 2 (CDX-2). This report describes a Korean woman diagnosed with ovarian cancer with subclavian lymph node metastasis revealed by FDG PET/CT and verified by an immunohistochemical staining. Differentiating between the primary ovarian lesion and the metastatic lesion will allow the initiation of an appropriate treatment and help predict the prognosis.
Lymphatic metastasis; Ovary cancer
Lynch syndrome is a genetic malignancy syndrome affecting the colon, endometrium, and other organs. It is difficult to find a Lynch syndrome patient without any family history of cancer. We have recently examined an endometrial cancer patient with a MSH2 gene mutation without a family history of cancer. A 55-year old Korean woman was admitted to a local clinic for vaginal bleeding. An endometrial biopsy revealed the presence of adenocarcinoma (endometrioid type, grade 1). After surgical staging, no further adjuvant therapy was required. Analysis of the tissue using immunohistochemistry (IHC) showed the endometrium stained negatively for MSH2. Microsatellite instability (MSI) was analyzed for five markers. The patient was scored as unstable. Further, additional gene sequencing revealed one missense mutation in c.23C > T (p.Thr8Met). This is the first case of Lynch syndrome endometrial cancer in Korea in which the patient does not have any family history of cancer.
Endometrial neoplasms; Lynch syndrome
We have recently experienced an endometrial cancer 12 years after the diagnosis of colon cancer with Lynch syndrome. A 49-year-old Korean woman had a family history of colon cancer. Her mother had colon cancer at 56-year-old, and her brother had colon cancer at 48 years old. The patient received surgery for endometrial cancer at the same hospital 12 years after being treated for colon cancer. Immunohistochemistry showed that her endometrial tissue stained negative for MSH2. A microsatellite instability test was performed and showed the presence of instability high microsatellite instability. An hMLH2 gene mutation was detected at codon 629 codon of exon 12, in which a glutamine was replaced with an arginine (1886A>G [p.Gln629Arg]). To our knowledge, this is the first case of metachronous cancer in a Lynch syndrome family in Korea with a gap of more than ten years between cancer diagnoses.
Colon cancer; Endometrial cancer; Lynch syndrome
Recent genetic screens of fly mutants and molecular analysis have revealed that the Hippo (Hpo) pathway controls both cell proliferation and cell death. Deregulation of its human counterpart (the MST pathway) has been implicated in human cancers. However, how this pathway is linked with the known tumor suppressor network remains to be established. RUNX3 functions as a tumor suppressor of gastric cancer, lung cancer, bladder cancer, and colon cancer. Here, we show that RUNX3 is a principal and evolutionarily conserved component of the MST pathway. SAV1/WW45 facilitates the close association between MST2 and RUNX3. MST2, in turn, stimulates the SAV1–RUNX3 interaction. In addition, we show that siRNA-mediated RUNX3 knockdown abolishes MST/Hpo-mediated cell death. By establishing that RUNX3 is an endpoint effector of the MST pathway and that RUNX3 is capable of inducing cell death in cooperation with MST and SAV1, we define an evolutionarily conserved novel regulatory mechanism loop for tumor suppression in human cancers.
Coronary artery perforation (CAP) after percutaneous coronary intervention is a rare, but serious complication. It can cause cardiac tamponade, acute myocardial infarction or death. The treatments of CAP involve prolonged balloon inflation, emergent surgery, coil embolization, and implantation of covered stent. We have successfully performed the emergent microcoil embolization in a patient with uncontrolled Ellis grade 3 guidewire-induced CAP resulting in delayed cardiac tamponade. Contrasting our usual expectation, the 1-year follow-up angiography showed a patent flow at the embolized site.
Embolization, therapeutic; Cardiac tamponade; Angioplasty, balloon, coronary
Diabetes is a risk factor for breast cancer development and is associated with poor prognosis for breast cancer patients. However, the molecular and biochemical mechanisms underlying the association between diabetes and breast cancer have not been fully elucidated. Here, we investigated estradiol response in MCF-7 breast cancer cells with or without chronic exposure to insulin. We found that insulin priming is necessary and specific for estradiol-induced cancer cell growth, and induces anaplerotic shunting of glucose into macromolecule biosynthesis in the estradiol treated cells. Treatment with ERK or Akt specific inhibitors, U0126 or LY294002, respectively, suppressed estradiol-induced growth. Interestingly, molecular analysis revealed that estradiol treatment markedly increases expression of cyclin A and B, and decreases p21 and p27 in the insulin-primed cells. In addition, estradiol treatment activated metabolic genes in pentose phosphate (PPP) and serine biosynthesis pathways in the insulin-primed cells while insulin priming decreased metabolic gene expression associated with glucose catabolism in the breast cancer cells. Finally, we found that anti-diabetic drug metformin and AMPK ligand AICAR, but not thiazolidinediones (TZDs), specifically suppress the estradiol-induced cellular growth in the insulin-primed cells. These findings suggest that estrogen receptor (ER) activation under chronic hyperinsulinemic condition increases breast cancer growth through the modulation of cell cycle and apoptotic factors and nutrient metabolism, and further provide a mechanistic evidence for the clinical benefit of metformin use for ER-positive breast cancer patients with diabetes.
breast cancer; diabetes; estrogen receptor; estradiol; iInsulin priming; MCF-7; metformin
The purpose of this study was to examine the inter- and intra-rater reliability of an electronic inspiratory loading device for the assessment of pulmonary functions: maximum inspiratory pressure, peak inspiratory flow, and vital capacity.
Subjects were 50 patient volunteers in a rehabilitation hospital who had experienced their first episode of unilateral stroke with hemiparesis during the previous 6 months (26 men, 24 women; mean age [±SD], 55.96 [±12.81] years), with no use of medications that could induce drowsiness, evidence of restrictive lung disease, history of asthma, use of psychotropic drugs, or alcohol consumption habit.
Maximum inspiratory pressure, peak inspiratory flow, and vital capacity for pulmonary functions were assessed using an electronic inspiratory loading device (PowerBreathe, K5, 2010) by 2 examiners, with patients in an unassisted sitting position, and 1 examiner re-assessed with same patients at the same time of a day after 1 week. Intra-class correlation coefficients were used to assess reliability.
Intra-rater reliability ranged from intra-class correlation coefficients (ICCs)=0.959 to 0.986 in variables. For the inter-rater reliability between 2 examiners, the ICCs ranged from 0.933 to 0.985. Intra-rater and inter-rater reliability were good in variables (maximal inspiratory pressure, peak inspiratory flow, and vital capacity).
The intra- and inter-examiner reliability of the pulmonary function measurements, maximum inspiratory pressure, peak inspiratory flow, and vital capacity, for the post-stroke patients was very high. The results suggest that the electronic inspiratory loading device would be useful for clinical rehabilitative assessment of pulmonary function.
Inhalation; Reproducibility of Results; Respiratory Function Tests; Stroke
This paper presents a fast multiple sampling method for low-noise CMOS image sensor (CIS) applications with column-parallel successive approximation register analog-to-digital converters (SAR ADCs). The 12-bit SAR ADC using the proposed multiple sampling method decreases the A/D conversion time by repeatedly converting a pixel output to 4-bit after the first 12-bit A/D conversion, reducing noise of the CIS by one over the square root of the number of samplings. The area of the 12-bit SAR ADC is reduced by using a 10-bit capacitor digital-to-analog converter (DAC) with four scaled reference voltages. In addition, a simple up/down counter-based digital processing logic is proposed to perform complex calculations for multiple sampling and digital correlated double sampling. To verify the proposed multiple sampling method, a 256 × 128 pixel array CIS with 12-bit SAR ADCs was fabricated using 0.18 μm CMOS process. The measurement results shows that the proposed multiple sampling method reduces each A/D conversion time from 1.2 μs to 0.45 μs and random noise from 848.3 μV to 270.4 μV, achieving a dynamic range of 68.1 dB and an SNR of 39.2 dB.
successive approximation register ADC; column parallel readout; CMOS image sensor
This study aimed to investigate the effects of dry aging on the quality of pork loin. Longissimus lumborum muscles were dissected from the right half of five pork carcasses and were used as the control samples. The left halves of the carcasses were aged at 2±1℃ and a relative humidity of 80% for 40 d. The total aerobic bacteria count was similar between the control and dry-aged pork loin (p>0.05). Lactic-acid bacteria was absent in both the control and dry-aged pork loins. Dry-aged pork loin contained low moisture and high protein and ash compared to the controls (p<0.05). The pH was higher and cooking loss was lower in dry-aged pork loin compared to that in the control (p<0.05). Flavor related compounds, such as total free amino acid, hypoxanthine, and inosine of pork loin were higher in dry-aged pork loin; whereas, inosine 5'-monophosphate and guanosine 5'-monophosphate were low in dry-aged pork loin than control (p<0.05). There was no difference in carnosine and anserine content between dry-aged pork loin and the control (p>0.05). Dry-aged pork loin had lower hardness and shear force and received higher core in sensory evaluation than the control (p<0.05). According to the results, dry aging improved textural and sensorial quality of pork loin.
dry aging; pork loin; tenderness; sensorial quality
This study was designed to investigate whether proton pump inhibitors (PPI, V-ATPase blocker) could increase the effect of cytotoxic agents in chemoresistant epithelial ovarian cancer (EOC). Expression of V-ATPase protein was evaluated in patients with EOC using immunohistochemistry, and patient survival was compared based on expression of V-ATPase mRNA from a TCGA data set. In vitro, EOC cell lines were treated with chemotherapeutic agents with or without V-ATPase siRNA or PPI (omeprazole) pretreatment. Cell survival and apoptosis was assessed using MTT assay and ELISA, respectively. In vivo experiments were performed to confirm the synergistic effect with omeprazole and paclitaxel on tumor growth in orthotopic and patient-derived xenograft (PDX) mouse models. Expression of V-ATPase protein in ovarian cancer tissues was observed in 44 patients (44/59, 74.6%). Higher expression of V-ATPase mRNA was associated with poorer overall survival in TCGA data. Inhibition of V-ATPase by siRNA or omeprazole significantly increased cytotoxicity or apoptosis to paclitaxel in chemoresistant (HeyA8-MDR, SKOV3-TR) and clear cell carcinoma cells (ES-2, RMG-1), but not in chemosensitive cells (HeyA8, SKOV3ip1). Moreover, the combination of omeprazole and paclitaxel significantly decreased the total tumor weight compared with paclitaxel alone in a chemoresistant EOC animal model and a PDX model of clear cell carcinoma. However, this finding was not observed in chemosensitive EOC animal models. These results show that omeprazole pretreatment can increase the effect of chemotherapeutic agents in chemoresistant EOC and clear cell carcinoma via reduction of the acidic tumor microenvironment.
epithelial ovarian cancer; microenvironment; V-ATPase; omeprazole; clear cell carcinoma
A cold-active β-1,4-d-mannanase from the Antarctic springtail C. antarcticus (CaMan) has been expressed, purified and crystallized. The CaMan crystal belonged to space group P212121, with unit-cell parameters a = 73.40, b = 83.81, c = 163.55 Å, and diffracted to 2.35 Å resolution. A 2.40 Å resolution data set was also collected from a CaMan–mannopentaose complex crystal.
The CaMan gene product from Cryptopygus antarcticus, which belongs to the glycoside hydrolase family 5 type β-1,4-d-mannanases, has been crystallized using a precipitant solution consisting of 0.1 M Tris–HCl pH 8.5, 25%(w/v) polyethylene glycol 3350 by the microbatch crystallization method at 295 K. The CaMan protein crystal belonged to space group P212121, with unit-cell parameters a = 73.40, b = 83.81, c = 163.55 Å. Assuming the presence of two molecules in the asymmetric unit, the solvent content was estimated to be about 61.29%. CaMan–mannopentaose (M5) complex crystals that were isomorphous to the CaMan crystals were obtained using the same mother liquor containing 1 mM M5.
β-1,4-d-mannanase; Cryptopygus antarcticus; glycoside hydrolase family 5
The objective of the present study was to investigate the effects of three different culture media on the development of canine somatic cell nuclear transfer (SCNT) embryos. Canine cloned embryos were cultured in modified synthetic oviductal fluid (mSOF), porcine zygote medium-3 (PZM-3), or G1/G2 sequential media. Our results showed that the G1/G2 media yielded significantly higher morula and blastocyst development in canine SCNT embryos (26.1% and 7.8%, respectively) compared to PZM-3 (8.5% and 0%) or mSOF (2.3% and 0%) media. In conclusion, this study suggests that blastocysts can be produced more efficiently using G1/G2 media to culture canine SCNT embryos.
canine; G1/G2 media; in vitro culture; somatic cell nuclear transfer embryo
Transgenic animals have become important tools for the production of therapeutic proteins in the domestic animal. Production efficiencies of transgenic animals by conventional methods as microinjection and retrovirus vector methods are low, and the foreign gene expression levels are also low because of their random integration in the host genome. In this study, we investigated the homologous recombination on the porcine β-casein gene locus using a knock-in vector for the β-casein gene locus. We developed the knock-in vector on the porcine β-casein gene locus and isolated knock-in fibroblast for nuclear transfer. The knock-in vector consisted of the neomycin resistance gene (neo) as a positive selectable marker gene, diphtheria toxin-A gene as negative selection marker, and 5′ arm and 3′ arm from the porcine β-casein gene. The secretion of enhanced green fluorescent protein (EGFP) was more easily detected in the cell culture media than it was by western blot analysis of cell extract of the HC11 mouse mammary epithelial cells transfected with EGFP knock-in vector. These results indicated that a knock-in system using β-casein gene induced high expression of transgene by the gene regulatory sequence of endogenous β-casein gene. These fibroblasts may be used to produce transgenic pigs for the production of therapeutic proteins via the mammary glands.
Knock-in; Homologous Recombination; Therapeutic Proteins; Porcine β-casein Gene
Although the age-adjusted Framingham risk score (AFRS), flow-mediated dilation (FMD), brachial-ankle pulse wave velocity (baPWV), high-sensitivity C-reactive protein (hsCRP), fibrinogen, homocysteine, and free fatty acid (FFA) can predict future cardiovascular events (CVEs), a comparison of these risk assessments for patients with stable angina has not been reported. We enrolled 203 patients with stable angina who had been scheduled for coronary angiography (CAG). After CAG, 134 patients showed significant coronary artery disease. During 4.2 yr follow-up, 36 patients (18%) showed CVEs, including myocardial infarction, de-novo coronary artery revascularization, in-stent restenosis, stroke, and cardiovascular death. ROC analysis showed that AFRS, FMD, baPWV, and hsCRP could predict CVEs (with AUC values of 0.752, 0.707, 0.659, and 0.702, respectively, all P<0.001 except baPWV P=0.003). A Cox proportional hazard analysis showed that AFRS and FMD were independent predictors of CVEs (HR, 2.945; 95% CI, 1.572-5.522; P=0.001 and HR, 0.914; 95% CI, 0.826-0.989; P=0.008, respectively). However, there was no difference in predictive power between combining AFRS plus FMD and AFRS alone (AUC 0.752 vs. 0.763; z=1.358, P=0.175). In patients with stable angina, AFRS and FMD are independent predictors of CVEs. However, there is no additive value of FMD on the AFRS in predicting CVEs.
Framingham Risk Score; Flow-Mediated Dilation; Cardiovascular Event
In order to investigate its structure and function, the medium-chain dehydrogenase/reductase superfamily YncB-like protein from V. vulnificus was expressed, purified and crystallized. X-ray diffraction analyses of YncB and the YncB–NADP(H) complex are reported at resolutions of 1.85 and 1.81 Å, respectively.
The yncB gene product from Vibrio vulnificus, which belongs to the medium-chain dehydrogenase/reductase (MDR) superfamily, was crystallized using the microbatch crystallization method at 295 K. Diffraction data sets were collected using synchrotron radiation. Crystals of selenomethionine-substituted YncB protein belonged to space group P212121, with unit-cell parameters a = 90.52, b = 91.56, c = 104.79 Å. Assuming the presence of two molecules in the asymmetric unit, the solvent content was estimated to be about 57%. Crystals of the YncB–NADP(H) complex belonged to space group P41212 or P43212, with unit-cell parameters a = b = 90.14, c = 105.61 Å. Assuming the presence of one molecule in the asymmetric unit, the solvent content was estimated to be about 56.42%.
MDR superfamily; Vibrio vulnificus; YncB