Serial cloning of pigs by somatic cell nuclear transfer: restoration of phenotypic normality during serial cloning.

Somatic cell nuclear transfer (scNT) is a useful way to create cloned animals. However, scNT clones exhibit high levels of phenotypic instability. This instability may be due to epigenetic reprogramming and/or genomic damage in the donor cells. To test this, we produced transgenic pig fibroblasts harboring the truncated human thrombopoietin (hTPO) gene and used them as donor cells in scNT to produce first-generation (G1) cloned piglets. In this study, 2,818 scNT embryos were transferred to 11 recipients and five G1 piglets were obtained. Among them, a clone had a dimorphic facial appearance with severe hypertelorism and a broad prominent nasal bridge. The other clones looked normal. Second-generation (G2) scNT piglets were then produced using ear cells from a G1 piglet that had an abnormal nose phenotype. We reasoned that, if the phenotypic abnormality of the G1 clone was not present in the G2 and third-generation (G3) clones, or was absent in the G2 clones but reappeared in the G3 clones, the phenotypic instability of the G1 clone could be attributed to faulty epigenetic reprogramming rather than to inherent/accidental genomic damage to the donor cells. Blastocyst rates, cell numbers in blastocyst, pregnancy rates, term placenta weight and ponderal index, and birth weight between G1 and G2 clones did not differ, but were significantly (P < 0.05) lower than control age- and sex-matched piglets. Next, we analyzed global methylation changes during development of the preimplantation embryos reconstructed by donor cells used for the production of G1 and G2 clones and could not find any significant differences in the methylation patterns between G1 and G2 clones. Indeed, we failed to detect the phenotypic abnormality in the G2 and G3 clones. Thus, the phenotypic abnormality of the G1 clone is likely to be due to epigenetic dysregulation. Additional observations then suggested that expression of the hTPO gene in the transgenic clones did not appear to be the cause of the phenotypic abnormality in the G1 clones and that the abnormality was acquired by only a few of the G1 clone's cells during its gestational development.     

Polymerase chain reaction (PCR) amplification for the detection of porcine parvovirus

A polymerase chain reaction (PCR) amplification method was developed and evaluated to detect porcine parvovirus (PPV). A pair of 20-base primers and an oligonucleotide probe were derived from the DNA sequences common to two isolates of PPV, NADL-8 and NADL-2. The primers flanked 118-bp nucleotides within the region coding for the major structural protein VP2. After DNA amplification of PPV replicative form (RF), a 158-bp fragment was detected in agarose gels. This amplified fragment was shown to be specific for PPV DNA after Southern transfer and hybridization to a 20-base internal probe. The amplified fragment also contained a single EcoRI cleavage site. Various conditions, such as number of cycles and annealing temperature, were examined to optimize the conditions for detecting viral DNAs from infected cell cultures and swine fetal tissues. Four different isolates of PPV, NADL-8, NADL-2, KBSH and Kresse, and two other viruses, canine parvovirus (CPV) and pseudorabies virus (PRV), were included to determine specificity of amplification. Slot blot hybridization with a radiolabeled probe was used to evaluate the sensitivity of PCR amplification. The optimized protocol was specific for PPV detecting equally all four strains of PPV, but failing to amplify CPV or PRV sequences. The PCR method could detect at least 100 fg of viral replicative form (RF) DNA or the equivalent of 1 PFU of infectious virus. The applications of this method include routine detection of PPV in clinical samples and as a contaminant in mammalian cell lines.     

Exon-specific DNA hypomethylation of the p53 gene of rat colon induced by dimethylhydrazine. Modulation by dietary folate.

Folate deficiency enhances colorectal carcinogenesis in dimethylhydrazine-treated rats. Folate is an important mediator of DNA methylation, an epigenetic modification of DNA that is known to be dysregulated in the early stages of colorectal cancer. This study investigated the effect of dimethylhydrazine on DNA methylation of the colonic p53 gene and the modulation of this effect by dietary folate. Sprague-Dawley rats were fed diets containing 0, 2, 8, or 40 mg of folate/kg of diet. Five weeks after diet initiation, dimethylhydrazine was injected weekly for fifteen weeks. Folate-depleted and folate-replete control animals did not receive dimethylhydrazine and were fed the 0- and 8-mg folate diets, respectively. The extent of p53 methylation was determined by a quantitative HpaII-polymerase chain reaction. In exons 6 and 7, significant p53 hypomethylation was observed in all dimethylhydrazine-treated rats relative to controls (P < 0.01), independent of dietary folate. In exon 8, significant p53 hypomethylation was observed only in the dimethylhydrazine-treated folate-depleted rats compared with controls (P = 0.038) and was effectively overcome by increasing levels of dietary folate (P = 0.008). In this model, dimethylhydrazine induces exon-specific p53 hypomethylation. In some exons, this occurs independent of dietary folate, and in others, increasing levels of dietary folate effectively override the induction of hypomethylation in a dose-responsive manner. This may be a mechanism by which increasing levels of dietary folate inhibit colorectal carcinogenesis.     

Detection of Rickettsia tsutsugamushi in Experimentally infected mice by PCR.

We developed a rapid procedure for the detection of Rickettsia tsutsugamushi DNA by the PCR technique. The primer pair used for the PCR was designed from the DNA sequence of the gene encoding a 120-kDa antigen, which was proven to be group specific by immunoblot analysis with mouse hyperimmune sera against various rickettsial strains. This PCR method was able to detect up to 10 ag of plasmid DNA (pKT12). Specific PCR products were obtained with DNAs from R. tsutsugamushi Kato, Karp, Gilliam, TA716, TA1817, and Boryong, but not with DNAs from other rickettsiae, such as R. prowazekii, R. typhi, R. akari, and strain TT118. In a study with experimentally infected mice, the PCR method could detect rickettsial DNA from 2 days after inoculation (DAI), whereas serum antibody against R. tsutsugamushi could be detected from 6 to 8 DAI by an immunofluorescence test. Although clinical manifestations subsided after 14 DAI, rickettsial DNA in blood samples could be detected by PCR for up to 64 DAI. These results suggest that this PCR method can be applied to the early diagnosis of scrub typhus and can also be used to detect the residual rickettsiae after clinical symptoms subside.     

Diminished adhesion of Anaplasma phagocytophilum-infected neutrophils to endothelial cells is associated with reduced expression of leukocyte surface selectin

Anaplasma phagocytophilum propagates within neutrophils and causes a disease marked by inflammatory tissue injury or complicated by opportunistic infections. We hypothesized that infection with A. phagocytophilum modifies the binding of neutrophils to endothelial cells and the expression of neutrophil adhesion molecules and studied these changes in vitro. Infected dimethyl sulfoxide-differentiated HL-60 cells and neutrophils showed reduced binding to cultured brain and systemic endothelial cells and lost expression of P-selectin glycoprotein ligand 1 (PSGL-1, CD162) and L-selectin (CD62L) (to 33 and 5% of control values, respectively), at a time when the levels of beta(2) integrin and immunoglobulin superfamily adhesion molecules and activation markers Mac-1 and intercellular adhesion molecule 1 increased (5 to 10 times that of the control). The loss of CD162 and CD62L expression was inhibited by EDTA, which suggests that neutrophil activation and sheddase cleavage occurred. The loss of selectin expression and the retained viability of the neutrophils persisted for at least 18 h with A. phagocytophilum infection, whereas Escherichia coli and Staphylococcus aureus rapidly killed neutrophils. The adhesion defect might increase the numbers of infected cells and their persistence in the blood prior to tick bites. However, decreased CD162 expression and poor endothelial cell binding may partly explain impaired host defenses, while simultaneous neutrophil activation may aggravate inflammation. These observations may help us to understand the modified biological responses, host inflammation, and immune response that occur with A. phagocytophilum infections.     

Factors associated with BMI, weight perceptions and trying to lose weight in African-American smokers

This study examined sociodemographic, behavioral and psychosocial factors associated with BMI, weight perceptions and trying to lose weight among African-American smokers (N=600, M=44.2 years, 70% female). Sixty-eight percent of the sample were overweight or obese (sample BMI M=28.0, SD=6.7). Three separate, simultaneous multivariable regression models were used to determine which factors were associated with BMI, weight perceptions and trying to lose weight. Poorer health, female gender and high-school education or higher were significantly associated with higher BMIs (p<0.05). Being female (OR=5.8, 95% CI=3.6-9.3) and having a higher BMI (OR=0.6, 95% CI=0.5-0.6) was associated with perception of overweight and smoking more cigarettes per day (OR=1.0, 95% CI=1.0-1.1), and perceiving oneself as overweight (OR=14.1, 95% CI=8.2-24.2) was associated with trying to lose weight. Participants somewhat underestimated their BMI in their weight perceptions. Those who perceived themselves as overweight were more likely to be trying to lose weight; therefore, increasing participant awareness of actual BMI status may lead to improved weight-control efforts in African-American smokers. Several expected associations with outcomes were not found, suggesting that BMI and weight constructs are not well-understood in this population.     

Associations of serum TRAIL concentrations, anthropometric variables, and serum lipid parameters in healthy adults

To investigate the relationships of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), anthropometric variables, and lipid parameters, we measured serum TRAIL concentrations, body mass index (BMI), total body fat (TBF), and serum lipid profiles in 207 healthy adults. There were no significant differences in serum TRAIL concentrations between men and women, nor between elderly persons and middle-aged subjects. However, men with TBF 16.4 kg (75th per centile) exhibited significantly higher serum TRAIL concentrations than those with TBF 11.2 kg (25th per centile) (69.7 +/- 15.1 pg/ml vs 50.2 +/- 14.3 pg/ml, p < 0.05). Serum TRAIL concentration averaged 76.2 +/- 16.1 pg/ml in women with low-density lipoprotein cholesterol (LDL-C) 165 mg/dl (75th per centile), which was significantly above the values (53.1 +/- 12.9 pg/ml, p < 0.05) in those with LDL-C 117 mg/dl (25th per centile), although no differences were observed on the basis of TBF (75th percentile vs 25th percentile). Serum TRAIL concentrations correlated significantlywithTBF (r = 0.31, p < 0.05) and lean body mass (r = -0.26, p <0.05) in men and LDL-C (r = 0.32, p < 0.05) and total cholesterol (r = 0.21, p < 0.05) in women. In conclusion, serum TRAIL concentrations are associated with serum lipid levels and body adiposity in healthy adults, but are unrelated to a subject's age or gender.     

Cloning, expression and characterization of a murine-human chimeric antibody with specificity for pre-S2 surface antigen of hepatitis B virus

The cloning, expression and characterization of a murine-human chimeric antibody with specificity for the pre-S2 surface antigen (Ag) of hepatitis B virus (HBV) is described. The heavy and light chain variable region (VH and VL) genes encoding the murine monoclonal antibody (mAb) were isolated and combined with human γ 1 and κ constant region genes, respectively. The expression vectors containing the chimeric heavy and light chain genes were sequentially electroporated into murine Sp2/0 hybridoma cells and transfectomas secreting chimeric antibody were isolated. The chimeric antibody was purified and characterized by ELISA, Western analysis and competition immunoassay, demonstrating that the transfectoma functionally express and secrete murine-human chimeric antibody which retained the specificity and affinity of the parental murine mAb.     

The distribution of fetal nuchal translucency thickness in normal Korean fetuses

The aim of present study was to establish normative data for the distribution of nuchal translucency (NT) thickness in normal Korean fetuses. The data were collected from pregnant women with singleton pregnancies in whom fetal ultrasound was performed and the fetal NT thickness was measured between 11 and 14 weeks of gestation. Among them, a total of 2,577 fetuses with a known normal outcome were included in this study. The distribution of multiple of median (MoM) values of the NT thickness with crown-rump length (CRL) in 10-mm intervals and the 95th percentile of MoM were calculated with the linear regression method. The present study showed that NT measurements increase with increasing CRL and a false positive rate increases with increasing gestational age. Therefore, a fixed cut-off point through the first trimester was not appropriate and each NT measurement should be examined according to the gestational age. The present study offers normative data of the fetal NT thickness in a Korean population, which can be used as reference for screening chromosomal aberrations or other congenital abnormalities in the first trimester.