Estimation of Wilson's disease incidence and carrier frequency in the Korean population by screening ATP7B major mutations in newborn filter papers using the SYBR green intercalator method based on the amplification refractory mutation system

Wilson's disease (WD), an autosomal recessive disorder of copper transport, is one of the most common inherited metabolic disorders in Korea. Despite its frequency, the incidence and carrier frequency of WD has not yet been estimated in the Korean population. We therefore screened for four major missense mutations (p.Arg778Leu, p.Ala874Val, p.Leu1083Phe, and p.Asn1270Ser) of the ATP7B gene in 476 newborn filter papers by real-time multiplex PCR and melting curve analysis using the SYBR Green intercalator method based on the amplification refractory mutation system test. Newborn filter papers with abnormal melting curves were subjected to subsequent sequence analysis. Three mutated alleles, one p.Arg778Leu and two p.Ala874Val, were detected among the 476 newborn filter papers (952 alleles). The carrier frequency and incidence of WD in the Korean population were determined as 1 in 88.2 and 30,778, respectively, by reversely calculating based on the Hardy-Weinberg law.     

A circularly permuted β-lactamase as a novel reporter for evaluation of protein cyclization efficiency

Split inteins have been used as a versatile tool in protein engineering to mediate efficient in vivo and in vitro trans-splicing of a protein. The trans-splicing ability of split inteins was also applied to the in vivo cyclization of a protein. However, cyclization efficiency is dependent upon the type of split inteins employed and the conditions under which cyclization occur. In this study, a novel reporter system that easily measures the cyclization efficiency of split inteins was developed. For this purpose TEM-1 beta-lactamase was divided into two fragments (24 approximately 215 and 216 approximately 286 amino acids) and circularly permuted. The circularly permuted beta-lactamase expressed in Escherichia coli showed little beta-lactamase activity, most likely due to the structural modification of the protein. However, when the circularly permuted beta-lactamase was cyclized by the Synechocystis sp. PCC6803 DnaB split mini-intein, beta-lactamase activity both in vitro and in vivo was recovered. These results suggest that the novel reporter system can be exploited to develop new inteins with high efficiency of in vivo protein cyclization.     

Stable expression and secretion of polyhydroxybutyrate depolymerase of <Emphasis Type="Italic">Paucimonas lemoignei</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

An efficient strategy for the expression and secretion of extracellular polyhydroxybutyrate depolymerase (PhaZ1) of Paucimonas lemoignei in Escherichia coli was developed by employing the signal peptide of PhaZ1 and a truncated ice nucleation protein anchoring motif (INPNC). Directly synthesized mature form of Phaz1 was present in the cytoplasm of host cells as inclusion bodies, while a construct containing Phaz1 and its own N-terminal signal peptide (PrePhaz1) enabled the secretion of active Phaz1 into the extracellular medium. However, the PrePhaz1 construct was harmful to the host cell and resulted in atypical growth and instability of the plasmid during the cultivation. In contrast, INPNC-Phaz1 and INPNC-PrePhaz1 fusion constructs did not affect growth of host cells. INPNC-Phaz1 was successfully displayed on the cell surface with its fusion form, but did not retain Phaz1 activity. In the case of INPNC-PrePhaz1, the initially synthesized fusion form was separated by precise cleavage of the signal peptide, and active Phaz1 was consequently released into the culture medium. The amount of Phaz1 derived from E. coli (INPNC-PrePhaz1) was almost twice as great as that directly expressed from E. coli (PrePhaz1), and was predominantly (approximately 85%) located in the periplasm when cultivated at 22°C but was efficiently secreted into the extracellular medium when cultivated at 37°C.     

Hepatitis C virus (HCV) genotyping by annealing reverse transcription-PCR products with genotype-specific capture probes

The genotype of the hepatitis C virus (HCV) strain infecting a given patient is an important predictive factor for the clinical outcome of chronic liver disease and its response to anti-viral therapeutic agents. We herein sought to develop a new easy, sensitive and accurate HCV genotyping method using annealing genotype-specific capture probes (AGSCP) in an automation-friendly 96-well plate format. The validation of our new AGSCP was performed using the Standard HCV Genotype Panel. We then used both our AGSCP and the commercially available INNO-LiPA assay to analyze the HCV genotypes from 111 Korean patients. Discordant results were analyzed by direct sequencing. AGSCP successfully genotyped the standard panel. The genotypes of 111 patient samples were also obtained successfully by AGSCP and INNO-LiPA. We observed a high concordance rate (93 matched samples, 83.8%) between the two assays. Sequencing analysis of the 18 discordant results revealed that the AGSCP had correctly identified 12 samples, whereas the INNO-LiPA had correctly identified only 6. These results collectively indicate that AGSCP assay is a convenient and sensitive method for large-scale genotyping, and it may be a promising tool for the determination of HCV and other genotypes in clinical settings.     

Temperature management strategy for efficient gene expression in a thermally inducible <Emphasis Type="Italic">Escherichia coli</Emphasis>/bacteriophage system

In a two-phase operation, E. coli containing λSNNU1 (Q ⁻ S ⁻) in the chromosome is typically cultured at 33°C and cloned gene expression is induced by elevating the temperature. At least 40°C is necessary for complete induction of cloned gene expression; however, temperatures above 40°C have been shown to inhibit cloned gene expression. This suggests that a three-phase operation, which has an induction phase between the growth and production phases, may result in higher gene expression. In this study, optimal temperature management strategies were investigated for the three-phase operation of cloned gene expression in thermally inducible E. coli/bacteriophage systems. The optimal temperature for the induction phase was determined to be 40°C. When the temperature of the production stage was 33°C, the optimal time period for the induction phase at 40°C was determined to be 60 min. In contrast, when the temperature of the production phase was 37°C, the optimal period for the induction phase at 40°C was 20∼30 min. When the three-phase temperature and temporal profile were set at a growth phase of 33°C, an induction phase at 40°C for 30 min, and a production phase at 37°C, the highest level of cloned gene expression was achieved.     

Construction and characterization of a recombinant whole-cell biocatalyst of Escherichia coli expressing styrene monooxygenase under the control of arabinose promoter

A recombinant Escherichia coli (pBAB1) containing styrene monooxygenase (SMO) was developed for the conversion of styrene to enantiopure (S)-styrene oxide that is an important chiral building block in organic synthesis. The styAB genes encoding SMO was cloned into a multicopy plasmid under the tightly regulated promoter of bacterial l-arabinose operon which is inducible by l-arabinose. The recombinant showed that expression level of StyA protein and whole-cell SMO activities were varied depending on the concentration of the inducer l-arabinose. The maximum SMO activity was 108 U/g cdw when the cells were induced with 0.2% l-arabinose. SDS-PAGE and Western blot analyses indicated that whole-cell SMO activity was strongly correlated with the expression level of StyA protein. Organic-aqueous two-phase experiment could yield 50 mM enantiopure (S)-styrene oxide in organic phase in 18 h, but the recombinant SMO activity was unstable during the reaction. The expression of styAB under the control of l-arabinose promoter was significantly repressed in the presence of glucose.     

Effect of maternal restraint stress on fetal development of ICR mice.

The present study was conducted to elucidate the susceptibility of embryos and fetuses at different gestational stages to the maternal stress in mice. Groups of pregnant ICR mice were subjected to daily 12-h restraint stress, taped in the supine position on a plastic board, on gestational days (GD) 1-4, 5-8, 9-12 and 13-16, respectively. Caesarean sections were performed on gestational day 18, and the fetuses were weighed and examined for morphological defects. During the daily restraint for 4 days, the maternal body weights markedly decreased. Although the body weights recovered gradually after termination of the stress, the recovery was not full until the final stage of pregnancy. Interestingly, restraint stress caused growth retardation of the fetuses, leading to a significant decrease in their body weights, and increased early and late resorptions of embryos and fetuses according to the stress periods. Although the preceding (GD1-4) and concurrent (GD5-8) stresses did not affect embryonic implantation, restraint stress on GD9-12 caused cleft palate. Whereas vertebral abnormalities, mainly bipartite ossification, were observed only in animals stressed on GD5-8, abnormalities of sternebrae, exhibiting asymmetric or bipartite ossification, were enhanced by the stress at all of the gestational stages. On the other hand, the incidence of other malformations including renal malposition and costal abnormalities was not increased by stress at any of the 4 stages. Taken together, the results suggest that intensive restraint stress influences the maternal body weight resulting in growth retardation and increased mortality of embryos and fetuses, in addition to gestational stage-specific ventricular dilatation, cleft palate and sternal abnormalities.     

Stable isotope labeling by amino acids in cell culture (SILAC) and proteome quantitation of mouse embryonic stem cells to a depth of 5,111 proteins

Embryonic stem (ES) cells are pluripotent cells isolated from mammalian preimplantation embryos. They are capable of differentiating into all cell types and therefore hold great promise in regenerative medicine. Here we show that murine ES cells can be fully SILAC (stable isotope labeling by amino acids in cell culture)-labeled when grown feeder-free during the last phase of cell culture. We fractionated the SILAC-labeled ES cell proteome by one-dimensional gel electrophoresis and by isoelectric focusing of peptides. High resolution analysis on a linear ion trap-orbitrap instrument (LTQ-Orbitrap) at sub-ppm mass accuracy resulted in confident identification and quantitation of more than 5,000 distinct proteins. This is the largest quantified proteome reported to date and contains prominent stem cell markers such as OCT4, NANOG, SOX2, and UTF1 along with the embryonic form of RAS (ERAS). We also quantified the proportion of the ES cell proteome present in cytosolic, nucleoplasmic, and membrane/chromatin fractions. We compared two different preparation approaches, cell fractionation followed by one-dimensional gel separation and in-solution digestion of total cell lysate combined with isoelectric focusing, and found comparable proteome coverage with no apparent bias for any functional protein classes for either approach. Bioinformatics analysis of the ES cell proteome revealed a broad distribution of cellular functions with overrepresentation of proteins involved in proliferation. We compared the proteome with a recently published map of chromatin states of promoters in ES cells and found excellent correlation between protein expression and the presence of active and repressive chromatin marks.     

Protocol for the recovery of in vivo matured canine oocytes based on once daily measurement of serum progesterone

The collection of in vivo matured canine oocytes relies on the accurate prediction of ovulation. The present study was designed to develop a protocol for the recovery of in vivo matured canine oocytes based on once daily measurements of serum progesterone (P(4)) concentrations. Blood samples (2 mL) were collected every day at 0900 h, and P(4) concentrations were analyzed using a DSL-3900 ACTIVE((R)) Progesterone Coated-Tube Radioimmunoassay Kit. The average number of oocytes at the metaphase II (M II) stage was significantly higher at or after 72 h (6.7 to 7.5) compared to 56 h (1.7) following ovulation. The highest numbers of corpora lutea, and therefore the highest numbers of oocytes, were recovered from bitches with initial ovulatory P(4) concentrations ranging from 6.0 to 8.0 ng/ mL (12.2 and 11.4, respectively) compared to from 4.0 to 4.9 ng/ mL (9.6 and 8.8, respectively; p < 0.05). The average number of M II oocytes recovered at 84 h from bitches with initial ovulatory P(4) levels of 5.0 to 5.9 ng/mL (7.7) was higher compared to bitches with P(4) levels of 4.0 to 4.9 ng/ mL (3.5) and 6.0 to 8.0 ng/ mL (4.8; p < 0.05). When oocyte recovery time was adjusted for initial ovulatory P(4) concentration, no significant difference in recovery rates or oocyte quality were observed. In conclusion, once daily measurements of P(4) can be used to predict ovulation in bitches, and oocyte recovery time should be adjusted for initial ovulatory serum P(4) concentrations.