Fetal gender determination in early pregnancy through qualitative and quantitative analysis of fetal DNA in maternal serum

Fetal DNA in maternal plasma and serum has been shown to be a useful material for fetal gender determination and for screening tests for abnormal pregnancies except during early gestational ages. Maternal serum samples were obtained from 81 pregnant women during the 5th-10th weeks of gestation. Fetal gender was determined by conventional polymerase chain reaction (PCR) to detect a Y-chromosomal sequence (DYS14) in maternal serum during early gestation and confirmed by examination of the newborns after delivery. Real-time quantitative analyses of the SRY and beta-globin genes were also performed in order to determine fetal gender and to quantify fetal DNA concentration in maternal serum during early gestation. When using conventional PCR, the total sensitivity of identifying a male fetus was 95%, but its sensitivity after the 7th week was 100%, whereas in real-time quantitative PCR, the total sensitivity after the 5th week was 100%. Quantitative analyses of the SRY gene revealed that the mean concentration of fetal DNA in maternal serum was 30.55 copies/ml, that fetal DNA concentration showed a tendency to increase with the progression of pregnancy, and that it had a wide normal range. Thus, we could confidently determine fetal gender by using maternal serum samples taken as early as the 7th week.     

Purification and characterization of bovine steroid 21-hydroxylase (P450c21) efficiently expressed in Escherichia coli

Steroid 21-hydroxylase, P450c21, is responsible for the conversion of progesterone and 17alpha-hydroxyprogesterone to their 21-hydroxylated derivatives. P450c21 has been poorly investigated because of difficulty in obtaining sufficient quantities of purified protein. To solve the problem, we have attempted to express the bovine P450c21 in Escherichia coli as a stable form. The N-terminal membrane anchor and basic regions of P450c21 were replaced by the basic region of CYP2C3. The engineered P450c21 was expressed at a level higher than 1.2micromol/L culture (>60mg/L) when coexpressed with molecular chaperones GroES/GroEL. Utilizing three steps of column chromatography, the protein was highly purified to the specific content 16.6nmol/mg (91.2% purity). The purified protein is a monomer in the presence of 1% sodium cholate as determined by gel filtration analysis, suggesting that this membrane anchor-truncated form of P450c21 is more soluble than the native form. The purified enzyme showed typical substrate-binding difference spectra and 21-hydroxylase activities for both progesterone and 17alpha-hydroxyprogesterone. Truncation of the membrane anchor increases solubility of P450c21 facilitating expression of this protein in E. coli yielding sufficient quantities for both biochemical and biophysical studies.     

Assembly mechanism of FCT region type 1 pili in serotype M6 Streptococcus pyogenes

The human pathogen Streptococcus pyogenes produces diverse pili depending on the serotype. We investigated the assembly mechanism of FCT type 1 pili in a serotype M6 strain. The pili were found to be assembled from two precursor proteins, the backbone protein T6 and ancillary protein FctX, and anchored to the cell wall in a manner that requires both a housekeeping sortase enzyme (SrtA) and pilus-associated sortase enzyme (SrtB). SrtB is primarily required for efficient formation of the T6 and FctX complex and subsequent polymerization of T6, whereas proper anchoring of the pili to the cell wall is mainly mediated by SrtA. Because motifs essential for polymerization of pilus backbone proteins in other Gram-positive bacteria are not present in T6, we sought to identify the functional residues involved in this process. Our results showed that T6 encompasses the novel VAKS pilin motif conserved in streptococcal T6 homologues and that the lysine residue (Lys-175) within the motif and cell wall sorting signal of T6 are prerequisites for isopeptide linkage of T6 molecules. Because Lys-175 and the cell wall sorting signal of FctX are indispensable for substantial incorporation of FctX into the T6 pilus shaft, FctX is suggested to be located at the pilus tip, which was also implied by immunogold electron microscopy findings. Thus, the elaborate assembly of FCT type 1 pili is potentially organized by sortase-mediated cross-linking between sorting signals and the amino group of Lys-175 positioned in the VAKS motif of T6, thereby displaying T6 and FctX in a temporospatial manner.     

Streptolysin S contributes to group A streptococcal translocation across an epithelial barrier

Group A Streptococcus pyogenes (GAS) is a human pathogen that causes local suppurative infections and severe invasive diseases. Systemic dissemination of GAS is initiated by bacterial penetration of the epithelial barrier of the pharynx or damaged skin. To gain insight into the mechanism by which GAS penetrates the epithelial barrier, we sought to identify both bacterial and host factors involved in the process. Screening of a transposon mutant library of a clinical GAS isolate recovered from an invasive episode allowed identification of streptolysin S (SLS) as a novel factor that facilitates the translocation of GAS. Of note, the wild type strain efficiently translocated across the epithelial monolayer, accompanied by a decrease in transepithelial electrical resistance and cleavage of transmembrane junctional proteins, including occludin and E-cadherin. Loss of integrity of intercellular junctions was inhibited after infection with a deletion mutant of the sagA gene encoding SLS, as compared with those infected with the wild type strain. Interestingly, following GAS infection, calpain was recruited to the plasma membrane along with E-cadherin. Moreover, bacterial translocation and destabilization of the junctions were partially inhibited by a pharmacological calpain inhibitor or genetic interference with calpain. Our data indicate a potential function of SLS that facilitates GAS invasion into deeper tissues via degradation of epithelial intercellular junctions in concert with the host cysteine protease calpain.     

Citrin/mitochondrial glycerol-3-phosphate dehydrogenase double knock-out mice recapitulate features of human citrin deficiency

Citrin is the liver-type mitochondrial aspartate-glutamate carrier that participates in urea, protein, and nucleotide biosynthetic pathways by supplying aspartate from mitochondria to the cytosol.Citrin also plays a role in transporting cytosolic NADH reducing equivalents into mitochondria as a component of the malate-aspartate shuttle. In humans, loss-of-function mutations in the SLC25A13 gene encoding citrin cause both adult-onset type II citrullinemia and neonatal intrahepatic cholestasis, collectively referred to as human citrin deficiency. Citrin knock-out mice fail to display features of human citrin deficiency. Based on the hypothesis that an enhanced glycerol phosphate shuttle activity may be compensating for the loss of citrin function in the mouse, we have generated mice with a combined disruption of the genes for citrin and mitochondrial glycerol 3-phosphate dehydrogenase. The resulting double knock-out mice demonstrated citrullinemia, hyperammonemia that was further elevated by oral sucrose administration, hypoglycemia, and a fatty liver, all features of human citrin deficiency. An increased hepatic lactate/pyruvate ratio in the double knock-out mice compared with controls was also further elevated by the oral sucrose administration, suggesting that an altered cytosolic NADH/NAD(+) ratio is closely associated with the hyperammonemia observed. Microarray analyses identified over 100 genes that were differentially expressed in the double knock-out mice compared with wild-type controls, revealing genes potentially involved in compensatory or downstream effects of the combined mutations. Together, our data indicate that the more severe phenotype present in the citrin/mitochondrial glycerol-3-phosphate dehydrogenase double knock-out mice represents a more accurate model of human citrin deficiency than citrin knock-out mice.     

Conversion of dechlorodauricumine into chlorinated alkaloids in Menispermum dauricum root culture

(15)N-Labeled dechlorodauricumine and dechloroacutumine were isolated from Menispermum dauricum roots cultured in a chloride-deficient medium, in which nitrogen-containing macro-components K(14)NO(3) and ((14)NH(4))(2)SO(4) were replaced by K(15)NO(3) and ((15)NH(4))(2)SO(4), respectively. These (15)N-labeled substrates were supplied independently to the roots cultured in a chloride-enriched medium. LC-ESI-MS analysis of alkaloids extracted from the roots, harvested 5 and 10 days after administering the (15)N-labeled substrates, revealed that the (15)N derived from dechlorodauricumine was much more effectively incorporated into chlorinated alkaloids than that derived from dechloroacutumine. These findings suggest that dechlorodauricumine is the principal precursor of the chlorinated alkaloids produced by M. dauricum roots.     

Application of PCR for Detection of Mycoplasma DNA and Pestivirus RNA in Human Live Viral Vaccines

PCR techniques were applied for the detection of mycoplasma DNA and pestivirus RNA to 43 lots of live viral vaccines (measles, mumps, rubella, and oral poliomyelitis) produced by six manufacturers in Japan. Although mycoplasma DNA was not detected in any of the vaccines tested, pestivirus RNA was detected in 12 lots (28%). The incidence of contamination among the four viral vaccines was in the range of 20 to 37%, and the incidence among the six manufacturers varied from 0 to 56%.     

The effects of intermittent exposure to hypoxia during endurance exercise training on the ventilatory responses to hypoxia and hypercapnia in humans

The present study was performed to investigate the effects of a combination of intermittent exposure to hypoxia during exercise training for short periods on ventilatory responses to hypoxia and hypercapnia (HVR and HCVR respectively) in humans. In a hypobaric chamber at a simulated altitude of 4,500 m (barometric pressure 432 mmHg), seven subjects (training group) performed exercise training for 6 consecutive days (30 min · day⁻¹), while six subjects (control group) were inactive during the same period. The HVR, HCVR and maximal oxygen uptake (V˙O_{2 max}) for each subject were measured at sea level before (pre) and after exposure to intermittent hypoxia. The post exposure test was carried out twice, i.e. on the 1st day and 1 week post exposure. It was found that HVR, as an index of peripheral chemosensitivity to hypoxia, was increased significantly (P < 0.05) in the control group after intermittent exposure to hypoxia. In contrast, there was no significant increase in HVR in the training group after exposure. The HCVR in both groups was not changed by intermittent exposure to hypoxia, while V˙O_{2 max} increased significantly in the training group. These results would suggest that endurance training during intermittent exposure to hypoxia depresses the increment of chemosensitivity to hypoxia, and that intermittent exposure to hypoxia in the presence or absence of exercise training does not induce an increase in the chemosensitivity to hypercapnia in humans. Accepted: 18 March 1998     

Adaptive changes in hypercapnic ventilatory response during training and detraining

To confirm the effects of physical training and detraining on CO2 chemosensitivity, we followed hypercapnic ventilatory response at rest in the same five subjects during pre-, post- and detraining for 6 years. They joined our university badminton teams as freshmen and participated regularly in their team's training for about 3 h a day, three times a week, for 4 years. After that they retired from their teams and stopped training in order to study in the graduate school for 2 years. Maximum pulmonary ventilation (VEmax) and maximal oxygen uptake (VO2max) for each subject were determined during maximal treadmill exercise. The slope (S) of ventilatory response to carbon dioxide at rest was measured by Read's rebreathing method. Mean values of VEmax increased statistically during training and decreased statistically during detraining. A similar tendency was observed in VO2max. The average value of S before training was 1.91 l.min-1.mmHg-1, (+/- ) SD 0.52 and it decreased gradually with increasing training periods; the difference between the S values before (1980) and after training (1982, 1983 and 1984) were all significant. Furthermore, the mean values of S increased significantly during detraining as compared with those obtained at the end of training (April 1984). We concluded that in normal subjects, long-term physical training increases aerobic work capacity and decreases CO2 ventilatory responsiveness, and that the ventilatory adaptations with training observed here are reversible through detraining.