CHIPS for genetic testing to improve a regional clinical genetic service.

In current practice of clinical genetics, molecular diagnosis has become more widely used than ever before. DNA diagnosis is important for appropriate medical care of the patient, and proper genetic counseling to the family. However, genetic testing of orphan disease cannot always be performed easily. In multiple congenital anomalies (MCA) syndromes by monogenic cause, the broad mutational spectrum and large size of responsible genes often make molecular diagnosis expensive and cumbersome. We solve this problem with on-demand genetic testing by CHIPS (CEL nuclease mediated heteroduplex incision with polyacrylamide gel electrophoresis and silver staining) technology, which is the ultimately conventional and economical mutation screening system. In this article, we show eight patients with MCA syndromes who were recently treated at our hospital, and demonstrate that CHIPS successfully offers efficient and inexpensive genetic testing and facilitates clinical genetic service in our local region.

Impact of valley polarization on the resistivity in two dimensions.

We examine the temperature dependence of resistivity in a two-dimensional electron system formed in a silicon-on-insulator quantum well. The device allows us to tune the valley splitting continuously in addition to the electron density. Our data provide a global picture of how the resistivity and its temperature dependence change with valley polarization. At the boundary between valley-polarized and partially polarized regions, we demonstrate that there is an insulating contribution from spin-degenerate electrons occupying the upper valley-subband edge.

[Does the bilateral internal thoracic artery harvesting increase wound infection?].

The aim of this study is to compare the frequency of wound infection between bilateral and single internal thoracic artery (ITA) harvesting in coronary artery bypass grafting (CABG) cases. Two hundreds and thirty-four consecutive CABG cases performed harvesting either bilateral ITA (BITA) or single ITA (SITA) from January 2004 to December 2008, with or without concomitant surgery were studied. Harmonic Scalpel was used for the harvesting with skeletonization technique. The cases were divided into 2 groups: BITA group (n = 180) and SITA group (n = 54). The frequencies of wound infection were 3.7% in SITA group and 6.1% in BITA group. As to deep sternal infection, they were 1.9% in SITA group and 1.1% in BITA group. There was no significant difference between the 2 groups. Multivariate analysis of all patients showed that emergency cases, hypertension, congestive heart failure, and reopening for bleeding were identified as independent risk factors for wound infection. There were 113 diabetes mellitus (DM) patients out of all patients ; SITA group (n = 22) and BITA group (n = 91). Their wound infection rates were 4.5% and 6.6%, and those of deep sternal infection were 0% and 2.2%, respectively. There was no significant difference between them. In conclusion, BITA harvesting with skeletonized technique may be used as safely as SITA harvesting even in DM patients.

Oxidative stress causes enhanced endothelial cell injury in human heme oxygenase-1 deficiency.

The first known human case of heme oxygenase-1 (HO-1) deficiency is presented in this report. The patient is a six-year-old boy with severe growth retardation. He has been suffering from persistent hemolytic anemia characterized by marked erythrocyte fragmentation and intravascular hemolysis, with paradoxical increase of serum haptoglobin and low bilirubin. An abnormal coagulation/fibrinolysis system, associated with elevated thrombomodulin and von Willebrand factor, indicated the presence of severe, persistent endothelial damage. Electron microscopy of renal glomeruli revealed detachment of endothelium, with subendothelial deposition of an unidentified material. Iron deposition was noted in renal and hepatic tissue. Immunohistochemistry of hepatic tissue and immunoblotting of a cadmium-stimulated Epstein-Barr virus-transformed lymphoblastoid cell line (LCL) revealed complete absence of HO-1 production. An LCL derived from the patient was extremely sensitive to hemin-induced cell injury. Sequence analysis of the patient's HO-1 gene revealed complete loss of exon-2 of the maternal allele and a two-nucleotide deletion within exon3 of the paternal allele. Growth retardation, anemia, iron deposition, and vulnerability to stressful injury are all characteristics observed in recently described HO-1 targeted mice. This study presents not only the first human case of HO-1 deficiency but may also provide clues to the key roles played by this important enzyme in vivo.

Selectively induced high MRP gene expression in multidrug-resistant human HL60 leukemia cells.

A subclone HL60/DOX was selected from a human leukemic HL60 cell line for resistance to doxorubicin (DOX) by exposure to stepwise increasing concentrations of the drug and coexposure to a potential P-glycoprotein (P-gp) inhibitor, cepharanthine (a biscoclaurine alkaloid). Compared with the parent HL60 cells, the HL60/DOX cells were 13.0-fold more resistant to DOX and showed multidrug-resistant (MDR) phenotype characterized by 4.6-fold, 2.3-fold, and 5.7-fold cross-resistance to vincristine, pirarubicin, and etoposide, respectively, but no cross-resistance to alkylating agent, cisplatin. Immunocytochemical analyses using the specific monoclonal antibody, MRPr1, and quantitative analyses using a competitive reverse transcription-polymerase chain reaction (CRT-PCR) confirmed overexpression of MRP gene products (about 8-fold determined by CRT-PCR) in this resistant clone. The P-gp expression was not detectable by the monoclonal antibody, C219, in the HL60/DOX cells, and that was consistent with extremely low levels of mdr1 mRNA expression determined by CRT-PCR in this clone. Drug accumulation and efflux studies demonstrated the significantly increased efflux rate of DOX compared to the parent HL60 cells. This enhancement of DOX efflux was reversed by the addition of 10 microM verapamil. To investigate the additional underlying mechanisms contributing to MDR phenotype in the HL60/DOX cells, the levels of DNA topoisomerases (Topo) including Topo I, Topo IIalpha, and Topo IIbeta, and gamma-glutamylcystein synthetase (y-GCS) expression were determined using CRT-PCR techniques. Normal expression of each enzyme at the transcriptional level was demonstrated in this resistant clone. Southern blot analysis of the gene organization in the HL60/DOX cells revealed the amplification of MRP gene. These results indicate that alteration of the drug accumulation from enhanced efflux appears to be a major mechanism(s) of MDR phenotype and attributable to high levels of MRP expression in the HL60/DOX cells. Overexpression of MRP in this clone is regulated by the genomic amplification of DNA and increased levels of the MRP mRNA, independently with the normal expression of Topo I, Topo IIalpha, Topo IIbeta, or gamma-GCS.

Valley polarization assisted spin polarization in two dimensions.

Valleytronics is rapidly emerging as an exciting area of basic and applied research. In two-dimensional systems, valley polarization can dramatically modify physical properties through electron-electron interactions as demonstrated by such phenomena as the fractional quantum Hall effect and the metal-insulator transition. Here, we address the electrons' spin alignment in a magnetic field in silicon-on-insulator quantum wells under valley polarization. In stark contrast to expectations from a non-interacting model, we show experimentally that less magnetic field can be required to fully spin polarize a valley-polarized system than a valley-degenerate one. Furthermore, we show that these observations are quantitatively described by parameter-free ab initio quantum Monte Carlo simulations. We interpret the results as a manifestation of the greater stability of the spin- and valley-degenerate system against ferromagnetic instability and Wigner crystalization, which in turn suggests the existence of a new strongly correlated electron liquid at low electron densities.

Age-related changes in the cerebral distribution of 99mTc-ECD from infancy to adulthood.

UNLABELLED: Although cerebral blood flow in infants differs from that in older individuals, the distribution of 99mTc-ethyl cysteinate dimer (ECD) in infants has not been well studied. This study compared 99mTc-ECD distribution in infants and children with that in young adults. METHODS: 99mTc-ECD SPECT was performed on 37 patients suspected of having epilepsy, ranging in age from 3 mo to 26 y. The patients were divided into two age-matched groups, a drug-free group (n = 19) and a drug-taking group (n = 18), according to their anticonvulsant medication status at the time of examination. 99mTc-ECD (100-740 MBq) was injected interictally, and SPECT data were acquired using a triple-head gamma camera. Mean whole-brain counts were obtained from 10 sequential SPECT images. Regions of interest were set bilaterally on five areas of the cerebral cortex and on the basal ganglia, thalamus and cerebellum. The brain perfusion index (BPI) was obtained as a ratio of the mean counts in each region of interest to the mean whole-brain counts. The relationship between BPI and age in each region in the drug-free and drug-taking groups was analyzed separately and together using linear regression. The relationship between five patient age groups (<1 y, n = 4; 1-4 y, n = 9; 5-9 y, n = 8; 10-15 y, n = 7; >15 y, n = 9) and BPI in each region was also examined using multiple comparison analyses. RESULTS: Significant positive correlations between BPI and age in the frontal cortex and cerebellum were confirmed in the drug-free group. Anticonvulsant drugs did not affect the regression lines of BPI in the frontal cortex and cerebellum. Significant differences in BPI between age groups were seen in the parietal cortex, frontal cortex, occipital cortex, basal ganglia, thalamus and cerebellum in all patients. CONCLUSION: Age-related changes in cerebral 99mTc-ECD distribution were confirmed and found to be unaffected by the administration of anticonvulsant drugs. 99mTc-ECD uptake in children and infants is different from cerebral blood flow glucose metabolism as previously reported, especially in the cerebellum.

Lactoferrin in middle ear effusion.

Lactoferrin (LF) level in the middle ear effusion of patients with otitis media with effusion (OME) was determined by using an Enzyme-linked immunosorbent assay (ELISA) to find the information on non-specific immunity in the middle ear cavity. Lactate dehydrogenase (LDH), an intracellular enzyme, was also measured by spectrophotometer. We investigated cytological findings of middle ear effusion and classified cellular findings into five classes; Neutrophil dominant type, Lymphocytic-monocytic type, Mixed type, Cellular remnants type, and Too few cells type. LF and LDH levels in average: Serum, LF 1.14 microgram/ml, LDH 330.4 IU/l; Few cells type, LF 45.25 micrograms/ml, LDH 2,727.5 IU/l; Lymphocytic-monocytic type, LF 107.11 micrograms/ml, LDH 10,197.8 IU/l; Neutrophil dominant type, LF 99.73 micrograms/ml, LDH 10,580 IU/l; Mixed type, LF 163.71 micrograms/ml, LDH 19,342.9 IU/l; Cellular remnants type, LF 127.6 micrograms/ml, LDH 9,122 IU/l. LF level is high when cellular factors are rich in middle ear effusion.

[Evaluation of chlamydial IgM antibodies for clinical diagnosis].

Chlamydia trachomatis (C. trachomatis), C. psittaci, and C. pneumoniae are now well established as pathogens of respiratory infections including pneumonia. Serum samples from 223 infants and children with pneumonia, 31 patients with adult inclusion conjunctivitis, 16 parents of babies with neonatal inclusion conjunctivitis and others were tested for IgM antibodies to Chlamydiae. Diagnostic kits for chlamydial IgM antibodies (SeroELISA and IPAzyme) have been also evaluated for their diagnostic value. It was found that detection of specific IgM antibodies with SeroELISA has a diagnostic value in chlamydial pneumonias.

[Activation of bradykinin formation cascade on receiving autologous blood transfusion through a white cell-reduction filter in a patient treated with an ACE inhibitor].

We have experienced a case of anaphylactoid reaction on receiving autologous blood transfusion through a WBC filter for packed red blood cell (PRBC). The patient was a 71-year-old man with a history of hypertension treated with oral antihypertensive drug; enalapril, an angiotensin converting enzyme (ACE) inhibitor, who received anesthesia for Y-graft replacement. Autologous blood was obtained after the induction of general anesthesia in the operating room. Upon starting to return the stored blood with an unintentional use of a WBC filter, arterial blood pressure (ABP) fell within the first minute of the transfusion. We obtained three blood samples; pre-filtered blood (PRE), postfiltered blood (POST) and arterial blood (CIRC) after the event, and analyzed concentrations of bradykinin (BK), high molecular weight kininogen (HMWK) and high molecular weight kininogen-light chain (HMWK-LC). BK was higher in POST than in PRE. HMWK was lower in POST than in PRE, while HMWK-LC was higher in POST than in PRE. HMWK in CIRC was lower than in PRE, and HMWK-LC was higher in CIRC than in PRE. HMWK and HMWK-LC changes after the event suggest that BK formation cascade in the patient was activated on receiving the transfusion. ACE inhibitors were reported to augment such activation. The WBC filter has the negatively charged surface on filteration material and may activate the cascade. While WBC filters can avoid transfusion related reactions, hemodynamic responses should be watched closely in patients treated with ACE inhibitors.

[A psychological burden on patients with clinical trials during anesthesia].

We investigated with questionnaires, psychological outline of 30 patients, who were requested to participate in clinical trials of anti-tachycardia drugs during anesthesia. Although 14 patients consented to the trial, the consent was not based on adequate understanding or volunteerism in 3 patients. Nine patients of the consented group were anxious about the possible use of the trial drug. Eight patients of the rejected group felt anxiety on surgery and anesthesia, which was the most common reason for rejection. Forty % of refused patients felt a guilty conscience or embarrassed. Although we tried to obtain patients' consent following governmental and institutional regulations and guidelines, not only the consented but also the refused patients suffer from psychological burden with the clinical trial. It is of concern that recruitment to the trial enhances anxiety of the patients as they already feel uneasiness, unrest, and insecurity facing anesthesia and surgery. To avoid entry of less informed or unwilling patients to the clinical trial, we must secure patients' veto, and recruitment should be performed by clinicians who are not involved in anesthesia practice.

Metallic behaviour in SOI quantum wells with strong intervalley scattering.

The fundamental properties of valleys are recently attracting growing attention due to electrons in new and topical materials possessing this degree-of-freedom and recent proposals for valleytronics devices. In silicon MOSFETs, the interest has a longer history since the valley degree of freedom had been identified as a key parameter in the observation of the controversial "metallic behaviour" in two dimensions. However, while it has been recently demonstrated that lifting valley degeneracy can destroy the metallic behaviour, little is known about the role of intervalley scattering. Here, we show that the metallic behaviour can be observed in the presence of strong intervalley scattering in silicon on insulator (SOI) quantum wells. Analysis of the conductivity in terms of quantum corrections reveals that interactions are much stronger in SOI than in conventional MOSFETs, leading to the metallic behaviour despite the strong intervalley scattering.

Evaluation of diagnostic assays for neonatal and infantile chlamydial infections.

Several diagnostic assays for neonatal and infantile chlamydial infections, isolation with tissue culture, antigen detection by enzyme immunoassay (EIA) (IDEIA Chlamydia test), a nonisotopic DNA probe (Gen-Probe PACE 2 assay), serum IgM antibody detection by EIA (SeroELISA Chlamydia TRUE IgM), and polymerase chain reaction (PCR), were evaluated. Of 210 clinical specimens (170 nasopharyngeal and 40 conjunctival swabs) from 53 neonates and 102 infants with respiratory insufficiency and respiratory tract infections which were suspected to be associated chlamydial infection, chlamydial antigens were detected in 30 by IDEIA Chlamydia. Of these 30 samples, C. trachomatis was isolated from 27 specimens. Samples from 15 neonates and 6 infants were culture-positive and IDEIA Chlamydia-positive. Of 30 samples, 27 were tested with PCR and 8 with DNA probe. Twenty-three of 27 specimens were positive with PCR, while only one specimen was positive with DNA probe. EIA can be used for the diagnosis and screening of neonatal and infantile chlamydial infections.

Pharmacokinetics of mefenamic acid in preterm infants with patent ductus arteriosus.

The pharmacokinetics of mefenamic acid (MA), 2 mg/kg, were studied in 17 preterm infants with symptomatic patent ductus arteriosus. They were given this dosage orally at 24 h intervals. There were marked inter-individual differences in some of the pharmacokinetic parameters after the first dose; peak plasma concentration (Cmax) varied from 1.2 to 6.1 micrograms/mL with a mean of 3.8 micrograms/mL, time to reach Cmax (tmax) varied from 2 to 18 h with a mean of 7.7 h and plasma half-life (t1/2) varied from 3.8 to 43.6 h with a mean of 18.7 h. The group of infants (10/17) who had ductus closure after the first dose had significantly lower clearance (P < 0.01), longer t1/2 (P < 0.01) and higher 24 h plasma concentration (P < 0.001) compared to the group of infants (7/17) who had no ductus closure after the first dose. It appeared that the plasma concentration of MA had to be above 2.0 micrograms/mL and maintained at this concentration for at least 12 h for MA associated with ductus closure in preterm infants to take effect. In view of the inter-individual variation of plasma MA concentration and the effective plasma concentration, we suggest that measurement of the plasma concentration should be done 24 h after the first dose. This might be useful for safe and effective therapy for infants with ductus closure failure after the first dose.

Remarkable changes in the plasma levels of pituitary protein "7B2" during childhood.

We measured plasma immunoreactive (IR)-7B2 concentrations in 96 children (57 males and 39 females) from the newborn period to 20 yr of age. Plasma IR-7B2 concentrations in infants less than 2 yr of age (range 175-580 pg/ml, n = 19) were much higher than those in adults (range 20-138 pg/ml). Plasma levels of IR-7B2 decreased with age during childhood to reach the adult level at 15-20 yr. Significant negative correlations were found between plasma levels of IR-7B2 and dehydroepiandrosterone sulfate (r = -0.4154, p less than 0.05, n = 27), luteinizing hormone (r = -0.4948, p less than 0.05, n = 20) and follicle-stimulating hormone (r = -0.4682, p less than 0.05, n = 20). The possibility of a relationship between the reduction of plasma IR-7B2 levels and pubertal development warrants attention.

Simple, rapid and sensitive method for the determination of indomethacin in plasma by high-performance liquid chromatography with ultraviolet detection.

A micro method for determination of indomethacin in plasma was developed. Following deproteinization of plasma with acetonitrile containing internal standard (mefenamic acid), the separation of indomethacin and internal standard was achieved by high-performance liquid chromatography using a 7 microm LiChrosorb-RP18 column (250x4 mm I.D.) at 50 degrees C. The mobile phase was 6 mM phosphoric acid-acetonitrile (50:50). The flow-rate was kept at 2.0 ml/min and the column effluent was monitored at 205 nm. The coefficients of variation of the method estimated at 0.2 and 1.0 microg/ml were 4.2 and 2.3%, and the detection limit of the drug was about 0.05 microg/ml (S/N=5). The method requires minimum pretreatment of the plasma with a small sample volume (25 microl), and is very suitable for therapeutic drug monitoring of indomethacin in premature infants with symptomatic patent ductus arteriosus.

Changes in cerebral oxygenation in children undergoing surgical repair of ventricular septal defects.

There have been few published studies on changes in cerebral oxygenation during paediatric cardiac surgery as measured by conventional near-infrared spectroscopy. We studied changes in cerebral oxygenation in 16 children undergoing surgical repair of ventricular septal defects. Fifteen of the patients showed similar patterns of changes: brain tissue concentrations of oxyhaemoglobin decreased significantly during cardiopulmonary bypass, whereas there was no significant change in brain tissue concentrations of deoxyhaemoglobin. In the remaining patient, who suffered decreased blood flow to the lower body during surgery, the pattern of changes was different to that of the other subjects. This patient suffered postoperative respiratory and renal failure. This study suggests that conventional near-infrared spectroscopy may be useful for clinical monitoring during ventricular septal defect repair.

Diffuse brain damage caused by acute twin-twin transfusion during late pregnancy.

BACKGROUND: Monoamniotic twinning is a relatively rare event with increased antenatal and perinatal mortality. We describe a brain damage detected in a surviving monoamniotic twin after intrauterine death of the co-twin at 37 weeks of gestation. RESULTS: Severe entanglement and knotting of the umbilical cords was apparent at the time of delivery and a portion of the cord to the dead twin was narrowed significantly. It was suggested that transfer of blood occurred across placental anastomoses from the survivor to the dead fetus, resulting in transient but severe hypovolemia in the survivor. It is difficult to prevent this type of brain damage because the course of acute twin-twin transfusion is very rapid and the damage has already occurred by the time the death of the twin is diagnosed. CONCLUSIONS: We suggest that elective delivery should be considered in cases of monoamniotic twin pregnancies with additional risk factors.

Microsatellite analysis of childhood leukemia: correlation of 9p and 12p chromosome abnormalities with expression of related genes.

BACKGROUND: Loss of heterozygosity (LOH) and DNA replication error (RER) have been thought to be involved in carcinogenesis, but have not been investigated in childhood leukemia and lymphoma. METHODS: Eighty samples from 65 patients with childhood leukemia and lymphoma were examined using seven different microsatellite markers for RER analysis. Additionally, LOH in two chromosome regions (9p and 12p) was investigated. Furthermore, expression of the TEL, TEL/AML1 and p27(KIP1) genes on 12p and the p16 gene on 9p were detected by reverse transcriptase polymerase chain reaction. RESULTS: Replication errors were detected in 5/65 patients (7.7%). Most (4/5 patients) RER were preferentially located in the 9p and 12p regions. There were two patients who had DNA abnormalities in both 9p and 12p, one with common acute lymphoblastic leukemia (ALL) showed 9p LOH and the TEL/AML1 fusion gene on 12p and the other with common ALL and 12p RER had diminished expression of both the p27(KIP1) gene on 12p and the p16 gene on 9p. CONCLUSIONS: Combined DNA alterations on 9p and 12p, involving LOH, RER and/or gene mutation and chromosomal translocation, were found in childhood acute leukemia, especially in common ALL.