Single-stranded conformational polymorphism analysis using automated capillary array electrophoresis apparatuses.

We describe a new environment of a single-stranded conformational polymorphism (SSCP) analysis using automated capillary array sequencers (e.g., ABI Prism 3100 and 3700). In this environment, electrophoretic conditions, settings for instrument management, and software for data analysis are adjusted for SSCP analysis. Highly reproducible results are obtained with this new system, and fragments with mutations and/or polymorphisms in different capillaries or different runs can be reliably detected. The relative peak heights between alleles are quantitative and reproducible between runs, and so allele frequencies of single nucleotide polymorphisms can be accurately estimated by a pooled DNA strategy. The method allows unattended, low-cost, and quantitative SSCP analysis using instruments that are widely accessible.

Precise estimation of allele frequencies of single-nucleotide polymorphisms by a quantitative SSCP analysis of pooled DNA.

We show that single-nucleotide polymorphisms (SNPs) of moderate to high heterozygosity (minor allele frequencies >10%) can be efficiently detected, and their allele frequencies accurately estimated, by pooling the DNA samples and applying a capillary-based SSCP analysis. In this method, alleles are separated into peaks, and their frequencies can be reliably and accurately quantified from their peak heights (SD <1.8%). We found that as many as 40% of publicly available SNPs that were analyzed by this method have widely differing allele frequency distributions among groups of different ethnicity (parents of Centre d'Etude Polymorphisme Humaine families vs. Japanese individuals). These results demonstrate the effectiveness of the present pooling method in the reevaluation of candidate SNPs that have been collected by examination of limited numbers of individuals. The method should also serve as a robust quantitative technique for studies in which a precise estimate of SNP allele frequencies is essential-for example, in linkage disequilibrium analysis.

[The false positive reaction of the Triage panel drug-of-abuse by herbal drugs ma-huang (Ephedra sinica (Ephedraceae))].

We investigated false-positive reactions obtained from a drug screening test using a Triage panel. We detected 2 cases giving false-positive reaction for AMP (amphetamine, methamphetamine) during the screening of 187 normal subjects. Subsequent follow up testing by high-performance liquid chromatography (HPLC), showed both to be false-positive reactions. As both cases have a history of ingesting the herbal drug, Ma-huang (Ephedra sinica (Ephedraceae)), containing ephedrine, we examined the relationship between false-positive reactions on Triage and Ma-huang. All urine samples collected from 7 healthy volunteers following administration of Ma-huang indicated AMP positive on Triage. Also a high ratio of AMP positives was observed in the patients who were administered Ma-huang-containing drugs at the hospital. However, none of them were identified as true-positives by HPLC or gas chromatography mass spectrometry (GC/MS) analysis. The extract of Ma-huang contained in herbal drugs, which otherwise contain neither amphetamine nor its derivatives, gives (AMP) positive indications on Triage. We speculate that unidentified components of Ma-huang cause the false-positive reactions. We suggest that follow-up tests by GC/MS or HPLC are needed wherever a positive result is obtained from a screening test by Triage. Furthermore, it will be established to continue collecting information on prescribed and non-prescribed drugs.

[Indication and effectiveness of endoscopic percutaneous gastrostomy as a route of parenteral alimentation for the home care patient].

We are managing 8 home care patients who have a gastrostomy made using an endoscopic percutaneous technique as a route of parenteral alimentation. Based on our experience, the preconditions for an endoscopic percutaneous gastrostomy as a route of parenteral alimentation are 1. normal gastrointestinal function, 2. difficulty in swallowing, 3. possibility that the caregiver can manage the gastrostomy. When we performed an endoscopic percutaneous gastrostomy as a route of parenteral alimentation for 8 home care patients, we obtained the several advantages mentioned below. 1. Swallowing pneumonia was prevented. 2. Adequate amount of alimental liquid could be infused. 3. Patient could take a bath or shower with the gastrostomy, and good QOL was realized. 4. The home care patient with the gastrostomy could have a satisfactorily long life.

Differential decreases in c-fos and aldolase C mRNA expression in the rat cerebellum after repeated administration of methamphetamine.

The effects of repeated methamphetamine administration on c-fos mRNA and aldolase C (Zebrin) mRNA expression in the rat cerebellum were investigated. A single dose of methamphetamine induced c-fos mRNA expression in granule and Purkinje cells of both anterior and posterior lobes. In the posterior lobe, in particular, c-fos mRNA signals were distributed in a parasagittal organization, like Zebrin bands. Repeated methamphetamine injections reduced methamphetamine-induced c-fos mRNA signals in the anterior hemisphere and in part of the posterior vermis (lobule VII) and posterior hemisphere. Aldolase C mRNA signals in Purkinje cells decreased only in lobules where methamphetamine-induced c-fos signals were not reduced (lobules VI and IX). Therefore, differential decreases in c-fos mRNA and aldolase C mRNA expression after repeated methamphetamine administration depend upon the localization of Purkinje cells in the cerebellum. Since c-fos mRNA and aldolase C mRNA expressions are markers of excitability and the metabolic state of Purkinje cells, respectively, hypofunction of inhibitory Purkinje cells could be induced if methamphetamine is repeatedly injected. Since repeated methamphetamine administration in this experimental paradigm increased horizontal movement and the rearing activity of rats, the hemisphere of the cerebellum may be involved in development of methamphetamine-induced motor behavioral sensitization in addition to the striatum and the nucleus accumbens.

Molecular basis of hereditary methaemoglobinaemia, types I and II: two novel mutations in the NADH-cytochrome b5 reductase gene.

Hereditary methaemoglobinaemia, caused by deficiency of NADH-cytochrome b5 reductase (b5R), has been classified into two types, an erythrocyte (type I) and a generalized (type II). We analysed the b5R gene of two Thai patients and found two novel mutations. The patient with type II was homozygous for a C-to-T substitution in codon 8 3 that changes Arg (CGA) to a stop codon (TGA), resulting in a truncated b5R without the catalytic portion. The patient with type I was homozygous for a C-to-T substitution in codon 178 causing replacement of Ala (GCG) with Val (GTG). To characterize effects of this missense mutation, we investigated enzymatic properties of mutant b5R (Ala 178 Val). Although the mutant enzyme showed normal catalytic activity, less stability and different spectra were observed. These results suggest that this substitution influenced enzyme stability due to the slight change of structure. In conclusion, the nonsense mutation led to type II because of malfunction of the truncated protein. On the other hand, the missense mutation caused type I, due to degradation of the unstable mutant enzyme with normal activities in patient's erythrocytes, because of the lack of compensation by new protein synthesis during the long life-span of erythrocytes.