Stochastic formulation for a partial neural circuit of <Emphasis Type="Italic">C. elegans</Emphasis>

We present a stochastic formulation for a partial neural circuit of Caenorhabditis elegans. This study is concerned with how to reduce the degree of freedom in a large neural circuit. In the presented formulation, neurons in the whole neural circuit are divided into two complementary groups. One is the neurons which are mainly associated with a certain behavior, and the other is the remaining neurons of C. elegans. In an ordinary study on a partial neural circuit, the influence of the latter (the remaining neurons) on the former (the associated neurons) is completely neglected. In the presented formulation, however, the influence is expressed by a stochastic variable. The structure of the ensemble for the stochastic variable is appropriately evaluated by the neural connectivity of C. elegans since the neural connectivity of C. elegans has been completely determined. In this way, the degree of freedom is effectively reduced. We apply the presented formulation to determine the synaptic signs in the touch sensitivity circuit of C. elegans. The synaptic signs are determined to satisfy the locomotory behaviors in C. elegans. We find that the influence of the remaining neurons on the touch sensitivity circuit is important to determine the synaptic signs.     

Effect of valproic acid on the urinary metabolic profile of a patient with succinic semialdehyde dehydrogenase deficiency

The metabolic changes in a patient with succinic semialdehyde dehydrogenase deficiency were investigated following valproate administration using urease pretreatment and gas chromatography-mass spectrometry. A stable isotope dilution technique was used for quantification of urinary 4-hydroxybutyrate. Urinary levels of 4-hydroxybutyrate were 4-fold higher after 1-month valproate therapy. 4,5-Dihydrohexanoate, 2-deoxytetronate and 3-deoxytetronate were also 1.7-2.7-fold higher. The urinary excretions of 4-hydroxybutyrate in valproate non-medicated controls were age dependence and decreased with age. Relationships between 4-hydroxybutyrate excretion and 4-hydroxyvalproate or 5-hydroxyvalproate excretion were observed in valproate medicated controls. It seems that 4-hydroxyvalproate and 5-hydroxyvalproate as well as valproate are involved with increased excretion of 4-hydroxybutyrate following valproate administrations.     

In Situ Hybridization at the Electron Microscopic Level Using a Bromodeoxyuridine Labeled DNA Probe

A bromodeoxyuridine (BrdU) labeled DNA probe was used for in situ hybridization at the electron microscopic (EM) level. A BrdU labeled DNA probe was hybridized in situ to cryostat sections of paraformaldehyde fixed OCT compound embedded cultured HL-60 cells. After hybridization, some sections were incubated with FITC-conjugated anti-BrdU monoclonal antibody for fluorescence microscopy (FM). and others were embedded in Quetol for electron microscopy (EM). The ultrathin sections of Quetol-embedded specimens were incubated with the anti-BrdU monoclonal antibody and the immunoglobulin: gold colloid. In both FM and EM studies, the signals were concentrated in the rough endoplasmic reticulum. Moreover, some label was arranged from the nucleus to the cytoplasm at the EM level. Relatively simple methods using the BrdU labeled DNA probe for the detection of the defined nucleic acid sequence with reasonable tissue preservation and high resolution are described here. This method may be useful for developmental and disease related studies of specific mRNA in cells and tissues.     

Simultaneous analysis of pipecolic acid with proline in the brain by selected ion-monitoring technique

A method for the simultaneous analysis of pipecolic acid and proline in the brain is developed. The qualification and quantification of pipecolic acid and proline are accomplished with gas chromatography/mass spectrometry including a selected ion-monitoring technique by using deuterium-labeled proline as an internal standard, after the amino and carboxylic groups of these cyclic amino acids are derivatized with boron trifluoride methanol complex and heptafluorobutyric anhydride. The lower limit of quantification for the method is picomole levels and the concentration of pipecolic acid and proline in rat whole brain is determined to be 1.05 and 71.50 nmol/g of tissue, respectively.     

Application of selected ion monitoring technique for quantitative determination of picomole levels of pyrrolidine in the brain

A specific and sensitive method for analysis of brain pyrrolidine, a volatile amine with potent synaptotropic actions on the peripheral and central nervous systems, was devised. The method involves the isolation of volatile amines by steam distillation and the qualification and quantification of pyrrolidine by gas chromatography/mass spectrometry ($\text{gc}\text{ms}$) including a selected ion monitoring technique with deuterium-labeled pyrrolidine as an internal standard. The lower limit of quantification for the method was 2 pmol, and the mean concentration of pyrrolidine in the rat whole brain was determined to be 95 pmol/g of tissue.     

Analysis of organic acids in the hearts of patients with idiopathic cardiomyopathy by gas chromatography—mass spectrometry

Organic acids in the hearts of patients with idiopathic cardiomyopathy, obtained by biopsy, were studied using gas chromatography—mass spectrometry. The profiling of organic acids was compared among eight cases of hypertrophic cardiomyopathy, three cases of congestive cardiomyopathy, and nine cases of other heart diseases, which were regarded as controls.It was found that almost all organic acids, especially deoxyaldonic acids of 2-deoxytetronic acid, 2,3-dideoxypentonic acid, 3-deoxy-2-C-(hydroxymethyl)tetronic acid, 3-deoxyerythropentonic acid and 3-deoxy-2-C-(hydroxymethyl)erythropentonic acid, were accumulated in large amounts in the heart in congestive cardiomyopathy, while these acids were decreased in hypertrophic cardiomyopathy. It was therefore suggested that deoxyaldonic acid metabolism in the heart in congestive cardiomyopathy is quite different from that in hypertrophic cardiomyopathy.     

Comparative mutability of the Ames tester strains of Salmonella typhimurium by ultraviolet radiation and by 4-nitroquinoline 1-oxide

A standard method for determining mutant frequencies per survivor was used to study the detailed kinetics of reverse mutations of Ames tester strains of Salmonella typhimurium induced by UV and by 4N1O. After UV irradiation, strain TA1538 was non-mutable, but its plasmid-containing derivative TA98 was mutable, whereas TA1535 was mutable and its plasmid-bearing derivative TA100 was about 10-fold more mutable. After treatment with 4NQO, TA98 was less mutable than TA1538, whereas TA100 was more mutable than TA1535 by a factor of 10–50. TA1537 was slightly less mutable than TA1535 by either UV or 4NQO. The differential mutabilities of these strains are briefly discussed in relation to the “hot spot” base sequences for reversion and the nature of DNA damage caused by UV and 4NQO.     

Genome-Wide Screening of Salt Tolerant Genes by Activation-Tagging Using Dedifferentiated Calli of Arabidopsis and Its Application to Finding Gene for Myo-Inositol-1-P-Synthase

Salinity represents a major abiotic stress factor that can adversely limit the production, quality and geographical distribution of crops. In this study we focused on dedifferentiated calli with fundamental cell functions, the salt tolerance of which had not been previously examined. The experimental approach was based on activation tagging without regeneration of plants for the identification of salt-tolerant mutants of Arabidopsis. Among 62,000 transformed calli that were screened, 18 potential mutants resistant to 150 mM NaCl were obtained. Thermal asymmetric interlaced (TAIL)-PCR was performed to determine the location of T-DNA integration in the genome. In one line, referred to as salt tolerant callus 1 (stc1), expression of a gene [At4g39800: myo-inositol-1-P-synthase 1 (MIPS1)] was considerably enhanced in calli. Plants regenerated from calli showed tolerance to salt in germination and subsequent growth. Retransformation of wild-type Arabidopsis with MIPS1 conferred salt tolerance, indicating that MIPS1 is the causal gene. The over-expression of MIPS1 increased the content of total inositol. The involvement of MIPS1 in salt tolerance through the fundamental cell growth has been proved in Arabidopsis.     

Simultaneous Copy Number Losses within Multiple Subtelomeric Regions in Early-Onset Type2 Diabetes Mellitus

Genetic factors play very important roles in the onset and progression of type 2 diabetes mellitus (T2DM). However, the genetic factors correlating with T2DM onset have not as yet been fully clarified. We previously found that copy number losses in the subtelomeric region on chromosome 4p16.3 were detected in early-onset Japanese T2DM patients (onset age <35 years) at a high frequency. Herein, we additionally found two novel copy number losses within the subtelomeric regions on chromosomes 16q24.2-3 and 22q13.31-33, which have significant associations with early-onset Japanese T2DM. The associations were statistically significant by Fisher''s exact tests with P values of 5.19×10⁻³ and 1.81×10⁻³ and odds ratios of 5.7 and 4.4 for 16q24.2-3 and 22q13.31-33, respectively. Furthermore, copy number variation (CNV) analysis of the whole genome using the CNV BeadChip system verified simultaneous copy number losses in all three subtelomeric regions in 11 of our 100 T2DM subjects, while none of 100 non-diabetic controls showed the copy number losses in all three regions. Our results suggest that the mechanism underlying induction of CNVs is involved in the pathogenesis of early-onset T2DM. Thus, copy number losses within multiple subtelomeric regions are strongly associated with early-onset T2DM and examination of simultaneous CNVs in these three regions may lead to the development of an accurate and selective procedure for detecting genetic susceptibility to T2DM.