Rapid Detection of SNP (c.309T>G) in the MDM2 Gene by the Duplex SmartAmp Method

Background

Genetic polymorphisms in the human MDM2 gene are suggested to be a tumor susceptibility marker and a prognostic factor for cancer. It has been reported that a single nucleotide polymorphism (SNP) c.309T>G in the MDM2 gene attenuates the tumor suppressor activity of p53 and accelerates tumor formation in humans.

Methodology

In this study, to detect the SNP c.309T>G in the MDM2 gene, we have developed a new SNP detection method, named “Duplex SmartAmp,” which enabled us to simultaneously detect both 309T and 309G alleles in one tube. To develop this new method, we introduced new primers i.e., nBP and oBPs, as well as two different fluorescent dyes that separately detect those genetic polymorphisms.

Results and Conclusions

By the Duplex SmartAmp method, the genetic polymorphisms of the MDM2 gene were detected directly from a small amount of genomic DNA or blood samples. We used 96 genomic DNA and 24 blood samples to validate the Duplex SmartAmp by comparison with results of the conventional PCR-RFLP method; consequently, the Duplex SmartAmp results agreed totally with those of the PCR-RFLP method. Thus, the new SNP detection method is considered useful for detecting the SNP c.309T>G in the MDM2 gene so as to judge cancer susceptibility against some cellular stress in the clinical setting, and also to handle a large number of samples and enable rapid clinical diagnosis.     

Isolation and Identification of 3-(2-Furoyl)alanine and l-Pipecolic Acid from Green Gram Seeds

4-Hydroxy-3,7-dimethyl-2,6-octadienal (5-Hydroxycitral) (2) was prepared from 3,7-dimethyl-2,6-octatrienal (citral) (1) via bromoaldehyde (4) and acetoxyaldehyde (6). 3,7-Dimethyl-2,4,6-octadienal (7) was also obtained as a byproduct. 4-Hydroxy-3,7-dimethyl-2,6-octadienal (2) showed similar growth inhibition activity to that of 1 against Sarcoma 180.     

Generation of (2-Nitroethyl)benzene and related benzenoids from L-Phenylalanine; flower scents of the Japanese Loquat Eriobotrya japonica [Rosales: Rosaceae]

ABSTRACT

(2-Nitroethyl)benzene, methyl 4-methoxybenzoate and 4-methoxybenzaldehyde have been known as major scent components in flowers of the Japanese loquat Eriobotrya japonica [Rosales: rosaceae], together with 13 related benzenoids, including Z- and E-2-phenylacetaldoxime and benzyl alcohol. The scents air-trapped from a flowering panicle during 24 h incubation with d₈-L-phenylalanine were composed of 15 deuterium labeled compounds {d₆-styrene, d₅-benzaldehyde, d₇-2-phenylacetaldehyde, methyl d₅-benzoate, d_{7 ?}2-phenylethanol, d₇-2-phenylacetonitrile, d₄-1,4-dimethoxybenzene, d₇-Z-2-phenylacetaldoxime, d₄-4-methoxybenzaldehyde, d₇-E-2-phenylacetaldoxime, d₄-4-methoxybenzyl alcohol, d₇-(2-nitroethyl)benzene, methyl d₄-4-methoxybenzoate, methyl d₆-cinnamate and ethyl d₄-4-methoxybenzoate}. On the other hand, hexane extracts of the flower petal incubate with a mixture of d₅-Z- and d₅-E-2-phenylacetaldoxime after 24 h indicated generation of six d₅-labeld components {d₅-benzaldehyde, d₅-benzyl alcohol, d₅-2-phenylacetaldehyde, methyl d₅-benzoate, d₅-2-phenylethanol, and d₅-(2-nitroethyl)benzene}. By comparing those results, (2-nitroethyl)benzene was concluded as a product directly generated from a mixture of Z- and E-2-phenylacetaldoxime together with six minor benzenoids, while two major compounds (4-methoxybenzaldehyde and methyl 4-methoxybenzoate) together with three minors from L-phenylalanine, presumably via L-tyrosine. The other two minor components were derived from L-phenylalanine.     

Ect2, an Ortholog of <Emphasis Type="Italic">Drosophila’s</Emphasis><Emphasis Type="Italic">Pebble</Emphasis>, Negatively Regulates Neurite Outgrowth in Neuroblastoma × Glioma Hybrid NG108-15 Cells

To identify genes required for brain development, we previously performed in vivo RNA interference (RNAi) screening in Drosophila embryos. We identified pebble as a gene that disrupts development of the Drosophila nervous system. Although pebble has been shown to be involved in neuronal development of Drosophila in several screens, the involvement of Ect2, a mammalian ortholog of pebble, in mammalian neuronal development has not been addressed. To examine the role of Ect2 in neuronal differentiation, we performed Ect2 RNAi in the mouse neuroblastoma × rat glioma NG108-15 cell line. Depletion of Ect2 resulted in an increased proportion of binucleate cells and morphological differentiation of NG108-15 cells characterized by the outgrowth of neurites. These morphological changes were correlated with an increased level of acetylcholine esterase mRNA. In addition, expression of Ect2 was decreased in differentiated NG108-15 cells induced by dibutyryl cyclic AMP. These findings indicate that Ect2 negatively regulates the differentiation of NG108-15 cells and suggest that Ect2 may play a role in neuronal differentiation and brain development in vivo.     

Driving forces enable high-titer anaerobic 1-butanol synthesis in Escherichia coli

1-Butanol, an important chemical feedstock and advanced biofuel, is produced by Clostridium species. Various efforts have been made to transfer the clostridial 1-butanol pathway into other microorganisms. However, in contrast to similar compounds, only limited titers of 1-butanol were attained. In this work, we constructed a modified clostridial 1-butanol pathway in Escherichia coli to provide an irreversible reaction catalyzed by trans-enoyl-coenzyme A (CoA) reductase (Ter) and created NADH and acetyl-CoA driving forces to direct the flux. We achieved high-titer (30 g/liter) and high-yield (70 to 88% of the theoretical) production of 1-butanol anaerobically, comparable to or exceeding the levels demonstrated by native producers. Without the NADH and acetyl-CoA driving forces, the Ter reaction alone only achieved about 1/10 the level of production. The engineered host platform also enables the selection of essential enzymes with better catalytic efficiency or expression by anaerobic growth rescue. These results demonstrate the importance of driving forces in the efficient production of nonnative products.     

Comparison of surface electromyographic (sEMG) activity of submental muscles between the head lift and tongue press exercises as a therapeutic exercise for pharyngeal dysphagia

Objective: The present study compared surface electromyographic (sEMG) activity obtained from the submental muscle group for a tongue press and a head lift exercise as potential therapeutic exercises for dysphagic elderly. Materials and methods: Fifty‐three healthy volunteers with a mean age of 35.3 participated in this study. Subjects were required to perform an isometric task, pressing their tongue against the hard palate, and an isotonic task requiring sustained lingual force against the hard palate. Pressure sensors were used to measure the amount of lingual pressure against the hard palate. Submental sEMG data from these tasks were compared with those obtained from the isometric and isotonic aspects of a head lift exercise. Results: No sEMG differences were identified between the isometric tongue press task and head lift exercise. Isotonic tongue press exercises resulted in significantly higher maximum and mean sEMG values compared with the isotonic head lift exercise (p < 0.05). The submental sEMG activity from the tongue press exercise was equal (isometric) to, or greater (isotonic) than comparable muscle activation obtained during the head lift exercise. Conclusions: The tongue press exercise may be less strenuous than the head lift exercise while achieving the same therapeutic effect.     

Identification of crinosterol from astigamatid mites

A 24-alkylsterol, crinosterol [(24S)-24-methylcholesta-5,22(E)-dien-3beta-ol] has been isolated from sea-dwelling animals, protists and plants. Here, we identified crinosterol from nine species of mites (Acari). The compound was identified by using (1)H-NMR analysis and GCMS spectral data along with the HPLC retention time by comparing with those of the synthesized compound. As far as we know, this is the first report on the identification of crinosterol from arthropods. Furthermore, after Rhizoglyphus robini were fed on artificial diets with d(3)-methionine, d(2)-crinosterol was detected from the mite's extracts. The incorporation of two deuterium atoms into the sterol indicated that a d(3)-methyl group was introduced into the C24 of the side chain to form crinosterol. Although the details of the biosynthesis of crinosterol remain unknown, the discovery of crinosterol in the mites implies the existence of interesting sterol metabolisms in the animals.