We have developed real-time PCR systems to quantitate feline cytokine gene expression. The method is based on the cleavage of fluorescent dye-labelled probes by the 5'-3' exonuclease activity of the Taq DNA polymerase during PCR and measurement of fluorescence intensity by a Sequence Detection System. The feline-specific TaqMan probes were designed to encompass an intron, thus allowing differen...

BACKGROUND Tetrahydrobiopterin (BH4) is an essential cofactor of nitric oxide synthase, and GTP cyclohydrolase I (GCHI) is a rate-limiting enzyme in the biosynthesis of BH4. The expression of inducible nitric oxide synthase (iNOS) was earlier demonstrated in the ventricles of patients with dilated cardiomyopathy (DCM) although that of GCHI was not clarified. The present study was designed to de...

Quantitative real-time polymerase chain reaction (qPCR) is an efficient and widely used technique to monitor gene expression. Housekeeping genes (HKGs) are often empirically selected as the reference genes for data normalization. However, the suitability of HKGs used as the reference genes has been seldom validated. Here, six HKGs were chosen (actin A3, actin A1, GAPDH, G3PDH, E2F, rp49) in fou...

Quantitative real-time PCR (qPCR) is a powerful and reproducible method of gene expression analysis in which expression levels are quantified by normalization against reference genes. Therefore, to investigate the potential biomarkers and therapeutic targets for epithelial ovarian cancer by qPCR, it is critical to identify stable reference genes. In this study, twelve housekeeping genes (ACTB, ...

BACKGROUND Because of recent advances in the molecular diagnosis of cancer patients, tissue quality has become more important in daily practice. METHODS To evaluate the effects of fixative, duration of fixation, decalcification, and storage periods on nucleic acid integrity, DNA and RNA were extracted from gastrointestinal cancer tissue. The yield and purity were analyzed, and polymerase chai...

We recently reported a cell death cascade whereby cellular stressors activate nitric oxide formation leading to S-nitrosylation of GAPDH that binds to Siah and translocates to the nucleus. The nuclear GAPDH/Siah complex augments p300/CBP-associated acetylation of nuclear proteins, including p53, which mediate cell death. We report a 52 kDa cytosolic protein, GOSPEL, which physiologically binds ...

Two cDNA clones, encoding cytosolic and chloroplast glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from mustard (Sinapis alba), have been identified and sequenced. Comparison of the deduced amino acid sequences with one another and with the GAPDH sequences from animals, yeast and bacteria demonstrates that nucleus-encoded subunit A of chloroplast GAPDH is distinct from its cytosolic counterp...

Dihydropyrazine (DHP), which is produced during the Maillard reaction, generates radicals that not only cause breakage of chromosomal DNA leading to mutagenic lesions but also induce oxidative damage to cellular proteins. In the present study, we show that three DHP derivatives, which generated superoxide anions, caused inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). SH-compound...

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) ac tivity and mKNA contenÃis altered in the androgen-responsive human prostate curcinoma cell line LNCaP after exposure to the synthetic androgcn RISSI. Elevation in GAPDH activity is noted as early as 24 h after treatment with 1 n\i KISS) and lasts at least 96 h. RISSI has no effect on the activity of GAPDH in androgcn-independent D...

D-Serine is an essential coagonist with glutamate for stimulation of N-methyl-D-aspartate (NMDA) glutamate receptors. Although astrocytic metabolic processes are known to regulate synaptic glutamate levels, mechanisms that control D-serine levels are not well defined. Here we show that d-serine production in astrocytes is modulated by the interaction between the D-serine synthetic enzyme serine...