Nerve growth factor, neural stem cells and Alzheimer's disease

The protein family of the neurotrophins (NTs) comprises structurally and functionally related molecules such as nerve growth factor (NGF) which influences the proliferation, differentiation, survival and death of neuronal cells. In addition to their established functions for cell survival, NTs also mediate higher brain activities such as learning and memory. Changes in NT expression levels have thus been implicated in neurological diseases such as Alzheimer's disease (AD), an age-related neurodegenerative disorder that is characterized by progressive loss of memory and deterioration of higher cognitive functions. The present review provides an overview of the functional role of NGF in neural stem cells and AD while pointing to a potential application of this peptide for the treatment of AD.     

Chemical structure-dependent gene expression of proteasome subunits via regulation of the antioxidant response element

Antioxidants possess potent ability to regulate gene expression beyond their specific antioxidant activity. Genomic analysis reveals that three phenolic antioxidants, probucol, BO-653, and tBHQ, all of which have a phenoxyl group with one or two tert-butyl groups at the ortho-position, inhibit both the mRNA and protein levels of proteasome alpha-subunits in human endothelial cells. The chemical structure required for the gene regulation was studied by using derivatives of BO-653 and other antioxidants. It was found that the phenoxyl group and tert-butyl group at the ortho-position of the compounds were critical for down-regulation of the proteasome gene. Two antioxidant responsive elements (AREs) were identified in the promoter region of proteasome alpha subunit 3 (PSMA3). Results from promoter truncation analysis revealed that the proximal ARE region was necessary for the down-regulation of the expression of PSMA3. Electrophoretic mobility shift assays revealed that BO-653-mediated induction of DNA-binding to an upstream promoter region of PSMA3 containing the ARE motif was blocked by antibody against c-Jun but not Nrf2. These results indicate that the suppression of the proteasome alpha subunits expression by phenolic antioxidants is strictly dependent on both their chemical structure and the ARE consensus region in the promoter, which may be negatively regulated by AP-1.     

Impaired flow-dependent control of vascular tone and remodeling in P2X4-deficient mice

The structure and function of blood vessels adapt to environmental changes such as physical development and exercise. This phenomenon is based on the ability of the endothelial cells to sense and respond to blood flow; however, the underlying mechanisms remain unclear. Here we show that the ATP-gated P2X4 ion channel, expressed on endothelial cells and encoded by P2rx4 in mice, has a key role in the response of endothelial cells to changes in blood flow. P2rx4(-/-) mice do not have normal endothelial cell responses to flow, such as influx of Ca(2+) and subsequent production of the potent vasodilator nitric oxide (NO). Additionally, vessel dilation induced by acute increases in blood flow is markedly suppressed in P2rx4(-/-) mice. Furthermore, P2rx4(-/-) mice have higher blood pressure and excrete smaller amounts of NO products in their urine than do wild-type mice. Moreover, no adaptive vascular remodeling, that is, a decrease in vessel size in response to a chronic decrease in blood flow, was observed in P2rx4(-/-) mice. Thus, endothelial P2X4 channels are crucial to flow-sensitive mechanisms that regulate blood pressure and vascular remodeling.     

Changes in melatonin binding sites under artificial light-dark, constant light and constant dark conditions in the masu salmon brain

To test whether the affinity (Kd) and total binding capacity (Bmax) of melatonin receptors exhibit daily and circadian changes in teleost fish whose melatonin secretion is not regulated by intra-pineal clocks, we examined the changes in melatonin binding sites in the brains of underyearling masu salmon Oncorhynchus masou under artificial light-dark (LD), constant light (LL) and constant dark (DD) conditions. In Experiment 1, fish were reared under a long (LD 16:8) or short (LD 8:16) photoperiod for 69 days. Blood and brains were sampled eight times at 3 h intervals. Plasma melatonin levels were high during the dark phase and low during the light phase in both photoperiodic groups. The Bmax exhibited no daily variations. Although the Kd slightly, but significantly, changed under LD 8:16, this may be of little physiological significance. In Experiment 2, fish reared under LD 12:12 for 27 days were exposed to LL or DD from the onset of the dark phase under LD 12:12. Blood and brains were sampled 13 times at 4 h intervals for two complete 24 h cycles. Plasma melatonin levels were constantly high in the DD group and low in the LL group. No significant differences were observed in the Kd and the Bmax between the two groups, and the Kd and the Bmax exhibited no circadian variation either in the LL or DD groups. These results indicate that light conditions have little effect on melatonin binding sites in the masu salmon brain.     

Overexpression of salt-tolerant glutaminase from Micrococcus luteus K-3 in Escherichia coli and its purification

A high-expression plasmid, pKSGHE3-1, containing the salt-tolerant glutaminase (EC 3.5.1.2) from marine bacterium Micrococcus luteus K-3 was constructed. pKSGHE3-1 was made by inserting the DNA fragment (1.43 kb) containing the structural gene synthesized by polymerase chain reaction into the downstream region of the tac promoter of expression vector pKK223-3. The translational start codon was located 10 bases downstream of the Shine-Dalgarno sequence (AGGA) of pKK223-3. Escherichia coli JM109 transformed with pKSGHE3-1 exhibited more than 190-fold higher glutaminase activity than M. luteus K-3 under optimal culture conditions. The enzyme was purified to homogeneity through three column chromatography steps with a final yield of 17.1%. The recombinant enzyme showed the same enzymatic properties, including salt tolerance, as those of M. luteus K-3. This glutaminase expression system allows the production of sufficient quantities of glutaminase for basic structure-function studies including chemical modification and future X-ray crystallization analysis.