Enzyme-based choline and L-glutamate biosensor electrodes on silicon microprobe arrays.

Brain-implantable microprobe arrays, 6.5 mm shaft-length, incorporating several recessed Pt microelectrodes (50 µm×150 µm) and an integrated Ag/AgCl reference electrode fabricated by silicon micromachining dry etching techniques (DRIE) are described. The microelectrodes are coated by an enzyme membrane and a semi-permeable m-phenylenediamine layer for the selective detection of the neurotransmitters choline and L-glutamate at physiologically relevant concentrations. The functionalisation is based on electrochemically aided adsorption (EAA) combined with chemical co-cross-linking using glutaraldehyde and electrochemical polymerisation, respectively. These deposition methods are fully compatible with the fabricated microprobe arrays for the simultaneous detection of several analytes in different brain target areas. They are spatially controlled and allow fabricating biosensors on several microelectrodes in parallel or providing a cross-talk-free coating of closely spaced microelectrodes with different enzyme membranes. A sensitivity of 132±20 µA mM(-1) cm(-2) for choline and 95±20 µA mM(-1) cm(-2) for L-glutamate with limits of detections below 0.5 µM was obtained. The results of in vitro and in vivo experiments confirm the functional viability of the choline and l-glutamate biosensors.

Genome-wide analysis of vaccinia virus protein-protein interactions.

To detect interactions between proteins of vaccinia virus, we carried out a comprehensive two-hybrid analysis to assay every pairwise combination. We constructed an array of yeast transformants that contained each of the 266 predicted viral ORFs as Gal4 activation domain hybrid proteins. The array was individually mated to transformants containing each ORF as a Gal4-DNA-binding domain hybrid, and diploids expressing the two-hybrid reporter gene were identified. Of the approximately 70,000 combinations, we found 37 protein-protein interactions, including 28 that were previously unknown. In some cases, e.g., late transcription factors, both proteins were known to have related roles although there was no prior evidence of physical associations. For some other interactions, neither protein had a known role. In the majority of cases, however, one of the interacting proteins was known to be involved in DNA replication, transcription, virion structure, or host evasion, thereby providing a clue to the role of the other uncharacterized protein in a specific process.

Degradation of the transcription factor Gcn4 requires the kinase Pho85 and the SCF(CDC4) ubiquitin-ligase complex.

Gcn4, a yeast transcriptional activator that promotes the expression of amino acid and purine biosynthesis genes, is rapidly degraded in rich medium. Here we report that SCF(CDC4), a recently characterized protein complex that acts in conjunction with the ubiquitin-conjugating enzyme Cdc34 to degrade cell cycle regulators, is also necessary for the degradation of the transcription factor Gcn4. Degradation of Gcn4 occurs throughout the cell cycle, whereas degradation of the known cell cycle substrates of Cdc34/SCF(CDC4) is cell cycle regulated. Gcn4 ubiquitination and degradation are regulated by starvation for amino acids, whereas the degradation of the cell cycle substrates of Cdc34/SCF(CDC4) is unaffected by starvation. We further show that unlike the cell cycle substrates of Cdc34/SCF(CDC4), which require phosphorylation by the kinase Cdc28, Gcn4 degradation requires the kinase Pho85. We identify the critical target site of Pho85 on Gcn4; a mutation of this site stabilizes the protein. A specific Pho85-Pcl complex that is able to phosphorylate Gcn4 on that site is inactive under conditions under which Gcn4 is stable. Thus, Cdc34/SCF(CDC4) activity is constitutive, and regulation of the stability of its various substrates occurs at the level of their phosphorylation.

Effect of fetal ethanol exposure on the in vitro release of growth hormone, somatostatin and growth hormone-releasing factor induced by clonidine and growth hormone feedback in male and female rats.

This study was designed to examine the effect of fetal ethanol (ETOH) exposure on the sensitivity of the hypothalamic-growth hormone (GH) axis to clonidine (an alpha 2-adrenoreceptor agonist) stimulation and GH feedback. During gestation, dams were fed either a liquid diet in which 36% of the calories were derived from ETOH, or pair-fed an isocaloric control liquid diet without ETOH. A second set of controls were fed lab chow ad libitum. After birth, offspring of ETOH-fed dams were cross-fostered to a separate group of ad libitum control dams. The hypothalami and pituitaries of 10-, 20-, 30-, and 50-day-old offspring were separated by age, diet, and sex; pooled 6 to 8 per chamber; and tested in a hypothalamic-pituitary coperifusion system. Chambers were perifused with either clonidine (2 x 10(-8) M) alone, which mimics the endogenous trigger for GH release, or clonidine in combination with human GH (2 x 10(-9) M) to determine sensitivity of tissue to feedback regulation. Both stimuli act at the hypothalamic level and indirectly modulate GH release via effects on hypothalamic factors. Results of this study indicate that tissue from control male rats is responsive to the clonidine-induced GH surge by 10 days of age and to feedback depression of GH release by 20 days of age. This sensitivity persists after puberty and is associated with corresponding changes in somatotropin-release inhibiting factor (SRIF) and GH-releasing factor (GRF) release (i.e., clonidine inhibits SRIF and stimulates GRF release, and human GH reverses this pattern). Fetal ETOH exposure depresses GH sensitivity to both stimuli in male pups (age x diet x drug: p < 0.002), and this depressed sensitivity is expressed by 30 days of age by reduced responses to alpha 2-adrenergic stimulation and GH feedback (drug x diet: p < 0.002 and p < 0.001 for 30 and 50 days of age, respectively). This effect of ETOH on GH release was associated with feedback insensitivity of SRIF (drug x diet: p < 0.003, at 50 days of age) and GRF [drug x diet; p < 0.044 at 30 days; clonidine vs. clonidine and GH: p > 0.05 (NS) at 50 days of age for ETOH pups]. The depressed response of GH to clonidine after puberty may be attributable to a combination of the trends toward decreased sensitivity of both SRIF and GRF at this age. The female GH axis was both less sensitive to stimuli and less effected by ETOH than corresponding tissue from male rats (sex x age x drug x diet: p < 0.011). GH release from control female pituitaries was sensitive to clonidine before, but not after, puberty and insensitive to GH feedback at both developmental stages. On the other hand, there was a specific effect of ETOH on SRIF release at 10 days of age (diet x drug: p < 0.014), and SRIF release remained sensitive to clonidine in pups from all diet groups after puberty. Because GH release was not influenced by these changes in SRIF, these findings suggest that GH release is less sensitive to SRIF in females. In conclusion, this study suggests that fetal ETOH exposure interferes with the development of the sensitivity of the GH axis to alpha 2-adrenergic stimulation and feedback in males. Thus, the male GH axis is both more sensitive to the stimuli tested in this study and more effected by ETOH than the female axis. Furthermore, the effects of ETOH on these mechanisms do not alter GH release in males until the peripubertal period. It is likely, therefore, that the GH regulatory mechanism examined in this study does not contribute to growth retardation before puberty. If the effects of ETOH on GH release contributes to growth retardation in prepubertal males and in females, it most likely involves other regulatory mechanisms. On the other hand, because the adult pattern of GH release is programmed during development, the influence of ETOH on these developmental events may influence the male pattern of GH release and GH activity in adulthood.