Site-specific H/D exchange of p-methoxyphenol studied by resonant two-photon ionization and mass-analyzed threshold ionization spectroscopy.

The origin of the S(1) <-- S(0) transition (E(1)) and the adiabatic ionization energy (IE) of cis-p-methoxyphenol-d(1)-OD are determined to be 33 660 and 62 302 cm(-1), whereas those of cis-p-methoxyphenol-d(1)-OCH(2)D are 33 669 and 62 323 cm(-1), respectively. Similarly, the E(1) and IE of trans-p-methoxyphenol-d(1)-OD are determined to be 33 563 and 62 191 cm(-1) and those of trans-p-methoxyphenol-d(1)-OCH(2)D are 33 575 and 62 216 cm(-1), respectively. Comparing these data with those of p-methoxyphenol suggests that the H/D exchange on the OH substituent gives rise to a red shift in both the E(1) and IE, whereas that on the OCH(3) group yields a blue shift. The mass-analyzed threshold ionization spectra of the selected isomers can be used as the fingerprints for molecular identification. Analysis of these cation spectra shows that the substituent-sensitive in-plane C-OH and C-OCH(3) bending (mode 9b) and breathing (mode 1) vibrations are active for all of these isomeric cations.

Inhibition of histone acetyltransferase function of p300 by PKCdelta.

Protein kinase Cdelta (PKCdelta) is one of the functionally distinct isoforms in PKC family. p300 is a histone acetyltransferase/transcription coactivator. They share certain properties, such as ubiquitous expression, growth and tumor suppression, and ability to enhance differentiation and apoptosis. In this study, we found that PKCdelta but not classical PKC, specifically phosphorylates p300 at serine 89 in vitro and in vivo. This phosphorylation causes inhibition of p300 intrinsic HAT activity. Subsequently, the targeted acetylation of nucleosomal histones is markedly reduced, which causes repression of p300 transcription coactivator function. These findings identify a new signal transduction pathway by which PKCdelta may inhibit cell growth and promote cellular differentiation.

Elevated serum endogenous inhibitor of nitric oxide synthase and endothelial dysfunction in aged rats.

1. The purpose of the present study was to determine the relationship between endothelial dysfunction and the endogenous inhibitor of nitric oxide synthase NG,NG'-asymmetric dimethylarginine (ADMA) in aged rats. 2. Studies were performed in male adult Sprague-Dawley rats (6 months old; n = 8) and in aged rats (20 months old; n = 8). Serum levels of ADMA and L-arginine were measured by high-performance liquid chromatography and responses of endothelium-intact aortic rings to acetylcholine (ACh) were tested. Nitric oxide synthase activity in kidney tissue and serum concentrations of nitrite, a stable end-product of nitric oxide, were assayed and serum contents of malondialdehyde, derived from lipid peroxidation and serum lipid and creatinine level were determined. 3. Serum levels of ADMA increased significantly in aged rats compared with adult rats (P < 0.01), whereas serum levels of L-arginine were similar in both groups (P = NS). Accordingly, the ratio of L-arginine/ADMA in old rats was lower than that in young rats (P < 0.01). Endothelium-dependent relaxation responses to ACh in aortic rings from aged rats were impaired and these impaired responses were improved by pre-incubation of aortic rings with L-arginine. 4. Nitric oxide synthase activity in the kidney, together with serum concentration of nitrite, was significantly decreased and serum contents of malondialdehyde, cholesterol and triglycerides were increased in old compared with young rats. However, the serum creatinine level was not significantly different between adult and aged rats. 5. Endogenous ADMA may be a contributor to age-related endothelial dysfunction and increases in endogenous ADMA may be linked to lipid peroxidation in aged rats.

Histone acetylation by p300 is involved in CREB-mediated transcription on chromatin.

Signal transduction through cAMP to activate gene expression via the cAMP-responsive element (CRE) is one of the most intensively studied transcription pathways. In this pathway, transcription factor CRE-binding protein (CREB) recognizes the CRE enhancer on DNA. The CREB protein is activated via phosphorylation at serine 133 by protein kinase A and then is able to recruit coactivator CREB-binding protein (CBP) and its homologue p300. This recruitment of CBP/p300 is required for transcription activation. The mechanism for CBP/p300 to participate in this transcription process is still unclear. CBP and p300 are histone acetyltransferases (HAT) and able to associate with other HAT proteins. It has been reported that the regulation of nuclear receptor-mediated transcription initiation by p300 requires chromatin and its HAT function. The data shown here indicate that the requirements for chromatin and p300 HAT activity also apply to the activation of CREB-mediated transcription. Serine 133-phosphorylated CREB recruits p300 onto chromatin for efficient acetylation of nucleosomes. This targeted acetylation by p300 is essential to CREB-dependent transcription pathway.

Phosphorylation of p300 at serine 89 by protein kinase C.

CREB-binding protein (CBP)/p300 plays an important role in the connection of many different signal transduction pathways and the promotion of certain differentiation and proliferation processes. This role depends upon the ability of CBP/p300 to serve as coactivator for transcription factors. It has been suggested that CBP/p300 is regulated by phosphorylation, but the nature of the phosphorylation, the responsible kinase in vivo, and its physiological significance are still unclear. Here, we demonstrate the first identification of an in vivo phosphorylation site, conserved serine 89, in p300. Signal-dependent protein kinase C is able to phosphorylate serine 89 and mediates this phosphorylation event in vivo. Different from other phosphorylation observed so far in CBP/p300, this serine 89-specific phosphorylation represses the transcriptional activity of p300. This phosphorylation-mediated regulation of p300 function represents a previously unrecognized signal transduction pathway for protein kinase C to regulate cell growth and differentiation.

Distance-independence of mitotic intrachromosomal recombination in Saccharomyces cerevisiae.

Many genetic studies have shown that the frequency of homologous recombination depends largely on the distance in which recombination can occur. We have studied the effect of varying the length of duplicated sequences on the frequency of mitotic intrachromosomal recombination in Saccharomyces cerevisiae. We find that the frequency of recombination resulting in the loss of one of the repeats and the intervening sequences reaches a plateau when the repeats are short. In addition, the frequency of recombination to correct a point mutation contained in one of these repeats is not proportional to the size of the duplication but rather depends dramatically on the location of the mutation within the repeated sequences. However, the frequency of mitotic interchromosomal reciprocal recombination is dependent on the distance separating the markers. The difference in the response of intrachromosomal and interchromosomal mitotic recombination to increasing lengths of homology may indicate there are different rate-limiting steps for recombination in these two cases. These findings have important implications for the maintenance and evolution of duplicated sequences.