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N G Brown

Publications and source records attributed to N G Brown.

8 recordsLinked to original sources
PubMed2000

Identification of sites phosphorylated by the vaccinia virus B1R kinase in viral protein H5R.

BACKGROUND: Vaccinia virus gene B1R encodes a serine/threonine protein kinase. In vitro this protein kinase phosphorylates ribosomal proteins Sa and S2 and vaccinia virus protein H5R, proteins that become phosphorylated during infection. Nothing is known about the sites phosphorylated on these proteins or the general substrate specificity of the kinase. The work described is the first to address these questions. RESULTS: Vaccinia virus protein H5R was phosphorylated by the B1R protein kinase in vitro, digested with V8 protease, and phosphopeptides separated by HPLC. The N-terminal sequence of one radioactively labelled phosphopeptide was determined and found to correspond to residues 81-87 of the protein, with Thr-84 and Thr-85 being phosphorylated. A synthetic peptide based on this region of the protein was shown to be a substrate for the B1R protein kinase, and the extent of phosphorylation was substantially decreased if either Thr residue was replaced by an Ala. CONCLUSIONS: We have identified the first phosphorylation site for the vaccinia virus B1R protein kinase. This gives important information about the substrate-specificity of the enzyme, which differs from that of other known protein kinases. It remains to be seen whether the same site is phosphorylated in vivo.

Binding Sites↗
PubMed1997

The environmental estrogen bisphenol A stimulates prolactin release in vitro and in vivo.

Environmental estrogens (xenoestrogens) are a diverse group of chemicals that mimic estrogenic actions. Bisphenol A (BPA), a monomer of plastics used in many consumer products, has estrogenic activity in vitro. The pituitary lactotroph is a well established estrogen-responsive cell. The overall objective was to examine the effects of BPA on PRL release and explore its mechanism of action. The specific aims were to: 1) compare the potency of estradiol and BPA in stimulating PRL gene expression and release in vitro; 2) determine whether BPA increases PRL release in vivo; 3) examine if the in vivo estrogenic effects are mediated by PRL regulating factor from the posterior pituitary; and 4) examine if BPA regulates transcription through the estrogen response element (ERE). BPA increased PRL gene expression, release, and cell proliferation in anterior pituitary cells albeit at a 1000- to 5000-fold lower potency than estradiol. On the other hand, BPA had similar efficacy to estradiol in inducing hyperprolactinemia in estrogen-sensitive Fischer 344 (F344) rats; Sprague Dawley (SD) rats did not respond to BPA. Posterior pituitary cells from estradiol- or BPA-treated F344 rats strongly increased PRL gene expression upon coculture with GH3 cells stably transfected with a reporter gene. Similar to estradiol, BPA induced ERE activation in transiently transfected anterior and posterior pituitary cells. We conclude that: a) BPA mimics estradiol in inducing hyperprolactinemia in genetically predisposed rats; b) the in vivo action of estradiol and BPA in F344 rats is mediated, at least in part, by increasing PRL regulating factor activity in the posterior pituitary; c) BPA appears to regulate transcription through an ERE, suggesting that it binds to estrogen receptors in both the anterior and posterior pituitaries. The possibility that BPA and other xenoestrogens have adverse effects on the neuroendocrine axis in susceptible human subpopulations is discussed.

Animals↗
PubMed1994

Interactions among three proteins that specifically activate translation of the mitochondrial COX3 mRNA in Saccharomyces cerevisiae.

The PET54, PET122, and PET494 proteins, which are associated with the yeast inner mitochondrial membrane, specifically activate translation of the mitochondrially encoded COX3 mRNA. We used the two-hybrid system to test whether pairs of these proteins, when fused to either the GAL4 DNA-binding or transcriptional activating domain, can physically associate as measured by the expression of the GAL4-dependent reporter, lacZ. PET54 and PET122 interacted in this system, and an amino-terminally truncated PET494 fragment showed an interaction with PET54. We also detected functional interactions between PET54 and PET122 genetically: a pet54 missense substitution (Phe to Gly at position 244) that caused a severe respiratory defect was suppressed both by a missense substitution affecting PET122 (Gly to Val at position 211) and by overproduction of wild-type PET122. Both Gly and Ala, substituted at PET54 position 244, disrupted the two-hybrid interactions with PET122 and PET494. While Ala at PET54 position 244 caused only a modest respiratory phenotype alone, it caused a severe respiratory defect when combined with a cold-sensitive mitochondrial mutation affecting the COX3 mRNA 5' leader. This synthetic defect was suppressed by a missense substitution in PET122 and by overproduction of wild-type PET122, indicating functional interactions among PET54, PET122, and the mRNA. Taken together with previous work, these data suggest that a complex containing PET54, PET122, and PET494 mediates the interaction of the COX3 mRNA with mitochondrial ribosomes at the surface of the inner membrane.

Alleles↗
PubMed1993

Cooperativity during multiple phosphorylations catalyzed by rhodopsin kinase: supporting evidence using synthetic phosphopeptides.

Rhodopsin kinase is a key component in the shutdown of visual transduction. The phosphorylation of rhodopsin's C-terminus was evaluated using synthetic peptides derived from the last 12 amino acids (337-348) as substrates and their phosphorylated counterparts as inhibitors. It was found that synthetic peptides were phosphorylated at the serine residue corresponding to Ser-343 in the primary sequence of bovine rhodopsin. The phosphopeptides were prepared by incorporating into the peptide chain a trityl-protected serine derivative at the site destined to contain the phosphoryl group. The trityl group was selectively released with 20% (v/v) dichloroacetic acid; the free hydroxyl group was then phosphitylated with di-tert-butyl N,N-diethylphosphoramidite, and the resulting phosphite derivative was oxidized with m-chloroperoxybenzoic acid. The phosphopeptides were found to have a greater affinity for the kinase compared with their nonphosphorylated counterparts; for the peptides corresponding to residues 337-348 of rhodopsin the affinity increased in the order VSKTETSQVAPA < VSKTETS[PO3H2]QVAPA < VS[PO3H2]KTETS[PO3H2]QVAPA. The results are interpreted to support the cooperativity hypotheses proposed previously [Wilden, U., & Kühn, H. (1982) Biochemistry 21, 3014-3022; Aton, B. R., Litman, B. J., & Jackson, M. L. (1984) Biochemistry 23, 1737-1741].

Amino Acid Sequence↗
PubMed1992

Mechanistic studies on rhodopsin kinase. Light-dependent phosphorylation of C-terminal peptides of rhodopsin.

The phosphorylation of a synthetic peptide, corresponding to the C-terminal 11 amino acids of bovine rhodopsin (VII, residues 338-348), was studied under different conditions. The peptide was only phosphorylated in the presence of photoactivated rhodopsin. Using the same protocol, 12 other peptides, mapping in the rhodopsin C-terminal, were screened for their effectiveness as substrates for rhodopsin kinase. It was found that the peptides became poorer substrates with increasing length, and the best substrates comprised the most C-terminal 9-12 amino acids as opposed to other parts of the C-terminus. It was noted that the absence of the two-terminal residues Pro347 and Ala348 impaired peptide phosphorylation. The effect of the decay of metarhodopsin II on the phosphorylation of rhodopsin and the peptides was determined, and it was found that the rhodopsin and peptide phosphorylations decayed with half times of approximately 33 min and 28 min, respectively. The sites of phosphorylation on the peptides were determined and in all cases the phosphorylation was found to be predominantly on serine residues. Only the 11-residue peptide (VII, residues 338-348) contained significant threonine phosphorylation, which was about 25% that on serine residues. Cumulatively, the results suggest that Ser343 is the preferred site of phosphorylation in vitro. The reason for the poor substrate effectiveness of the larger peptides was examined by competitive experiments in which it was shown that a poorly phosphorylated larger peptide successfully inhibited the phosphorylation of a 'good' peptide substrate. The studies above support a mechanism for rhodopsin kinase that we have termed the 'kinase-activation hypothesis'. This requires that the kinase exists in an inactive form and is activated only after binding to photoactivated rhodopsin.

Amino Acid Sequence↗
PubMed1985

Strikebound: a dispute involving hospital linen.

On September 6, 1984, industrial bans were placed on the movement of linen within The Royal Melbourne Hospital. Initially, linen was stored in ward areas and, later, on hospital balconies. The dispute was not settled for 14 days; by this time, 25 beds and the Emergency Department had been closed. Stockpiled "soiled" and "infectious" linen bags posed a major safety risk. Once the bans were lifted, linen was removed and laundered under supervision, according to a carefully planned programme, in order to minimize the exposure of patients and staff members to potential cross-infection. Recommendations are made to cover infection control aspects associated with industrial disputes of this nature.

Australia↗