Activation of multiple signal transduction pathways by glucocorticoids: protection of ovarian follicular cells against apoptosis

We have recently demonstrated that glucocorticoids protect against serum-deprivation, cAMP-, TNFalpha-, and p53-induced apoptosis in ovarian follicular cells involved in up-regulation of Bcl-2. We demonstrated that dexamethasone, which enhances steroidogenesis by up-regulation of the p450scc enzyme system, stimulates the MAPK cascade by phosphorylation of ERK1, ERK2 as well as by Akt phosphorylation within 1-5min with no effect on p38 MAPK phosphorylation. Moreover, glucocorticoids enhance expression of connexin 43, formation of gap junctions, expression of cadherins, and formation of adherence junctions within 24h of hormone stimulation of ovarian granulosa cells. It is suggested that the protective effects of glucocorticoids against apoptosis are mediated by both genomic and non-genomic mechanisms. Moreover, for the first time we show that protein phosphorylation, cell-cell contact, and intracellular communication are important mediators in glucocorticoid protection against apoptosis in ovarian follicular cells.     

p19ARF-independent induction of p53 and cell cycle arrest by Raf in murine keratinocytes

In tumorigenesis of the skin, activated Ras co-operates with mutations that inactivate the tumour suppressor p53, but the molecular basis for this co-operation remains unresolved. Here we show that activation of the Raf/MAP kinase pathway in primary mouse keratinocytes leads to a p53 and p21^Cip1-dependent cycle arrest and to terminal differentiation. Raf activation in keratinocytes lacking p53 or p21^Cip1 genes leads to expression of differentiation markers, but the cells do not cease to proliferate. Thus, loss of p53 or p21^Cip1 function is necessary to disable growth-inhibitory Raf/MAP kinase signalling. Activation of oncogenes, including Ras, has been reported to stabilize and activate p53 via induction of the tumour suppressor p19^ARF. However, the response to Raf in p19^ARF–/– keratinocytes was indistinguishable from wild-type controls. Thus, p19^ARF is not essential for Raf-induced p53 induction and cell cycle arrest in keratinocytes, indicating that oncogenes engage p53 activity via multiple mechanisms.     

Mechanistic studies of catechol generation from secondary quinone amines relevant to indole formation and tyrosinase activation

The biological significance of the spontaneous cyclization and redox reactions of ortho-quinone amines is that these appear to be the mechanism of formation of the indolic components of melanin and are also involved in the autoactivation of tyrosinase. We have previously shown that activation of tyrosinase is prevented by the formation of a cyclic betaine from a tertiary amine analogue. Evidence is presented to show that cyclization of ortho-quinones by Michael addition also occurs in the oxidation of secondary catecholamines. Three varieties of cyclic product have been detected and their formation is influenced by the nature of the N-substituent. Five-membered betaine rings form directly and, although six- and seven-membered rings also form, a transient spiro isomer of the ortho-quinone was in some cases detected as an intermediate. The heterocyclic products formed as betaines undergo redox exchange with residual quinone to form the corresponding aminochromes. We have established the kinetic constants of these reactions, either directly by pulse radiolysis measurements or by inference using a computer model of the reaction pathway to fit the observed data. To investigate the potential biological applications of this chemistry the system was also examined by tyrosinase-catalysed oxidation of the catecholamine substrates in which there is re-oxidation of the catechol formed by the redox exchange reaction and enables measurement of oxygen utilization stoichiometry. We show that the redox exchange reaction is unaffected by side-chain modification whereas cyclization is dependent on both electronic and steric factors. In the light of these studies we conclude that the failure of tertiary amine-derived betaines to undergo redox exchange, and thus block in vitro activation of tyrosinase, is due to the absence of a second exchangeable proton.     

Reactions of e(-)(aq), CO(2)(*)(-), HO(*), O(2)(*)(-) and O(2)((1)delta(g)) with a dendro[60]fullerene and C(60)[C(COOH)(2)](n) (n = 2-6)

Using pulse radiolysis and laser flash photolysis, we have investigated the reactions of the deleterious species, e(-)(aq), HO&z.rad;, O(2)(*)(-) and O(2)((1)Delta(g)) with 10 water-soluble cyclopropyl-fused C(60) derivatives including a mono-adduct dendro[60]fullerene (d) and C(60) derivatives based on C(60)[C(COOH)(2)](n=2-6), some of which are known to be neuroprotective in vivo. The rate constants for reactions of e(-)(aq) and HO&z.rad; lie in the range 0.5-3.3 x 10(10) M(-1) s(-1). The d and bis-adduct monoanion radicals display sharp absorption peaks around 1000 nm (epsilon = 7 000-11 500 M(-1) cm(-1)); the anions of the tris-, tetra-, and penta-adduct derivatives have broader, weaker absorptions. The monohydroxylated radicals have their most intense absorption maxima around 390-440 nm (epsilon = 1000-3000 M(-1) cm(-1)). The anion and hydroxylated radical absorption spectra display a blue-shift as the number of addends increases. The radical anions react with oxygen (k approximately 10(7)-10(9) M(-1) s(-1)). The reaction of O(2)(*)(-) with the C(60) derivatives does not occur via an electron transfer. The rate constants for singlet oxygen reaction with the dendrofullerene and eee-derivative in D(2)O at pH 7.4 are k approximately 7 x 10(7) and approximately 2 x 10(7) M(-1) s(-1) respectively, in contrast to approximately 1.2 x 10(5) M(-1) s(-1) for the reaction with C(60) in C(6)D(6). The large acceleration of the rates for electron reduction and singlet oxygen reactions in water is due to a solvophobic process.     

Use of isolated digestive-gland cells in the study of biochemical and physiological processes in gastropod molluscs

We describe a method for preparation and maintenance of isolated digestive-gland cells in the abalone, Haliotis kamtschatkana. Viability of the isolated cells was confirmed by the fact that 18 h after preparation the cells exhibited less than 5% staining with trypan blue and actively synthesized glycogen following the addition of glucose substrate. Use of the method in a 15-month study of metabolic activity of the digestive gland of H. kamtschatkana showed significant differences in oxygen consumption of isolated-cell preparations correlated with seasonal differences in somatic and gametogenetic growth, and with relative size of the digestive gland.     

Urea production and transport in teleost fishes

Teleosts appear to have retained the genes for the urea cycle enzymes. A few species express the full complement of enzymes and are ureotelic (e.g., Lake Magadi tilapia) or ammoniotelic (e.g., largemouth bass), whereas most species have low or non-detectable enzyme activities in liver tissue and excrete little urea (e.g., adult rainbow trout). It was surprising, therefore, to find the expression of four urea cycle enzymes during early life stages of rainbow trout. The urea cycle may play a role in ammonia detoxification during a critical time of development. Exposure to alkaline water (pH 9.0-9.5) or NH4Cl (0.2 mmol/l) increased urea excretion by several-fold in trout embryos, free embryos and alevin. Urea transport is either by passive simple diffusion or via carried-mediated transport proteins. Molecular studies have revealed that a specialised urea transport protein is present in kidney tissue of elasmobranchs, similar to the facilitated urea transporter found in the mammalian inner medulla of the kidney.     

A Mutation in the Mitochondrial Fission Gene Dnm1l Leads to Cardiomyopathy

Mutations in a number of genes have been linked to inherited dilated cardiomyopathy (DCM). However, such mutations account for only a small proportion of the clinical cases emphasising the need for alternative discovery approaches to uncovering novel pathogenic mutations in hitherto unidentified pathways. Accordingly, as part of a large-scale N-ethyl-N-nitrosourea mutagenesis screen, we identified a mouse mutant, Python, which develops DCM. We demonstrate that the Python phenotype is attributable to a dominant fully penetrant mutation in the dynamin-1-like (Dnm1l) gene, which has been shown to be critical for mitochondrial fission. The C452F mutation is in a highly conserved region of the M domain of Dnm1l that alters protein interactions in a yeast two-hybrid system, suggesting that the mutation might alter intramolecular interactions within the Dnm1l monomer. Heterozygous Python fibroblasts exhibit abnormal mitochondria and peroxisomes. Homozygosity for the mutation results in the death of embryos midway though gestation. Heterozygous Python hearts show reduced levels of mitochondria enzyme complexes and suffer from cardiac ATP depletion. The resulting energy deficiency may contribute to cardiomyopathy. This is the first demonstration that a defect in a gene involved in mitochondrial remodelling can result in cardiomyopathy, showing that the function of this gene is needed for the maintenance of normal cellular function in a relatively tissue-specific manner. This disease model attests to the importance of mitochondrial remodelling in the heart; similar defects might underlie human heart muscle disease.