GTP-dependent segregation of H-ras from lipid rafts is required for biological activity

Different sites of plasma membrane attachment may underlie functional differences between isoforms of Ras. Here we show that palmitoylation and farnesylation targets H-ras to lipid rafts and caveolae, but that the interaction of H-ras with these membrane subdomains is dynamic. GTP-loading redistributes H-ras from rafts into bulk plasma membrane by a mechanism that requires the adjacent hypervariable region of H-ras. Release of H-ras-GTP from rafts is necessary for efficient activation of Raf. By contrast, K-ras is located outside rafts irrespective of bound nucleotide. Our studies identify a novel protein determinant that is required for H-ras function, and show that the GTP/GDP state of H-ras determines its lateral segregation on the plasma membrane.

Heritability and gene-environment interactions for melanocytic nevus density examined in a U.K. adolescent twin study

Risk factors for melanoma include environmental (particularly ultraviolet exposure) and genetic factors. In rare families, susceptibility to melanoma is determined by high penetrance mutations in the genes CDKN2A or CDK4, with more common, less penetrant genes also postulated. A further, potent risk factor for melanoma is the presence of large numbers of melanocytic nevi so that genes controlling nevus phenotype could be such melanoma susceptibility genes. A large Australian study involving twins aged 12 y of predominantly U.K. ancestry showed strong evidence for genetic influence on nevus number and density. We carried out essentially the same study in the U.K. to gain insight into gene-environment interactions for nevi. One hundred and three monozygous (MZ) and 118 dizygous (DZ) twin pairs aged 10-18 y were examined in Yorkshire and Surrey, U.K. Nevus counts were, on average, higher in boys (mean = 98.6) than girls (83.8) (p = 0.009) and higher in Australia (110.4) than in the U.K. (79.2, adjusted to age 12 y, p < 0.0001), and nevus densities were higher on sun-exposed sites (92 per m(2)) than sun-protected sites (58 per m(2)) (p < 0.0001). Correlations in sex and age adjusted nevus density were higher in MZ pairs (0.94, 95% CI 0.92-0.96) than in DZ pairs (0.61, 95%CI 0.49-0.72), were notably similar to those of the Australian study (MZ = 0.94, DZ = 0.60), and were consistent with high heritability (65% in the U.K., 68% in Australia). We conclude that emergence of nevi in adolescents is under strong genetic control, whereas environmental exposures affect the mean number of nevi.

Derivation of the probability distribution function for the local density of states of a disordered quantum wire via the replica trick and supersymmetry

We consider the statistical properties of the local density of states of a one-dimensional Dirac equation in the presence of various types of disorder with Gaussian white-noise distribution. It is shown how either the replica trick or supersymmetry can be used to calculate exactly all the moments of the local density of states.' Careful attention is paid to how the results change if the local density of states is averaged over atomic length scales. For both the replica trick and supersymmetry the problem is reduced to finding the ground state of a zero-dimensional Hamiltonian which is written solely in terms of a pair of coupled spins which are elements of u(1, 1). This ground state is explicitly found for the particular case of the Dirac equation corresponding to an infinite metallic quantum wire with a single conduction channel. The calculated moments of the local density of states agree with those found previously by Al'tshuler and Prigodin [Sov. Phys. JETP 68 (1989) 198] using a technique based on recursion relations for Feynman diagrams. (C) 2001 Elsevier Science B.V. All rights reserved.

Time-dependent master equation simulation of complex elementary reactions in combustion: Application to the reaction of (CH2)-C-1 with C2H2 from 300-2000 K

Computational simulations of the title reaction are presented, covering a temperature range from 300 to 2000 K. At lower temperatures we find that initial formation of the cyclopropene complex by addition of methylene to acetylene is irreversible, as is the stabilisation process via collisional energy transfer. Product branching between propargyl and the stable isomers is predicted at 300 K as a function of pressure for the first time. At intermediate temperatures (1200 K), complex temporal evolution involving multiple steady states begins to emerge. At high temperatures (2000 K) the timescale for subsequent unimolecular decay of thermalized intermediates begins to impinge on the timescale for reaction of methylene, such that the rate of formation of propargyl product does not admit a simple analysis in terms of a single time-independent rate constant until the methylene supply becomes depleted. Likewise, at the elevated temperatures the thermalized intermediates cannot be regarded as irreversible product channels. Our solution algorithm involves spectral propagation of a symmetrised version of the discretized master equation matrix, and is implemented in a high precision environment which makes hitherto unachievable low-temperature modelling a reality.

The plant isoflavenoid genistein activates p53 and Chk2 in an ATM-dependent manner

Genistein is an isoflavenoid that is abundant in soy beans. Genistein has been reported to have a wide range of biological activities and to play a role in the diminished incidence of breast cancer in populations that consume a soy-rich diet. Genistein was originally identified as an inhibitor of tyrosine kinases; however, it also inhibits topoisomerase II by stabilizing the covalent DNA cleavage complex, an event predicted to cause DNA damage. The topoisomerase II inhibitor etoposide acts in a similar manner. Here we show that genistein induces the up-regulation of p53 protein, phosphorylation of p53 at serine 15, activation of the sequence-specific DNA binding properties of p53, and phosphorylation of the hCds1/Chk2 protein kinase at threonine 68. Phosphorylation and activation of p53 and phosphorylation of Chk2 were not observed in ATM-deficient cells. In contrast, the topoisomerase II inhibitor etoposide induced phosphorylation of p53 and Chk2 in ATM-positive and ATM-deficient cells. In addition, genistein-treated ATM-deficient cells were significantly more susceptible to genistein-induced killing than were ATM-positive cells. Together our data suggest that ATM is required for activation of a DNA damage-induced pathway that activates p53 and Chk2 in response to genistein.

Stimulation of protein kinase C-dependent and -independent signaling pathways by bistratene A in intestinal epithelial cells

The marine toxin bistratene A (BisA) potently induces cytostasis and differentiation in a variety of systems. Evidence that BisA is a selective activator of protein kinase C (PKC) delta implicates PKC delta signaling in the negative growth-regulatory effects of this agent. The current study further investigates the signaling pathways activated by BisA by comparing its effects with those of the PKC agonist phorbol 12-myristate 13-acetate (PMA) in the IEC-18 intestinal crypt cell line. Both BisA and PMA induced cell cycle arrest in these cells, albeit with different kinetics. While BisA produced sustained cell cycle arrest in G(o)/G(1) and G(2)/M, the effects of PMA were transient and involved mainly a G(o)/G(1), blockade. BisA also produced apoptosis in a proportion of the population, an effect not seen with PMA. Both agents induced membrane translocation/activation of PKC, with BisA translocating only PKC delta and PMA translocating PKC alpha, delta, and epsilon in these cells. Notably, while depletion of PKC alpha, delta, and epsilon abrogated the cell cycle-specific effects of PMA in IEC-18 cells, the absence of these PKC isozymes failed to inhibit BisA-induced G(o)/G(1), and G(2)/M arrest or apoptosis. The cell cycle inhibitory and apoptotic effects of BisA, therefore, appear to be PKC-independent in IEG-18 cells. On the other hand, BisA and PMA both promoted PKC-dependent activation of Erk 1 and 2 in this system. Thus, intestinal epithelial cells respond to BisA through activation of at least two signaling pathways: a PKC delta -dependent pathway, which leads to activation of mitogen-activated protein kinase and possibly cytostasis in the appropriate context, and a PKC-independent pathway, which induces both cell cycle arrest in G(o)/G(1) and G(2)/M and apoptosis through as yet unknown mechanisms. (C) 2001 Elsevier Science Inc. All rights reserved.

Cloning of a catalytic subunit of cAMP-dependent protein kinase from the honeybee (Apis mellifera) and its localization in the brain

In the honeybee the cAMP-dependent signal transduction cascade has been implicated in processes underlying learning and memory, The cAMP-dependent protein kinase (PKA) is the major mediator of cAMP action. To characterize the PKA system in the honeybee brain we cloned a homologue of a PKA catalytic subunit from the honeybee,The deduced amino acid sequence shows 80-94% identity with catalytic subunits of PKA from Drosophila melanogaster, Aplysia californica and mammals. The corresponding gene is predominantly expressed in the mushroom bodies, a structure that is involved in learning and memory processes. However, expression can also be found in the antennal and optic lobes,The level of expression varies within all three neuropiles.

Expression and purification of enzymatically active recombinant RNA-dependent RNA polymerase (NS5) of the flavivirus Kunjin

The NS5 protein of the flavivirus Kunjin (KUN) contains conserved sequence motifs characteristic of RNA-dependent RNA polymerase (RdRp) activity. To investigate this activity in vitro, recombinant NS5 proteins with C-terminal (NS5CHis) and N-terminal (NS5NHis) hexahistidine tags were produced in baculovirus-infected insect cells and purified to near homogeneity by nickel affinity chromatography. Purified NS5CHis exhibited RdRp activity with both specific (9 kb KUN replicon) and non-specific (8.3 kb Semliki Forest virus replicon) RNA templates; this activity did not require the presence of additional viral and/or cellular cofactors. RdRp activity of purified NS5NHis protein was reduced in comparison to NS5CHis, while purified NS5NHis incorporating a GDD -> GVD mutation within the polymerase active site (NS5GVD) lacked RdRp activity. RNase A digestion of the RdRp reaction products indicated that they were double-stranded and of a similar size to the KUN replicative form produced in Vero cells, thus demonstrating that the KUN NS5 protein has an intrinsic, albeit low and non-specific RdRp activity in vitro, similar to that reported for recombinant RdRp of other flaviviruses. However, in contrast to RNA polymerases of other Flavivirus species, purified KUN NS5 polymerase produced a single, full-length replicon RNA product, thus demonstrating efficient processivity. (C) 2001 Elsevier Science B.V. All rights reserved.

'Neighbourhood' size, dispersal and density estimates in the prickly forest skink (Gnypetoscincus queenslandiae) using individual genetic and demographic methods

Dispersal, or the amount of dispersion between an individual's birthplace and that of its offspring, is of great importance in population biology, behavioural ecology and conservation, however, obtaining direct estimates from field data on natural populations can be problematic. The prickly forest skink, Gnypetoscincus queenslandiae, is a rainforest endemic skink from the wet tropics of Australia. Because of its log-dwelling habits and lack of definite nesting sites, a demographic estimate of dispersal distance is difficult to obtain. Neighbourhood size, defined as 4 piD sigma (2) (where D is the population density and sigma (2) the mean axial squared parent-offspring dispersal rate), dispersal and density were estimated directly and indirectly for this species using mark-recapture and microsatellite data, respectively, on lizards captured at a local geographical scale of 3 ha. Mark-recapture data gave a dispersal rate of 843 m(2)/generation (assuming a generation time of 6.5 years), a time-scaled density of 13 635 individuals * generation/km(2) and, hence, a neighbourhood size of 144 individuals. A genetic method based on the multilocus (10 loci) microsatellite genotypes of individuals and their geographical location indicated that there is a significant isolation by distance pattern, and gave a neighbourhood size of 69 individuals, with a 95% confidence interval between 48 and 184. This translates into a dispersal rate of 404 m(2)/generation when using the mark-recapture density estimation, or an estimate of time-scaled population density of 6520 individuals * generation/km(2) when using the mark-recapture dispersal rate estimate. The relationship between the two categories of neighbourhood size, dispersal and density estimates and reasons for any disparities are discussed.

Self-reported calcium intake and bone mineral content in children and adolescents

Objective: We examined the relationship between self-reported calcium (Cal intake and bone mineral content (BMC) in children and adolescents. We hypothesized that an expression of Ca adjusted for energy intake (El), i.e., Ca density, would be a better predictor of BMC than unadjusted Ca because of underreporting of EI. Methods: Data were obtained on dietary intakes (repeated 24-hour recalls) and BMC (by DEXA) in a cross-section of 227 children aged 8 to 17 years. Bivariate and multivariate analyses were used to examine die relationship between Ca, Ca density, and the dependent variables total body BMC and lumbar spine BMC. Covariates included were height, weight, bone area, maturity age, activity score and El. Results: Reported El compared to estimated basal metabolic rate suggested underreporting of El. Total body and lumbar spine BMC were significantly associated with El, but not Ca or Ca density, in bivariate analyses. After controlling for size and maturity, multiple linear regression analysis revealed unadjusted Ca to be a predictor of BMC in males in the total body (p = 0.08) and lumbar spine (p = 0.01). Unadjusted Ca was not a predictor of BMC at either site in females. Ca density was not a better predictor of BMC at either site in males or females. Conclusions: The relationship observed in male adolescents in this study between Ca intake and BMC is similar to that seen in clinical trials. Ca density did not enable us to see a relationship between Ca intake and BMC in females, which may reflect systematic reporting errors or that diet is not a limiting factor in this group of healthy adolescents.

ENCORE: The effect of nutrient enrichment on coral reefs. Synthesis of results and conclusions

Coral reef degradation resulting from nutrient enrichment of coastal waters is of increasing global concern. Although effects of nutrients on coral reef organisms have been demonstrated in the laboratory, there is little direct evidence of nutrient effects on coral reef biota in situ. The ENCORE experiment investigated responses of coral reef organisms and processes to controlled additions of dissolved inorganic nitrogen (N) and/or phosphorus (P) on an offshore reef(One Tree Island) at the southern end of the Great Barrier Reef, Australia. A multi-disciplinary team assessed a variety of factors focusing on nutrient dynamics and biotic responses. A controlled and replicated experiment was conducted over two years using twelve small patch reefs ponded at low tide by a coral rim. Treatments included three control reefs (no nutrient addition) and three + N reefs (NH4Cl added), three + P reefs (KH2PO4 added), and three + N + P reefs. Nutrients were added as pulses at each low tide (ca twice per day) by remotely operated units. There were two phases of nutrient additions. During the initial, low-loading phase of the experiment nutrient pulses (mean dose = 11.5 muM NH4+; 2.3 muM PO4-3) rapidly declined, reaching near-background levels (mean = 0.9 muM NH4+; 0.5 muM PO4-3) within 2-3 h. A variety of biotic processes, assessed over a year during this initial nutrient loading phase, were not significantly affected, with the exception of coral reproduction, which was affected in all nutrient treatments. In Acropora longicyathus and A. aspera, fewer successfully developed embryos were formed, and in A. longicyathus fertilization rates and lipid levels decreased. In the second, high-loading, phase of ENCORE an increased nutrient dosage (mean dose = 36.2 muM NH4+; 5.1 muM PO4-3 declining to means of 11.3 muM NH4+ and 2.4 muM PO4-3 at the end of low tide) was used for a further year, and a variety of significant biotic responses occurred. Encrusting algae incorporated virtually none of the added nutrients. Organisms containing endosymbiotic zooxanthellae (corals and giant clams) assimilated dissolved nutrients rapidly and were responsive to added nutrients. Coral mortality, not detected during the initial low-loading phase, became evident with increased nutrient dosage, particularly in Pocillopora damicornis. Nitrogen additions stunted coral growth, and phosphorus additions had a variable effect. Coral calcification rate and linear extension increased in the presence of added phosphorus but skeletal density was reduced, making corals more susceptible to breakage. Settlement of all coral larvae was reduced in nitrogen treatments, yet settlement of larvae from brooded species was enhanced in phosphorus treatments. Recruitment of stomatopods, benthic crustaceans living in coral rubble, was reduced in nitrogen and nitrogen plus phosphorus treatments. Grazing rates and reproductive effort of various fish species were not affected by the nutrient treatments. Microbial nitrogen transformations in sediments,were responsive to nutrient loading with nitrogen fixation significantly increased in phosphorus treatments and denitrification increased in all treatments to which nitrogen had been added. Rates of bioerosion and grazing showed no significant effects of added nutrients, ENCORE has shown that reef organisms and processes investigated ill situ were impacted by elevated nutrients. Impacts mere dependent on dose level, whether nitrogen and/or phosphorus mere elevated and were often species-specific. The impacts were generally sub-lethal and subtle and the treated reefs at the end of the experiment mere visually similar to control reefs. Rapid nutrient uptake indicates that nutrient concentrations alone are not adequate to assess nutrient condition of reefs. Sensitive and quantifiable biological indicators need to be developed for coral reef ecosystems. The potential bioindicators identified in ENCORE should be tested in future research on coral reef/nutrient interactions. Synergistic and cumulative effects of elevated nutrients and other environmental parameters, comparative studies of intact vs. disturbed reefs, offshore vs, inshore reefs, or the ability of a nutrient-stressed reef to respond to natural disturbances require elucidation. An expanded understanding of coral reef responses to anthropogenic impacts is necessary, particularly regarding the subtle, sub-lethal effects detected in the ENCORE studies. (C) 2001 Published by Elsevier Science Ltd.

Horizontal transmission of entomopathogenic fungi by the diamondback moth

The relative potential of the pathogenic fungi Beauveria bassiana and Zoophthora radicans for use as autodisseminated biological control agents of the diamondback moth (Plutella xylostella) was compared. The LC50 of B. bassiana conidia to third instar larvae was 499 conidia/mm(2) of leaf surface and individual cadavers of mycosed fourth instar larvae yielded a mean of 67.5 X 10(6) (+/- 7.5 x 10(6)) conidia. All concentrations of B. bassiana tested in inoculation chambers (0.24, 2.4, and 6.2 mug/mm(2)) induced 100% mortality in adult male moths within 7 days. The times to death and sporulation were concentration and exposure duration dependent. A standard procedure for inoculating male moths resulted in > 85% mortality from Z. radicans and > 93% mortality from B. bassiana. Pairing of inoculated males with clean moths of both sexes yielded higher rates of passive transmission of B. bassiana than Z. radicans, but there was no evidence for sexual transmission of either pathogen. Similarly, B. bassiana was more effectively transmitted from inoculated male moths to larvae foraging on whole plants. Single sporulating cadavers producing B. bassiana or Z. radicans conidia placed on plants infested with larvae resulted in a similar rate of transmission for both pathogens. However, an increase of the density of sporulating cadavers from one to three/plant increased Z. radicans transmission (greater than fourfold) but had no effect on B. bassiana transmission. Simultaneous inoculations of larvae with conidia of both fungi reduced the mortality induced by each pathogen, the reduction being most acute for B. bassiana-induced mortality. Inoculation of adults with both fungi showed that, at concentrations required for effective passive transmission to larvae, B. bassiana severely inhibited Z. radicans mycosis in adults. (C) 2001 Academic Press.

An ATP-sensitive K+ conductance in dissociated neurones from adult rat intracardiac ganglia

1. An ATP-sensitive K+ (K-ATP) conductance has been identified using the perforated patch recording configuration in a population (52%) of dissociated neurones from adult rat intracardiac ganglia. The presence of the sulphonylurea receptor in approximately half of the intracardiac neurones was confirmed by labelling with fluorescent glibenclamide-BODIPY. 2. Under current clamp conditions in physiological solutions, leveromakalim (10 muM) evoked a hyperpolarization, which was inhibited by the sulphonylurea drugs glibenclamide and tolbutamide. 3. Under voltage clamp conditions in symmetrical (140 mM) K+ solutions, hath application of levcromakalim evoked an inward current with a density of similar to8 pA pF(-1) at -50 mV and a slope conductance of similar to9 nS, which reversed close to the potassium equilibrium potential (E-K). Cell dialysis with an ATP-free intracellular solution also evoked an inward current, which was inhibited by tolbutamide. 4. Bath application of either glibenclamide (10 muM) or tolbutamide (100 muM) depolarized adult intracardiac neurones by 3-5 mV, suggesting that a K-ATP conductance is activated under resting conditions and contributes to the resting membrane potential. 5. Activation of a membrane current by levcromakalim leas concentration dependent, with an EC50 of 1.6 muM. Inhibition of the levcromakalim-activated current by glibenclamide leas also concentration dependent, with an IC50 of 55 nM. 6. Metabolic inhibition with 2,4-dinitrophenol and iodoacetic acid or superfusion with hypoxic solution (P-O2 similar to 16 mmHg) also activated a membrane current. These currents exhibited similar I-P characteristics to the levcroinakalim-induced current and were inhibited by glibenclamide. 7. Activation of K-ATP channels in mammalian intracardiac neurones may contribute to changes in neural regulation of the mature heart and. cardiac function during ischaemia-reperfusion.