Ras isoform abundance and signalling in human cancer cell lines.

The ubiquitously expressed major Ras isoforms: H-, K- and N-Ras, are highly conserved, yet exhibit different biological outputs. We have compared the relative efficiencies with which epidermal or hepatocyte growth factor activates Ras isoforms and the requirement for specific isoforms in the activation of downstream pathways. We find that the relative coupling efficiencies to each Ras isoform are conserved between stimuli. Furthermore, in both cases, inhibition of receptor endocytosis led to reduced N- and H-Ras activation, but K-Ras was unaffected. Acute knockdown of each isoform with siRNA allows endogenous Ras isoform function and abundance to be probed. This revealed that there is significant variation in the contribution of individual isoforms to total Ras across a panel of cancer cell lines although typically K> or =N>>H. Intriguingly, cancer cell lines where a significant fraction of endogenous Ras is oncogenically mutated showed attenuated activation of canonical Ras effector pathways. We profiled the contribution of each Ras isoform to the total Ras pool allowing interpretation of the effect of isoform-specific knockdown on signalling outcomes. In contrast to previous studies indicating preferential coupling of isoforms to Raf and PtdIns-3-kinase pathways, we find that endogenous Ras isoforms show no specific coupling to these major Ras pathways.

Ras proteins: paradigms for compartmentalised and isoform-specific signalling.

Ras GTPases mediate a wide variety of cellular processes by converting a multitude of extracellular stimuli into specific biological responses including proliferation, differentiation and survival. In mammalian cells, three ras genes encode four Ras isoforms (H-Ras, K-Ras4A, K-Ras4B and N-Ras) that are highly homologous but functionally distinct. Differences between the isoforms, including their post-translational modifications and intracellular sorting, mean that Ras has emerged as an important model system of compartmentalised signalling and membrane biology. Ras isoforms in different subcellular locations are proposed to recruit distinct upstream and downstream accessory proteins and activate multiple signalling pathways. Here, we summarise data relating to isoform-specific signalling, its role in disease and the mechanisms promoting compartmentalised signalling. Further understanding of this field will reveal the role of Ras signalling in development, cellular homeostasis and cancer and may suggest new therapeutic approaches.

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.

Detection of thiol modification following generation of reactive nitrogen species: analysis of synaptic vesicle proteins.

S-nitrosylation is an important means of regulating the activity of proteins. We have developed a method which allows unbiased identification of thiol modified proteins within a complex mixture following NO generation, by taking advantage of the fact that prior nitrosylation will block subsequent modification of cysteine residues with 1-biotinamido-4-[4'-(maleimidomethyl)-cyclohexane-carboxamid o] butane (biotin-BMCC). Thiol modified proteins are reduced in intensity when revealed by blotting and overlay with avidin-horseradish peroxidase. In the case of a purified synaptic vesicle fraction we observe a high degree of enrichment of specific biotinylated proteins relative to homogenate. We find that thiol modification of proteins in the presence of NO donors is widespread, occurring in the majority of proteins that will react with biotin-BMCC. In a further development of this technique we have depleted the biotinylated proteins from solubilised synaptic vesicles using avidin-agarose and analysed the supernatants with a panel of antibodies. This has allowed us to identify SNARE proteins (soluble NSF attachment protein receptors) as potential targets for S-nitrosylation.

H-ras but not K-ras traffics to the plasma membrane through the exocytic pathway.

Ras proteins must be localized to the inner surface of the plasma membrane to be biologically active. The motifs that effect Ras plasma membrane targeting consist of a C-terminal CAAX motif plus a second signal comprising palmitoylation of adjacent cysteine residues or the presence of a polybasic domain. In this study, we examined how Ras proteins access the cell surface after processing of the CAAX motif is completed in the endoplasmic reticulum (ER). We show that palmitoylated CAAX proteins, in addition to being localized at the plasma membrane, are found throughout the exocytic pathway and accumulate in the Golgi region when cells are incubated at 15 degrees C. In contrast, polybasic CAAX proteins are found only at the cell surface and not in the exocytic pathway. CAAX proteins which lack a second signal for plasma membrane targeting accumulate in the ER and Golgi. Brefeldin A (BFA) significantly inhibits the plasma membrane accumulation of newly synthesized, palmitoylated CAAX proteins without inhibiting their palmitoylation. BFA has no effect on the trafficking of polybasic CAAX proteins. We conclude that H-ras and K-ras traffic to the cell surface through different routes and that the polybasic domain is a sorting signal diverting K-Ras out of the classical exocytic pathway proximal to the Golgi. Farnesylated Ras proteins that lack a polybasic domain reach the Golgi but require palmitoylation in order to traffic further to the cell surface. These data also indicate that a Ras palmitoyltransferase is present in an early compartment of the exocytic pathway.

Glutamate uptake occurs at an early stage of synaptic vesicle recycling.

Rapid membrane recycling in nerve terminals is required to maintain rapid synaptic transmission. Following the fusion of synaptic vesicles with synaptic plasma membranes, recycling can occur via clathrin-coated vesicles (CCVs) [1-3]. The fate of these vesicles is uncertain: they could simply uncoat and acquire other proteins from the cytosol to regenerate synaptic vesicles or they may fuse with endosomal structures from which synaptic vesicles could then bud. We have purified both CCVs and synaptic vesicles from rat brain, and measured the ability of these vesicle fractions to take up the excitatory neurotransmitter glutamic acid. We found that the normalized levels of glutamate uptake by the two types of vesicle were very similar. For each vesicle fraction, uptake required ATP and Cl- and could be fully inhibited by the specific vacuolar proton pump (v-ATPase) inhibitor concanamycin. We suggest that this ability to refill vesicles with neurotransmitter at the earliest intermediate on the recycling pathway - the CCV - may allow uncoated vesicles to immediately enter the releasable pool without sacrificing the quantal nature of neurotransmitter release.

Mortality among New Zealand Maori and non-Maori Mormons.

Mortality rates for New Zealand Maori and non-Maori Mormons in the period 1970-77 have been compared with those for non-Mormons in the census year 1976 to measure the impact of the Mormon lifestyle on differences in mortality between Maoris and non-Maoris. Maori mortality was much lower among Mormons than non-Mormons suggesting that environmental, rather than genetic factors, play a predominant role in the relatively high overall Maori mortality. However the prevalence of smoking among Maori Mormons was not much lower than for the general Maori population. Reasons for the relative mortality advantage of Maori Mormons were therefore not clear, although attitudes to health and health services utilization, and the influence of strong social support networks, might be involved. Paradoxically, non-Maori Mormon mortality rates were similar to those for non-Mormons. A combination of factors appeared to contribute to this finding including the fact that 26% of non-Maori Mormons were of Pacific Island origin, non-Maori Mormons were of lower socioeconomic status than other non-Maoris, and part Maoris probably constitute a high, but unknown, proportion of Mormons classified as non-Maoris.

Are low cholesterol values associated with excess mortality?

The relation between cholesterol concentration and mortality was studied prospectively over 17 years in 630 New Zealand Maoris aged 25-74. The dead or alive state of each person was determined in 1981. The causes of death were divided into three categories: cancer, cardiovascular disease, and "other." Using univariate and both linear and non-linear multivariate methods of analysis for survivorship data, significant inverse relations with serum cholesterol were found for total mortality in women, for mortality from cancer in men and women, and for other causes of mortality in both men and women. The inverse and non-linear association with total mortality in women remained significant when deaths in the first five years of follow up were excluded. This suggests that the association was not explained by undetected illness causing low cholesterol concentrations at the time of initial examination.

Kava, alcohol and tobacco consumption among Tongans with urbanization.

The prevalence of kava (Piper methysticum), alcohol and tobacco consumption in Nuku'alofa (urban) and Foa (rural) are described. Current kava consumption was males 48% and females 1%. Prevalence was significantly higher among rural males. Current alcohol consumption was almost exclusive to the urban population and predominantly male, but only 2 (1%) rural males were current alcohol consumers. 169 (84.0%) of the rural males were irregularly or had ceased consuming alcohol. Tobacco consumption also showed a significant male predominance. There was a significantly higher total tobacco consumption in the urban population. Concordance of the three habits was evident among males with 27 concordant positive (expected = 5.98) and 46 concordant negative (expected = 6.60). It appears that kava, a traditional Pacific beverage may have lost ground to alcohol as urban Tongans adopt a more cosmopolitan life style. Kava, if proved relatively harmless, may be promoted as a less unhealthy alternative to tobacco and alcohol.

Age-associated change in QRS axis: intrinsic or extrinsic ageing?

Human ageing is most usefully conceptualised as an interaction between intrinsic and extrinsic processes. Comparison of age-associated trends in populations living under different environmental conditions provides a method of recognising the effects of extrinsic influences. This paper compares the age-associated trend in mean frontal plane QRS axis in the male adult population of Pukapuka with the trend observed in an age-matched sample from a British population. The Pukapukans show no rise in mean blood pressure with age and have other features placing them at low risk of coronary heart disease. Although the British population showed the shift to the left in mean QRS axis characteristic of economically advanced populations there was no change in mean QRS axis with age among the Pukapukans. The conclusion is drawn that the shift with age in QRS axis observed in advanced populations is extrinsic in origin but is not necessarily a manifestation of coronary heart disease.

Cholesterol and mortality in New Zealand Maoris.

The relation between serum cholesterol concentration and mortality was studied prospectively over 11 years in 630 New Zealand Maoris aged 25-74. Serum cholesterol concentration was measured at initial examination in 1962-3 in 94% of the subjects and whether each was dead or alive was determined in 1974. The causes of death were divided into three categories: cancer, cardiovascular disease, and "other." The Mantel-Haenszel method of analysis of survivorship data showed a significant inverse relation between serum cholesterol concentration and overall mortality in men (x 2/2 = 11.6; p = 0.003) and women (x 2/2 = 7.6; p = 0.02) with odds ratios of 2.3 and 1.9 respectively. Similar significant inverse relations were found for cancer and "other" causes of death. These relations remained significant when baseline age, systolic blood pressure, and the Quetelt index were controlled in Cox's proportional hazards regression model. The results of this study provide evidence for a potentially deleterious effect of low serum cholesterol concentration. Hence, further research is needed before indiscriminate efforts are made to lower serum cholesterol concentrations in New Zealand Maoris.

The Tokelau Island migrant study: prevalence of various conditions before migration.

The Tokelau Island Migrant Study has shown no important differences between those who subsequently left their home islands to migrate to New Zealand and those who remained, in key anthropometric and biochemical variables already reported. This comparison is now extended to various common diseases and conditions, and again no major difference emerges. The Tokelauans are compared with other Polynesians and shown to have less diabetes, hypertension, effort pain, chronic bronchitis and varicose veins than New Zealand Maoris, while resembling some Cook Island groups. Changes in prevalences of some conditions following migration are postulated.

Prevalence of coronary heart disease in samples of New Zealand Maoris and Pakehas.

This paper reports the prevalence of coronary heart disease (CHD) and its relationship with several standard risk factors in samples of New Zealand Maoris and Pakehas. The age standardised prevalence rates of CHD are: 16.1 percent, and 7.3 percent in Maori females and males respectively, and 11.5 percent and 6.5 percent in Pakeha females and males respectively. In Maori females only serum uric acid is associated with an increasing prevalence of CHD. The high prevalence of CHD in females as compared with the higher national mortality rates for males suggests that the case fatality rates for CHD are higher in males. The lack of association between CHD and the risk factors in Maori females suggests the possibility of two syndromes of CHD in Maori females.

Smoking behaviour and respiratory health in teenage sample: the rotorua lakes study, 1.

A survey in a New Zealand high school compared boys and girls, Maori and European, aged 13 to 15 years, and found differences in smoking habits. Smoking was most prevalent in Maori students of both sexes, and least in European girls, who appeared to be most influenced by home factors such as father's occupational status and older siblings' habits. Maori students and European boys showed relationships rather to the habits of the peer group. Findings were comparable with earlier studies suggesting little secular change in the smoking habits of this age group. Current smokers in all sex/race groups had higher current respiratory morbidity scores than non-smokers and ex-smokers, and smoking partly accounted for racial differences in morbidity.