A novel 14-kilodalton protein interacts with the mitogen-activated protein kinase scaffold MP1 on a late endosomal/lysosomal compartment

We have identified a novel, highly conserved protein of 14 kD copurifying with late endosomes/lysosomes on density gradients. The protein, now termed p14, is peripherally associated with the cytoplasmic face of late endosomes/lysosomes in a variety of different cell types. In a two-hybrid screen with p14 as a bait, we identified the mitogen-activated protein kinase (MAPK) scaffolding protein MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) partner 1 (MP1) as an interacting protein. We confirmed the specificity of this interaction in vitro by glutathione S-transferase pull-down assays and by coimmunoprecipitation, cosedimentation on glycerol gradients, and colocalization. Moreover, expression of a plasma membrane-targeted p14 causes mislocalization of coexpressed MP1. In addition, we could reconstitute protein complexes containing the p14-MP1 complex associated with ERK and MEK in vitro. The interaction between p14 and MP1 suggests a MAPK scaffolding activity localized to the cytoplasmic surface of late endosomes/lysosomes, thereby combining catalytic scaffolding and subcellular compartmentalization as means to modulate MAPK signaling within a cell.

The role of Ca2+ in insulin-stimulated glucose transport in 3T3-L1 cells

We have examined the requirement for Ca2+ in the signaling and trafficking pathways involved in insulin-stimulated glucose uptake in 3T3-LI adipocytes. Chelation of intracellular Ca2+, using 1,2-bis (o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM), resulted in >95% inhibition of insulin-stimulated glucose uptake. The calmodulin antagonist, W13, inhibited insulin-stimulated glucose uptake by 60%. Both BAPTA-AM and W13 inhibited Akt phosphorylation by 70-75%. However, analysis of insulin-dose response curves indicated that this inhibition was not sufficient to explain the effects of BAPTA-AM and W13 on glucose uptake. BAPTA-AM inhibited insulin-stimulated translocation of GLUT4 by 50%, as determined by plasma membrane lawn assay and subcellular fractionation. In contrast, the insulin-stimulated appearance of HA-tagged GLUT4 at the cell surface, as measured by surface binding, was blocked by BAPTA/AM.. While the ionophores A23187 or ionomycin prevented the inhibition of Akt phosphorylation and GLUT4 translocation by BAPTA-AM, they did not overcome the inhibition of glucose transport. Moreover, glucose uptake of cells pretreated with insulin followed by rapid cooling to 4 degreesC, to promote cell surface expression of GLUT4 and prevent subsequent endocytosis, was inhibited specifically by BAPTA-AM. This indicates that inhibition of glucose uptake by BAPTA-AM is independent of both trafficking and signal transduction. These data indicate that Ca2+ is involved in at least two different steps of the insulin-dependent recruitment of GLUT4 to the plasma membrane. One involves the translocation step. The second involves the fusion of GLUT4 vesicles with the plasma membrane. These data are consistent with the hypothesis that Ca2+/cahnodulin plays a fundamental role in eukaryotic vesicle docking and fusion. Finally, BAPTA-AM may inhibit the activity of the facilitative transporters by binding directly to the transporter itself.

Quantification of sulfur and sulfur-containing compounds in wastewaters by means of a combination of liquid chromatographic methods

Low-micromolar concentrations of sulfite, thiosulfate and sulfide, present in synthetic wastewater or anaerobic digester effluent, were quantified by means of derivatization with monobromobimane, followed by HPLC separation with fluorescence detection. The concentration of elemental sulfur was determined, after its extraction with chloroform from the derivatized sample, by HPLC with UV detection. Recoveries of sulfide (both matrices), and of thiosulfate and sulfite (synthetic wastewater) were between 98 and 103%. The in-run RSDs on separate derivatizations were 13 and 19% for sulfite (two tests), between 1.5 and 6.6% for thiosulfate (two tests) and between 4.1 and 7.7% for sulfide (three tests). Response factors for derivatives of sulfide and thiosulfate, but not sulfite, were steady over a 13-month period during which 730 samples were analysed. Dithionate and tetrathionate did not seem to be detectable with this method. The distinctness of the elemental sulfur and the derivatizing-agent peaks was improved considerably by detecting elution at 297 instead of 263 nm. (C) 2002 Elsevier Science B.V. All rights reserved.

Eating 'Green': Motivations behind organic food consumption in Australia

Central to the development of green lifestyles is the consumption of foods that by dint of their status as chemical-free, locally produced and/or free of genetically modified ingredients, reduce the environmental impact of food provision. Yet there are many other factors, such as health concerns, that may also encourage the consumption of 'green' foods. This paper explores the ways in which Australian consumers construct organic food-a sector of the food industry that is currently growing at between 20 and 50 percent per annum but is struggling to keep up with rising consumer demand. In order to examine the significance of 'green' signifiers in the consumption practices of Australian consumers a series of focus group interviews and a national consumer survey were conducted. These examined both those characteristics of food that were valued in general, and those meanings that were associated with organic food in particular. In very general terms, analysis reveals that while consumers believed organic foods to be healthy and environmentally sound-both of which were considered desirable-these characteristics were subsumed by an overarching concern with convenience. This does not mean that consumers did not hold genuinely positive environmental attitudes. Rather, it reflects a range of contradictory beliefs and practices that appeared to derive from the discursive conflict between conventional and organic food industries over environmental, health and safety claims. The paper concludes by identifying the barriers and opportunities for expanding the organic industry in Australia in the context of the ways organics is constructed by consumers.

Petrology and geochemistry of the Bandas del Sur Formation, Las Canadas Edifice, Tenerife (Canary Islands)

The Bandas del Sur Formation preserves a Quaternary extra-caldera record of central phonolitic explosive volcanism of the Las Canadas volcano at Tenerife. Volcanic rocks are bimodal in composition, being predominantly phonolitic pyroclastic deposits, several eruptions of which resulted in summit caldera collapse, alkali basaltic lavas erupted from many fissures around the flanks. For the pyroclastic deposits, there is a broad range of pumice glass compositions from phonotephrite to phonolite. The phonolite pyroclastic deposits are also characterized by a diverse, 7-8-phase phenocryst assemblage (alkali feldspar + biotite + sodian diopside + titanomagnetite + ilmenite + nosean-hauyne + titanite + apatite) with alkali feldspar dominant, in contrast to interbedded phonolite lavas that typically have lower phenocryst contents and lack hydrous phases. Petrological and geochemical data are consistent with fractional crystallization (involving the observed phenocryst assemblages) as the dominant process in the development of phonolite magmas. New stratigraphically constrained data indicate that petrological and geochemical differences exist between pyroclastic deposits of the last two explosive cycles of phonolitic volcanism. Cycle 2 (0.85-0.57 Ma) pyroclastic fall deposits commonly show a cryptic compositional zonation indicating that several eruptions tapped chemically, and probably thermally stratified magma systems. Evidence for magma mixing is most widespread in the pyroclastic deposits of Cycle 3 (0.37-0.17 Ma), which includes the presence of reversely and normally zoned phenocrysts, quenched mafic glass blebs in pumice, banded pumice, and bimodal to polymodal phenocryst compositional populations. Syn-eruptive mixing events involved mostly phonolite and tephriphonolite magmas, whereas a pre-eruptive mixing event involving basaltic magma is recorded in several banded pumice-bearing ignimbrites of Cycle 3. The periodic addition and mixing of basaltic magma ultimately may have triggered several eruptions. Recharge and underplating by basaltic magma is interpreted to have elevated sulphur contents (occurring as an exsolved gas phase) in the capping phonolitic magma reservoir. This promoted nosean-hauyne crystallization over nepheline, elevated SO3 contents in apatite, and possibly resulted in large, climatologically important SO2 emissions.

New W-isotope evidence for rapid terrestrial accretion and very early core formation

The short-lived Hf-182-W-182-isotope system is an ideal clock to trace core formation and accretion processes of planets. Planetary accretion and metal/silicate fractionation chronologies are calculated relative to the chondritic Hf-182-W-182-isotope evolution. Here, we report new high-precision W-isotope data for the carbonaceous chondrite Allende that are much less radiogenic than previously reported and are in good agreement with published internal Hf-W chronometry of enstatite chondrites. If the W-isotope composition of terrestrial rocks, representing the bulk silicate Earth, is homogeneous and 2.24 epsilon(182W) units more radiogenic than that of the bulk Earth, metal/silicate differentiation of the Earth occurred very early. The new W-isotope data constrain the mean time of terrestrial core formation to 34 million years after the start of solar system accretion. Early terrestrial core formation implies rapid terrestrial accretion, thus permitting formation of the Moon by giant impact while Hf-182 was still alive. This could explain why lunar W-isotopes are more radiogenic than the terrestrial value. Copyright (C) 2002 Elsevier Science Ltd.

Lamproites from Gaussberg, Antarctica: Possible transition zone melts of Archaean subducted sediments

Petrogenetic models for the origin of lamproites are evaluated using new major element, trace element, and Sr, Nd, and Pb isotope data for Holocene lamproites from the Gaussberg volcano in the East Antarctic Shield. Gaussberg lamproites exhibit very unusual Pb isotope compositions (Pb-206/Pb-204 = 17.44-17.55 and Pb-207/Pb-204 = 15.56-15.63), which in common Pb isotope space plot above mantle evolution lines and to the left of the meteorite isochron. Combined with very unradiogenic Nd, such compositions are shown to be inconsistent with an origin by melting of sub-continental lithospheric mantle. Instead, a model is proposed in which late Archaean continent-derived sediment is subducted as K-hollandite and other ultra-high-pressure phases and sequestered in the Transition Zone (or lower mantle) where it is effectively isolated for 2-3 Gyr. The high Pb-207/Pb-204 ratio is thus inherited from ancient continent-derived sediment, and the relatively low Pb-206/Pb-204 ratio is the result of a single stage of U/Pb fractionation by subduction-related U loss during slab dehydration. Sr and Nd isotope ratios, and trace element characteristics (e.g. Nb/Ta ratios) are consistent with sediment subduction and dehydration-related fractionation. Similar models that use variable time of isolation of subducted sediment can be derived for all lamproites. Our interpretation of lamproite sources has important implications for ocean island basalt petrogenesis as well as the preservation of geochemically anomalous reservoirs in the mantle.

High-frequency climatic oscillations recorded in a Holocene coral reef at Leizhou Peninsula, South China sea

A detailed study of the Goniopora reef profile at Dengloujiao, Xuwen County, Leizhou Peninsula, the northern coast of the South China Sea suggests that a series of high-frequency, large-amplitude and abrupt cold events occurred during the Holocene Hypsithermal, an unusual phenomenon termed Leizhou Events in this paper. This period (corresponding to C-14 age of 6.2 -6.7 kaBP or calendar age of 6.7-7.2 kaBP), when the climatic conditions were ideal for coral. reefs to develop, can be divided into at least nine stages. Each stage (or called a climate optimum), lasting about 20 to 50 a, was terminated by an abrupt cold nap and (or) a sea-level lowering event in winter, leading to widespread emergence and death of the Goniopora corals, and growth discontinuities on the coral surface. Such a cyclic process resulted in the creation of a > 4m thick Goniopora reef flat. During this period, the crust subsided periodically but the sea level was rising. The reef profile provides valuable archives for the study of decadal-scale mid-Holocene climatic oscillations in the tropical area of South China. Our results provide new evidence for high-frequency climate instability in the Holocene Hypsithermal, and challenge the traditional understanding of Holocene climate.

Isolation and characterisation of Indian Ocean ciguatoxin

We report the isolation and initial characterisation of Indian Ocean ciguatoxin (I-CTX) present in toxic lipid soluble extracts isolated from ciguateric fishes collected off the Republic of Mauritius in the Indian Ocean. Following i.p. injection of this extract, mice displayed symptoms that were similar, though not identical, to those produced by Pacific and Caribbean ciguatoxins (P-CTXs and C-CTXs). Using a radiolabelled brevetoxin (PbTx) binding assay and mouse bioassay guided fractionation, I-CTX was purified by Florisil, Sephadex LH-20 and TSK HW-40S chromatography with good recovery. Isolation to purity was not possible by preparative reversed phase high-performance liquid chromatography (HPLC) due to significant losses of toxicity. However, analytical reversed phase HPLC coupled to an electrospray mass spectrometry detector identified a [M + H](+) ion at m/z 1141.58 which co-eluted with activity that displaced [3 H]-PbTx binding to rat brain. This mass corresponded to C-CTX-1, but the fragmentation pattern of I-CTX showed a different ratio of pseudo molecular and product ions. I-CTX was found to elute later than P-CTX-1 but was practically indistinguishable from C-CTX-1 on reversed phase HPLC, while the TSK HW-40S column chromatography differentiated I-CTX from the later eluting C-CTX-1. Taken together, these results indicate that I-CTX is a new ciguatoxin (CTX) responsible for ciguatera caused by reef fish in the Indian Ocean. (C) 2002 Elsevier Science Ltd. All rights reserved.

Slowed conduction and ventricular tachycardia after targeted disruption of the cardiac sodium channel gene Scn5a

Voltage-gated sodium channels drive the initial depolarization phase of the cardiac action potential and therefore critically determine conduction of excitation through the heart. In patients, deletions or loss-of-function mutations of the cardiac sodium channel gene, SCN5A, have been associated with a wide range of arrhythmias including bradycardia (heart rate slowing), atrioventricular conduction delay, and ventricular fibrillation. The pathophysiological basis of these clinical conditions is unresolved. Here we show that disruption of the mouse cardiac sodium channel gene, Scn5a, causes intrauterine lethality in homozygotes with severe defects in ventricular morphogenesis whereas heterozygotes show normal survival. Whole-cell patch clamp analyses of isolated ventricular myocytes from adult Scn5a(+/-) mice demonstrate a approximate to50% reduction in sodium conductance. Scn5a(+/-) hearts have several defects including impaired atrioventricular conduction, delayed intramyocardial conduction, increased ventricular refractoriness, and ventricular tachycardia with characteristics of reentrant excitation. These findings reconcile reduced activity of the cardiac sodium channel leading to slowed conduction with several apparently diverse clinical phenotypes, providing a model for the detailed analysis of the pathophysiology of arrhythmias.

Long-term trends in sunshine duration and its association with schizophrenia birth rates and age at first registration - data from Australia and the Netherlands

Back ground. Based on the well-described excess of schizophrenia births in winter and spring, we hypothesised that individuals with schizophrenia (a) would be more likely to be born during periods of decreased perinatal sunshine, and (b) those born during periods of less sunshine would have an earlier age of first registration. Methods. We undertook an ecological analysis of long-term trends in perinatal sunshine duration and schizophrenia birth rates based on two mental health registers (Queensland. Australia n = 6630; The Netherlands n = 24, 474). For each of the 480 months between 1931 and 1970, the agreement between slopes of the trends in psychosis and long-term sunshine duration series were assessed. Age at first registration was assessed by quartiles of long-term trends in perinatal sunshine duration, Males and females were assessed separately. Results. Both the Dutch and Australian data showed a statistically significant association between falling long-term trends in sunshine duration around the time of birth and rising schizophrenia birth rates for males only. In both the Dutch and Australian data there were significant associations between earlier age of first registration and reduced long-term trends in sunshine duration around the time of birth for both males and females, Conclusions. A measure of long-term trends in perinatal sunshine duration was associated with two epidemiological features of schizophrenia in two separate data sets. Exposures related to sunshine duration warrant further consideration in schizophrenia research. (C) 2002 Elsevier Science B.V. All rights reserved.

Mathematical modelling of schistosomiasis japonica: comparison of control strategies in the People's Republic of China

We present the first mathematical model on the transmission dynamics of Schistosoma japonicum. The work extends Barbour's classic model of schistosome transmission. It allows for the mammalian host heterogeneity characteristic of the S. japonicum life cycle, and solves the problem of under-specification of Barbour's model by the use of Chinese data we are collecting on human-bovine transmission in the Poyang Lake area of Jiangxi Province in China. The model predicts that in the lake/marshland areas of the Yangtze River basin: (1) once-early mass chemotherapy of humans is little better than twice-yearly mass chemotherapy in reducing human prevalence. Depending on the heterogeneity of prevalence within the population, targeted treatment of high prevalence groups, with lower overall coverage, can be more effective than mass treatment with higher overall coverage. Treatment confers a short term benefit only, with prevalence rising to endemic levels once chemotherapy programs are stopped (2) depending on the relative contributions of bovines and humans, bovine treatment can benefit humans almost as much as human treatment. Like human treatment, bovine treatment confers a short-term benefit. A combination of human and bovine treatment will dramatically reduce human prevalence and maintains the reduction for a longer period of time than treatment of a single host, although human prevalence rises once treatment ceases; (3) assuming 75% coverage of bovines, a bovine vaccine which acts on worm fecundity must have about 75% efficacy to reduce the reproduction rate below one and ensure mid-term reduction and long-term elimination of the parasite. Such a vaccination program should be accompanied by an initial period of human treatment to instigate a short-term reduction in prevalence, following which the reduction is enhanced by vaccine effects; (4) if the bovine vaccine is only 45% efficacious (the level of current prototype vaccines) it will lower the endemic prevalence, but will not result in elimination. If it is accompanied by an initial period of human treatment and by a 45% improvement in human sanitation or a 30% reduction in contaminated water contact by humans, elimination is then possible. (C) 2002 Elsevier Science B.V. All rights reserved.

In vivo and in vitro models demonstrate a role for caveolin-1 in the pathogenesis of ischaemic acute renal failure

Caveolae and their proteins, the caveolins, transport macromolecules; compartmentalize signalling molecules; and are involved in various repair processes. There is little information regarding their role in the pathogenesis of significant renal syndromes such as acute renal failure (ARF). In this study, an in vivo rat model of 30 min bilateral renal ischaemia followed by reperfusion times from 4 h to 1 week was used to map the temporal and spatial association between caveolin-1 and tubular epithelial damage (desquamation, apoptosis, necrosis). An in vitro model of ischaemic ARF was also studied, where cultured renal tubular epithelial cells or arterial endothelial cells were subjected to injury initiators modelled on ischaemia-reperfusion (hypoxia, serum deprivation, free radical damage or hypoxia-hyperoxia). Expression of caveolin proteins was investigated using immunohistochemistry, immunoelectron microscopy, and immunoblots of whole cell, membrane or cytosol protein extracts. In vivo, healthy kidney had abundant caveolin-1 in vascular endothelial cells and also some expression in membrane surfaces of distal tubular epithelium. In the kidneys of ARF animals, punctate cytoplasmic localization of caveolin-1 was identified, with high intensity expression in injured proximal tubules that were losing basement membrane adhesion or were apoptotic, 24 h to 4 days after ischaemia-reperfusion. Western immunoblots indicated a marked increase in caveolin-1 expression in the cortex where some proximal tubular injury was located. In vitro, the main treatment-induced change in both cell types was translocation of caveolin-1 from the original plasma membrane site into membrane-associated sites in the cytoplasm. Overall, expression levels did not alter for whole cell extracts and the protein remained membrane-bound, as indicated by cell fractionation analyses. Caveolin-1 was also found to localize intensely within apoptotic cells. The results are indicative of a role for caveolin-1 in ARF-induced renal injury. Whether it functions for cell repair or death remains to be elucidated. Copyright (C) 2003 John Wiley Sons, Ltd.