Urinary Metabolic Phenotyping the slc26a6 (Chloride-Oxalate Exchanger) Null Mouse Model

The prevalence of renal stone disease is increasing, although it remains higher in men than in women when matched for age. While still somewhat controversial, several studies have reported an association between renal stone disease and hypertension, but this may be confounded by a shared link with obesity. However, independent of obesity, hyperoxaluria has been shown to be associated with hypertension in stone-formers, and the most common type of renal stone is composed of calcium oxalate. The chloride-oxalate exchanger slc26a6 (also known as CFEX or PAT-1), located in the renal proximal tubule, was originally thought to have an important role in sodium homeostasis and thereby blood pressure control, but it has recently been shown to have a key function in oxalate balance by mediating oxalate secretion in the gut. We have applied two orthogonal analytical platforms (NMR spectroscopy and capillary electrophoresis with UV detection) in parallel to characterize the urinary metabolic signatures related to the loss of the renal chloride-oxalate exchanger in slc26a6 null mice. Clear metabolic differentiation between the urinary profiles of the slc26a6 null and the wild type mice were observed using both methods, with the combination of NMR and CE-UV providing extensive coverage of the urinary metabolome. Key discriminating metabolites included oxalate, m-hydroxyphenylpropionylsulfate (m-HPPS), trimethylamine-N-oxide, glycolate and scyllo-inositol (higher in slc26a6 null mice) and hippurate, taurine, trimethylamine, and citrate (lower in slc26a6 null mice). In addition to the reduced efficiency of anion transport, several of these metabolites (hippurate, m-HPPS, methylamines) reflect alteration in gut microbial cometabolic activities. Gender-related metabotypes were also observed in both wild type and slc26a6 null groups. Urinary metabolites that showed a sex-specific pattern included trimethylamine, trimethylamine-N-oxide, citrate, spermidine, guanidinoacetate, and 2-oxoisocaproate. The gender-dependent metabolic expression of the consequences of slc26a6 deletion might have relevance to the difference in prevalence of renal stone formation in men and women. The different composition of microbial metabolites in the slc26a6 null mice is consistent with the fact that the slc26a6 transporter is found in a range of tissues, including the kidney and intestine, and provides further evidence for the "long reach" of the microbiota in physiological and pathological processes.     

Electron crystallography of proteins in membranes

Electron crystallography has played a vital role in advancing our understanding of proteins in membranes since the 'fluid mosaic model' was proposed in 1972. It is now an established technique to reveal the structures of proteins in their natural bilayer environment and makes possible the study of biological mechanisms through freeze-trapping of transitional states. Thus, images and diffraction patterns of well-ordered, planar and tubular protein-lipid crystals are yielding atomic models, which tell us how the proteins in situ are designed and carry out their membrane-specific tasks. Recent methodological advances and the inclusion of tomographic and cryo-sectioning techniques are enabling detailed information to be obtained from increasingly smaller and more disordered membrane assemblies, extending the potential of this approach.     

Agrobacterium-mediated transformation and assessment of factors influ-encing transgene expression in loblolly pine (Pinus taeda L.)

INTRODUCTIONRecombinant DNA technology is a powerful toolfor the introduction of foreign genes into longlivedperennials and fOr fundamelltal studies of gene expression. Using such techniques, we can overcomethe difficulties associated with the breeding of a long-lived perennial. At present, although considerablereseaxch effort has been devoted to the genetic en-gineering of fOrest trees, it has lagged behind ad-vances made in herbaceous crops due both to eco-nomics and the recalcitrant n…     

Evolution of chinook salmon (Oncorhynchus tshawytscha) populations in New Zealand: pattern,rate, and process

Chinook salmon, Oncorhynchus tshawytscha, from the Sacramento River, California, USA were introduced to New Zealand between 1901 and 1907, and colonized most of their present-day range within about 10 years. The New Zealand populations now vary in phenotypic traits typically used to differentiate salmon populations within their natural range: growth in freshwater and at sea, age at maturity, dates of return to fresh water and reproduction, morphology, and reproductive allocation. This paper reviews a large research program designed to determine the relative contributions of phenotypic plasticity and genetic adaptation to this variation, in an effort to understand the processes underlying the natural evolution of new populations. We found strong evidence of trait divergence between populations within at most 30 generations, particularly in freshwater growth rate, date of return, and reproductive output, with plausible adaptive bases for these differences. Importantly, we also demonstrated not only a genetic basis for post-release survival but higher survival, and hence fitness, of a population released from its established site compared to another population released from the same site. We conclude that divergence of salmon in different rivers probably resulted initially from phenotypic plasticity (e.g., habitat-specific growth rates, and effects of upriver migration on ovarian investment). Philopatry (homing to natal streams) combined with rapid evolution of distinct breeding periods to restrict gene flow, facilitating divergence in other traits. We also suggest that in addition to genetic divergence resulting from random founder effects, divergence may also arise during the very early stages of colonization when the original colonists are a non-random, pre-adapted subset of the source population. This favored founders effect immediately improves the fitness of the new population. Overall, this research reveals the complex interplay of environmental and genetic controls over behavior, physiology and life history that characterize the early stages of population differentiation, a process that has taken place repeatedly during the history of salmon populations.     

Serological and proteomic evaluation of antibody responses in the identification of tumor antigens in renal cell carcinoma

Renal cell carcinoma (RCC) is relatively resistant to conventional chemotherapy and radiotherapy. However, reports of spontaneous regression along with promising results in clinical trials suggest that immunotherapuetic strategies may be of clinical benefit. Few RCC related antigens have been identified to date, and the technical difficulty and time constraints of current antigen identification techniques preclude the screening of large numbers of patients. A comparatively rapid strategy has been used to identify components of tumors that elicit an antibody response in the patient - the serological and proteomic evaluation of antibody responses (SPEAR) approach. This combines two-dimensional polyarylamide gel electrophoresis of tumor and normal kidney samples with immunoblotting using autologous patient sera and protein identification by mass spectrometry. Using the SPEAR approach to screen RCC patients for naturally occurring antitumor antibody responses, a number of candidate immunogens have been identified in patients with high-grade disease and their relative expression levels in tumor tissue compared to normal tissue have been studied. These proteins include annexins I and IV, thymidine phosphorylase (TP), carbonic anhydrase I, Mn-superoxide dismutase and major vault protein (MVP). Downstream analysis of the tissue expression of some of these proteins shows that MVP is up-regulated in 2/4 of RCC tumors but is also expressed in normal kidney whereas TP is up-regulated in 100% (11/11) of RCC cases examined with no or minimal expression in normal kidney, indicating a potential use as a therapeutic target.     

Proteomic changes in renal cancer and co-ordinate demonstration of both the glycolytic and mitochondrial aspects of the Warburg effect

Renal cell carcinoma (RCC) is the tenth most common cancer although the incidence is increasing. The main clinical problems stem from the relatively late presentation of many patients due to the often asymptomatic nature of the illness, and the relative insensitivity of metastatic disease to conventional chemotherapy and radiotherapy. Despite increasing knowledge of some of the genetic changes underlying sporadic renal cancer such as those involving the Von Hippel Lindau (VHL) gene, many of the underlying pathophysiological changes are ill-defined and there remains a need for the identification of disease markers for use in diagnosis and prognosis or as potential therapeutic targets. This study has used a proteomic approach, based on two-dimensional gel electrophoresis and mass spectrometry, to compare the protein profiles of conventional RCC tissue with patient-matched normal kidney cortex. Sequencing of 32 protein spots with significantly increased expression in RCC samples (>/= 4/6 patients) and 41 proteins whose levels decreased (6/6 patients) confirmed several previously known RCC-associated changes such as increases in Mn-superoxide dismutase, lactate dehydrogenase-A, aldolase A and C, pyruvate kinase M2, and thymidine phosphorylase. Additionally, several previously unknown changes were identified, including increased expression of three members of the annexin family and increased levels of the actin depolymerisation factor cofilin. The Warburg effect was also demonstrated with the identification of increases in proteins involved in the majority of steps in the glycolytic pathway and decreases in the gluconeogenic reactions, together with a parallel decrease in several mitochondrial enzymes. A number of the alterations seen were further confirmed in additional samples by immunohistochemistry, Western blotting, and laser capture microdissection.     

Activation of the nicotinic acetylcholine receptor involves a switch in conformation of the alpha subunits

The nicotinic acetylcholine (ACh) receptor belongs to a superfamily of synaptic ion channels that open in response to the binding of chemical transmitters. Their mechanism of activation is not known in detail, but a time-resolved electron microscopic study of the muscle-type ACh receptor had suggested that a local disturbance in the ligand-binding region and consequent rotations in the ligand-binding alpha subunits, connecting to the transmembrane portion, are involved. A more precise interpretation of this structural change is given here, based on comparison of the extracellular domain of the ACh receptor with an ACh-binding protein (AChBP) to which a putative agonist is bound. We find that, to a good approximation, there are two alternative extended conformations of the ACh receptor subunits, one characteristic of either alpha subunit before activation, and the other characteristic of all three non-alpha subunits and the protomer of AChBP. Substitution in the three-dimensional maps of alpha by non-alpha subunits mimics the changes seen on activation, suggesting that the structures of the alpha subunits are modified initially by their interactions with neighbouring subunits and switch to the non-alpha form when ACh binds. This structural change, which entails 15-16 degrees rotations of the inner pore-facing parts of the alpha subunits, most likely acts as the trigger that opens the gate in the membrane-spanning pore.     

Role of vaccinations as risk factors for ill health in veterans of the Gulf war: cross sectional study

ObjectivesTo explore the relation between ill health after the Gulf war and vaccines received before or during the conflict. To test the hypothesis that such ill health is limited to military personnel who received multiple vaccines during deployment and that pesticide use modifies any effect.DesignCross sectional study of Gulf war veterans followed for six to eight years after deployment.Setting UK armed forces.ParticipantsMilitary personnel who served in the Gulf and who still had their vaccine records.Results The response rate for the original survey was 70.4% (n=3284). Of these, 28% (923) had vaccine records. Receipt of multiple vaccines before deployment was associated with only one of the six health outcomes (post-traumatic stress reaction). By contrast five of the six outcomes (all but post-traumatic stress reaction) were associated with multiple vaccines received during deployment. The strongest association was for the multisymptom illness (odds ratio 5.0; 95% confidence interval 2.5 to 9.8).ConclusionAmong veterans of the Gulf war there is a specific relation between multiple vaccinations given during deployment and later ill health. Multiple vaccinations in themselves do not seem to be harmful but combined with the “stress” of deployment they may be associated with adverse health outcomes. These results imply that every effort should be made to maintain routine vaccines during peacetime.