North American Indian childhood cirrhosis (NAIC)

North American Indian childhood cirrhosis is a distinct form of neonatal familial cholestasis. To date, it has only been described in aboriginal children from northwestern Quebec. The disease rapidly evolves into cirrhosis with early portal hypertension and bleeding from esophageal varices. Twelve of 36 children followed at l'H?pital Ste-Justine since 1970 received a liver transplant. As of now, there are 17 living NAIC patients, 6 of whom had liver transplantation. We mapped NAIC to chromosome 16q22, and identified mutations in CIRH1A in patients. All are homozygous for the R565W mutation in cirhin, a WD40 repeat protein of unknown function. We showed that cirhin is a resident in the nucleolus. Cirhin interacts with Cirip, a functional, alternative splice variant of the HIVEP1 protein. Their interaction indicates synergistic action. The complete inactivation of mouse homolog, tex292 is likely embryonic lethal. The continued collaboration between patients, their families, clinicians and researchers that has helped to identify the disease gene and to develop a diagnostic test now focuses on finding a new treatment for this unique disease affecting First Nations children from Québec.     

Manganese porphyrin reduces renal injury and mitochondrial damage during ischemia/reperfusion

Renal ischemia/reperfusion (I/R) injury often occurs as a result of vascular surgery, organ procurement, or transplantation. We previously showed that renal I/R results in ATP depletion, oxidant production, and manganese superoxide dismutase (MnSOD) inactivation. There have been several reports that overexpression of MnSOD protects tissues/organs from I/R-related damage, thus a loss of MnSOD activity during I/R likely contributes to tissue injury. The present study examined the therapeutic benefit of a catalytic antioxidant, Mn(III) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (MnTnHex-2-PyP(5+)), using the rat renal I/R model. This was the first study to examine the effects of MnTnHex-2-PyP(5+) in an animal model of oxidative stress injury. Our results showed that porphyrin pretreatment of rats for 24 h protected against ATP depletion, MnSOD inactivation, nitrotyrosine formation, and renal dysfunction. The dose (50 microg/kg) used in this study is lower than doses of various types of antioxidants commonly used in animal models of oxidative stress injuries. In addition, using novel proteomic techniques, we identified the ATP synthase-beta subunit as a key protein induced by MnTnHex-2-PyP(5+) treatment alone and complex V (ATP synthase) as a target of injury during renal I/R. These results showed that MnTnHex-2-PyP(5+) protected against renal I/R injury via induction of key mitochondrial proteins that may be capable of blunting oxidative injury.     

Differential attention-dependent response modulation across cell classes in macaque visual area V4

The cortex contains multiple cell types, but studies of attention have not distinguished between them, limiting understanding of the local circuits that transform attentional feedback into improved visual processing. Parvalbumin-expressing inhibitory interneurons can be distinguished from pyramidal neurons based on their briefer action potential durations. We recorded neurons in area V4 as monkeys performed an attention-demanding task. We find that the distribution of action potential durations is strongly bimodal. Neurons with narrow action potentials have higher firing rates and larger attention-dependent increases in absolute firing rate than neurons with broad action potentials. The percentage increase in response is similar across the two classes. We also find evidence that attention increases the reliability of the neuronal response. This modulation is more than two-fold stronger among putative interneurons. These findings lead to the surprising conclusion that the strongest attentional modulation occurs among local interneurons that do not transmit signals between areas.     

Novel mechanism of cyclic AMP mediated extracellular signal regulated kinase activation in an intestinal cell line

The extracellular signal regulated kinase (ERK1/2) signaling cascade has been implicated as both a pro-apoptotic and anti-apoptotic pathway depending on cell type and context. In the T84 intestinal epithelial cell line, cAMP activates ERK1/2 resulting in the inhibition of apoptosis. Cyclic-AMP signaling relies on the binding and activation of a cAMP binding protein. In most cell types, the majority of this signaling occurs through an isoform of protein kinase A (PKAI or PKAII). Despite evidence to the contrary, we hypothesized that ERK1/2 activation is through a PKA isoform. Pharmacological activators and inhibitors of PKA as well as siRNA were used to further interrogate this potential signaling pathway. Our results demonstrate that at doses sufficient to increase PKA activity, PKAII specific cAMP analogs activate ERK1/2 while PKAI analogs do not. Pharmacological inhibition of the PKAII regulatory subunit and catalytic subunit as well as siRNA knockdown of the catalytic subunit blocks ERK1/2 activation. We conclude that in the T84 cell line, cAMP binding to the PKAII regulatory subunit leads to the subsequent phosphorylation of ERK1/2 and provides insight into the mechanism of cAMP mediated survival signaling in the intestinal epithelium. These results directly implicate PKAII as a mediator of cell survival in T84 cells and provide evidence for an additional means by which cAMP can influence intestinal cell turnover.     

Receptor-coupling properties of the invertebrate visual guanine nucleotide binding protein iGqalpha

Invertebrate visual iG(q)alpha is homologous to mammalian mG(q)alpha in two of the three domains important for G protein interaction with receptors; the C-terminus and the linker regions that connect the helical and ras-like domains of the alpha subunit. The third receptor-interacting domain, the N-terminus, contains a six amino acid extension MTLESI in mG(q)alpha that is not present in iG(q)alpha. In co-expression studies we assessed the promiscuity and efficacy of receptor coupling to phospholipase C (PLC) by iG(q)alpha, a non-palmitoylated mutant iG(q)alpha(C3,4A), mG(q)alpha and G(15)alpha. The invertebrate G proteins and mG(q)alpha only coupled to G(q)-coupled receptors (m1 muscarinic acetylcholine receptor (mChR1), alpha(1A)-adrenergic receptor (alpha1-AR)) and not to the G(i/s)-coupled receptors (CCR1 receptor, beta2-adrenergic receptor or dopamine D1 receptor) while G(15)alpha coupled to all receptors. iG(q)alpha and iG(q)alpha(C3,4A) both had double the efficacy for PLC activation compared to the mammalian G proteins when co-expressed with mChR1 and alpha1-AR. The increased efficacy of iG(q)alpha compared to mG(q)alpha was also seen downstream of PLC with carbachol stimulation of the mitogen-activated protein kinase, ERK1/2. Addition of the MTLESI extension onto the N-terminus of iG(q)alpha decreased its efficacy by 35% whereas deletion of this sequence from mG(q)alpha increased its efficacy by 60% in the PLC and ERK1/2 assays. iG(q)alpha, iG(q)alpha(C3,4A) and mG(q)alpha all displayed similar receptor-independent AlF(4)(-)activation of PLC and guanosine triphosphate hydrolysis (GTPase) activity. iG(q)alpha, and iG(q)alpha(C3,4A) both had increased receptor-activated guanosine 5'-[gamma-[(35)S]thio]triphosphate ([(35)S]GTPgammaS) binding when compared to mG(q)alpha when co-expressed with the mChR1. These results demonstrate that G(q) protein efficacy is at least partially determined by the presence of the amino-terminal MTLESI extension. Comparison of [(35)S]GTPgammaS binding rates helps explain the increased efficacy of the invertebrate G proteins.     

The potential of greenhouse sinks to underwrite improved land management

The current agricultural systems of broad areas of Australia are unsustainable, with large projected increases in salinization, decreases in water quality, wind erosion, and losses of biodiversity. It is well known that these problems can be partially resolved by farmland reforestation; however, a major issue is financing the scale of activity required. The international response to global warming, the United Nations Framework Convention on Climate Change and its Kyoto Protocol, includes provisions that enable greenhouse sinks (sequestration of carbon in soils and vegetation) to be used by parties to fulfil their obligations. The Kyoto Protocol also allows for trading in emission reductions, and this opens the possibility that investment in carbon sinks may help underwrite broader natural resource management objectives. This paper examines the possibilities for improved land management in Western Australia arising from the development of carbon sinks by considering: (a) the likelihood of a carbon market developing and the likely depth of that market as a result of current national and international policies, (b) the data available to provide estimates on different types of sinks, and (c) the likely benefits of wide-scale sink investment.It was estimated that the total amount of carbon that could be sequestered by reforesting 16.8 Mha of cleared farmland is 2200 Mt CO₂-e, and between 290 and 1170 Mt CO₂-e by destocking 94.8 Mha of rangelands. There were insufficient data to produce estimates of sequestration following changes in tillage practice in cropping systems or the revegetation of already salinized land. We conclude that carbon sinks are only likely to become profitable as a broad-scale stand-alone enterprise when carbon prices reach AUD$15/t CO₂-e, with this threshold value varying with carbon yield and project costs. Below this price, their value can be significant as an adjunct to reforestation schemes that are aimed at providing other products (wood, pulp, bioenergy) and land and water conservation benefits. Irrespective of this, carbon sinks provide an opportunity to both sequester carbon in a least-cost fashion and improve soil and watershed management.     

Opposing gradients of Gli repressor and activators mediate Shh signaling along the dorsoventral axis of the inner ear

Organization of the vertebrate inner ear is mainly dependent on localized signals from surrounding tissues. Previous studies demonstrated that sonic hedgehog (Shh) secreted from the floor plate and notochord is required for specification of ventral (auditory) and dorsal (vestibular) inner ear structures, yet it was not clear how this signaling activity is propagated. To elucidate the molecular mechanisms by which Shh regulates inner ear development, we examined embryos with various combinations of mutant alleles for Shh, Gli2 and Gli3. Our study shows that Gli3 repressor (R) is required for patterning dorsal inner ear structures, whereas Gli activator (A) proteins are essential for ventral inner ear structures. A proper balance of Gli3R and Gli2/3A is required along the length of the dorsoventral axis of the inner ear to mediate graded levels of Shh signaling, emanating from ventral midline tissues. Formation of the ventral-most otic region, the distal cochlear duct, requires robust Gli2/3A function. By contrast, the formation of the proximal cochlear duct and saccule, which requires less Shh signaling, is achieved by antagonizing Gli3R. The dorsal vestibular region requires the least amount of Shh signaling in order to generate the correct dose of Gli3R required for the development of this otic region. Taken together, our data suggest that reciprocal gradients of GliA and GliR mediate the responses to Shh signaling along the dorsoventral axis of the inner ear.     

Inhibition of protein glycation by skins and seeds of the muscadine grape

The formation of advanced glycation end products (AGEs) leading to protein glycation and cross-linking is associated with the pathogenesis of diabetic complications. The inhibition of protein glycation by phenolic and flavonoid antioxidants demonstrates that the process is mediated, in part, by oxidative processes. In this study, the effects of seed and skin extracts of the muscadine grape on AGEs formation were examined. Seeds and skins were extracted (10% w/v) with 50% ethanol and incubated at 37 degrees C with a solution containing 250 mM fructose and 10 mg/ml albumin. After 72 h, fluorescence was measured at the wavelength pair of 370 and 440 nm as an index of the formation of AGEs. Both seed and skin extracts were found to be efficacious inhibitors of AGE formation. A 1:300 dilution of the seed extract decreased fluorescence by approximately 65%, whereas muscadine grape skin extract produced a 40% lowering. This difference correlates with the greater antioxidant activity found in muscadine seeds in comparison to skins, however, on a mass basis, the inhibitory activities of the seeds and skins were found to be nearly equivalent. Gallic acid, catechin and epicatechin, the three major polyphenols in the seeds, all significantly decreased the AGE product related fluorescence at a concentration of 50 microM. Neither muscadine seed extract nor skin extract inhibited the methylglyoxal-mediated glycation of albumin. These results suggest that consumption of the muscadine grape may have some benefit in altering the progression of diabetic complications.