Alpha-conotoxin OmIA is a potent ligand for the acetylcholine-binding protein as well as alpha3beta2 and alpha7 nicotinic acetylcholine receptors

The molluskan acetylcholine-binding protein (AChBP) is a homolog of the extracellular binding domain of the pentameric ligand-gated ion channel family. AChBP most closely resembles the alpha-subunit of nicotinic acetylcholine receptors and in particular the homomeric alpha7 nicotinic receptor. We report the isolation and characterization of an alpha-conotoxin that has the highest known affinity for the Lymnaea AChBP and also potently blocks the alpha7 nAChR subtype when expressed in Xenopus oocytes. Remarkably, the peptide also has high affinity for the alpha3beta2 nAChR indicating that alpha-conotoxin OmIA in combination with the AChBP may serve as a model system for understanding the binding determinants of alpha3beta2 nAChRs. alpha-Conotoxin OmIA was purified from the venom of Conus omaria. It is a 17-amino-acid, two-disulfide bridge peptide. The ligand is the first alpha-conotoxin with higher affinity for the closely related receptor subtypes, alpha3beta2 versus alpha6beta2, and selectively blocks these two subtypes when compared with alpha2beta2, alpha4beta2, and alpha1beta1deltaepsilon nAChRs.     

Patterns of tree growth in relation to environmental variability in the tropical dry deciduous forest at Mudumalai, southern India

Tree diameter growth is sensitive to environmental fluctuations and tropical dry forests experience high seasonal and inter-annual environmental variation. Tree growth rates in a large permanent plot at Mudumalai, southern India, were examined for the influences of rainfall and three intrinsic factors (size, species and growth form) during three 4-year intervals over the period 1988–2000. Most trees had lowest growth during the second interval when rainfall was lowest, and skewness and kurtosis of growth distributions were reduced during this interval. Tree diameter generally explained <10% of growth variation and had less influence on growth than species identity or time interval. Intraspecific variation was high, yet species identity accounted for up to 16% of growth variation in the community. There were no consistent differences between canopy and understory tree growth rates; however, a few subgroups of species may potentially represent canopy and understory growth guilds. Environmentally-induced temporal variations in growth generally did not reduce the odds of subsequent survival. Growth rates appear to be strongly influenced by species identity and environmental variability in the Mudumalai dry forest. Understanding and predicting vegetation dynamics in the dry tropics thus also requires information on temporal variability in local climate.     

Structural basis for molecular recognition and presentation of histone H3 by WDR5

Histone methylation at specific lysine residues brings about various downstream events that are mediated by different effector proteins. The WD40 domain of WDR5 represents a new class of histone methyl-lysine recognition domains that is important for recruiting H3K4 methyltransferases to K4-dimethylated histone H3 tail as well as for global and gene-specific K4 trimethylation. Here we report the crystal structures of full-length WDR5, WDR5Delta23 and its complexes with unmodified, mono-, di- and trimethylated histone H3K4 peptides. The structures reveal that WDR5 is able to bind all of these histone H3 peptides, but only H3K4me2 peptide forms extra interactions with WDR5 by use of both water-mediated hydrogen bonding and the altered hydrophilicity of the modified lysine 4. We propose a mechanism for the involvement of WDR5 in binding and presenting histone H3K4 for further methylation as a component of MLL complexes.     

Role of pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during starvation

The PDC (pyruvate dehydrogenase complex) is strongly inhibited by phosphorylation during starvation to conserve substrates for gluconeogenesis. The role of PDHK4 (pyruvate dehydrogenase kinase isoenzyme 4) in regulation of PDC by this mechanism was investigated with PDHK4-/- mice (homozygous PDHK4 knockout mice). Starvation lowers blood glucose more in mice lacking PDHK4 than in wild-type mice. The activity state of PDC (percentage dephosphorylated and active) is greater in kidney, gastrocnemius muscle, diaphragm and heart but not in the liver of starved PDHK4-/- mice. Intermediates of the gluconeogenic pathway are lower in concentration in the liver of starved PDHK4-/- mice, consistent with a lower rate of gluconeogenesis due to a substrate supply limitation. The concentration of gluconeogenic substrates is lower in the blood of starved PDHK4-/- mice, consistent with reduced formation in peripheral tissues. Isolated diaphragms from starved PDHK4-/- mice accumulate less lactate and pyruvate because of a faster rate of pyruvate oxidation and a reduced rate of glycolysis. BCAAs (branched chain amino acids) are higher in the blood in starved PDHK4-/- mice, consistent with lower blood alanine levels and the importance of BCAAs as a source of amino groups for alanine formation. Non-esterified fatty acids are also elevated more in the blood of starved PDHK4-/- mice, consistent with lower rates of fatty acid oxidation due to increased rates of glucose and pyruvate oxidation due to greater PDC activity. Up-regulation of PDHK4 in tissues other than the liver is clearly important during starvation for regulation of PDC activity and glucose homoeostasis.     

Projected US distributions of transgenic and wildtype zebra danios, Danio rerio, based on temperature tolerance data

A genetically modified version of the south Asian, zebra danio, Danio rerio, a common aquarium fish, has become the first transgenic pet sold in the USA. Mean chronic lethal maxima of wildtype (39.8 °C, n=16$n=16$) and transgenic (39.3 °C, n=10$n=10$) zebra danios initially acclimated to 30 °C were statistically (but not dramatically) different as were mean chronic lethal minima of wildtype (5.3 °C, n=16$n=16$) and transgenic (5.6 °C, n=20$n=20$) zebra danios initially acclimated to 20 °C. These temperature tolerance values were used to estimate potential geographic distributions of the two varieties in the USA. Distributions of these D. rerio varieties in the USA should not be limited by their upper temperature tolerances, and low-temperature tolerance data suggest that both varieties are capable of overwintering in some southern and western US waters.     

Validation of high-throughput methods for measuring blood urea nitrogen and urinary albumin concentrations in mice

Chronic kidney disease is a substantial medical and economic burden. Animal models, including mice, are a crucial component of kidney disease research; however, recent studies disprove the ability of autoanalyzer methods to accurately quantify plasma creatinine levels, an established marker of kidney disease, in mice. Therefore, we validated autoanalyzer methods for measuring blood urea nitrogen (BUN) and urinary albumin concentrations, 2 common markers of kidney disease, in samples from mice. We used high-performance liquid chromatography to validate BUN concentrations measured using an autoanalyzer, and we utilized mouse albumin standards to determine the accuracy of the autoanalyzer over a wide range of albumin concentrations. We observed a significant, linear correlation between BUN concentrations measured by autoanalyzer and high-performance liquid chromatography. We also found a linear relationship between known and measured albumin concentrations, although the autoanalyzer method underestimated the known amount of albumin by 3.5- to 4-fold. We confirmed that plasma and urine constituents do not interfere with the autoanalyzer methods for measuring BUN and urinary albumin concentrations. In addition, we verified BUN and albuminuria as useful markers to detect kidney disease in aged mice and mice with 5/6-nephrectomy. We conclude that autoanalyzer methods are suitable for high-throughput analysis of BUN and albumin concentrations in mice. The autoanalyzer accurately quantifies BUN concentrations in mouse plasma samples and is useful for measuring urinary albumin concentrations when used with mouse albumin standards.     

Regulation of gap junction coupling through the neuronal connexin Cx35 by nitric oxide and cGMP

Gap-junctional coupling among neurons is subject to regulation by a number of neurotransmitters including nitric oxide. We studied the mechanisms by which NO regulates coupling in cells expressing Cx35, a connexin expressed in neurons throughout the central nervous system. NO donors caused potent uncoupling of HeLa cells stably transfected with Cx35. This effect was mimicked by Bay 21-4272, an activator of guanylyl cyclase. A pharmacological analysis indicated that NO-induced uncoupling involved both PKG-dependent and PKG-independent pathways. PKA was involved in both pathways, suggesting that PKG-dependent uncoupling may be indirect. In vitro, PKG phosphorylated Cx35 at three sites: Ser110, Ser276, and Ser289. A mutational analysis indicated that phosphorylation on Ser110 and Ser276, sites previously shown also to be phosphorylated by PKA, had a significant influence on regulation. Ser289 phosphorylation had very limited effects. We conclude that NO can regulate coupling through Cx35 and that regulation is indirect in HeLa cells.     

A novel RNAi library based on partially randomized consensus sequences of nuclear receptors: identifying the receptors involved in amyloid beta degradation

Combinatorial gene inactivation using an RNAi library is a powerful approach to discovering novel functional genes. However, generation of a comprehensive RNAi library remains technically challenging. In this report, we describe a simple and novel approach to designing gene-family-specific RNAi libraries by targeting conserved motifs using degenerate oligonucleotides. We created an siRNA library in the pHUMU vector using partially randomized sequences targeting the consensus region in the ZnF_C4 signature motif of the nuclear hormone receptors and thus against the entire receptor superfamily. For proof of principle, we adapted a reporter assay to screen this library for receptors that might be involved in reducing amyloid beta peptide accumulation. We modified a previously described luciferase reporter assay to measure the amyloid beta precursor cleavages occurring only between beta- and gamma-secretase cleavage sites, thus excluding the major gamma-secretase activities that could generate neurotoxic Abeta peptides. Our screen using this assay identified siRNA vectors that specifically increase the Abeta40/42 cleavage and pointed to a potential receptor target, ROR-gamma. SiRNAs targeting other regions of ROR-gamma not only confirmed the observed reporter activity but also reduced the level of the toxic Abeta peptides. The results demonstrated a general principle for the creation and application of this RNAi library approach for functional gene discovery within a predefined protein family. The discovered negative effect of ROR-gamma on the degradation of the toxic Abeta peptides may also provide a potential drug target or targetable pathway for intervention of Alzheimer disease.