High-resolution crystal structures of villin headpiece and mutants with reduced F-actin binding activity

Villin-type headpiece domains are approximately 70 amino acid modular motifs found at the C terminus of a variety of actin cytoskeleton-associated proteins. The headpiece domain of villin, a protein found in the actin bundles of the brush border epithelium, is of interest both as a compact F-actin binding domain and as a model folded protein. We have determined the high-resolution crystal structures of chicken villin headpiece (HP67) at 1.4 A resolution as well as two mutants, R37A and W64Y, at 1.45 and 1.5 A resolution, respectively. Replacement of R37 causes a 5-fold reduction in F-actin binding affinity in sedimentation assays. Replacement of W64 results in a much more drastic reduction in F-actin binding affinity without significant changes in headpiece structure or stability. The detailed comparison of these crystal structures with each other and to our previously determined NMR structures of HP67 and the 35-residue autonomously folding subdomain in villin headpiece, HP35, provides the details of the headpiece fold and further defines the F-actin binding site of villin-type headpiece domains.     

Nutrient-stimulated insulin secretion in mouse islets is critically dependent on intracellular pH

BACKGROUND: Many mechanistic steps underlying nutrient-stimulated insulin secretion (NSIS) are poorly understood. The influence of intracellular pH (pHi) on insulin secretion is widely documented, and can be used as an investigative tool. This study demonstrates previously unknown effects of pHi-alteration on insulin secretion in mouse islets, which may be utilized to correct defects in insulin secretion. METHODS: Different components of insulin secretion in mouse islets were monitored in the presence and absence of forced changes in pHi. The parameters measured included time-dependent potentiation of insulin secretion by glucose, and direct insulin secretion by different mitochondrial and non-mitochondrial secretagogues. Islet pHi was altered using amiloride, removal of medium Cl-, and changing medium pH. Resulting changes in islet pHi were monitored by confocal microscopy using a pH-sensitive fluorescent indicator. To investigate the underlying mechanisms of the effects of pHi-alteration, cellular NAD(P)H levels were measured using two-photon excitation microscopy (TPEM). Data were analyzed using Student's t test. RESULTS: Time-dependent potentiation, a function normally absent in mouse islets, can be unmasked by a forced decrease in pHi. The optimal range of pHi for NSIS is 6.4-6.8. Bringing islet pHi to this range enhances insulin secretion by all mitochondrial fuels tested, reverses the inhibition of glucose-stimulated insulin secretion (GSIS) by mitochondrial inhibitors, and is associated with increased levels of cellular NAD(P)H. CONCLUSIONS: Pharmacological alteration of pHi is a potential means to correct the secretory defect in non-insulin dependent diabetes mellitus (NIDDM), since forcing islet pHi to the optimal range enhances NSIS and induces secretory functions that are normally absent.     

Synthesizing and databasing fossil calibrations: divergence dating and beyond

Divergence dating studies, which combine temporal data from the fossil record with branch length data from molecular phylogenetic trees, represent a rapidly expanding approach to understanding the history of life. National Evolutionary Synthesis Center hosted the first Fossil Calibrations Working Group (3-6 March, 2011, Durham, NC, USA), bringing together palaeontologists, molecular evolutionists and bioinformatics experts to present perspectives from disciplines that generate, model and use fossil calibration data. Presentations and discussions focused on channels for interdisciplinary collaboration, best practices for justifying, reporting and using fossil calibrations and roadblocks to synthesis of palaeontological and molecular data. Bioinformatics solutions were proposed, with the primary objective being a new database for vetted fossil calibrations with linkages to existing resources, targeted for a 2012 launch.     

A prion protein epitope selective for the pathologically misfolded conformation

Conformational conversion of proteins in disease is likely to be accompanied by molecular surface exposure of previously sequestered amino-acid side chains. We found that induction of beta-sheet structures in recombinant prion proteins is associated with increased solvent accessibility of tyrosine. Antibodies directed against the prion protein repeat motif, tyrosine-tyrosine-arginine, recognize the pathological isoform of the prion protein but not the normal cellular isoform, as assessed by immunoprecipitation, plate capture immunoassay and flow cytometry. Antibody binding to the pathological epitope is saturable and specific, and can be created in vitro by partial denaturation of normal brain prion protein. Conformation-selective exposure of Tyr-Tyr-Arg provides a probe for the distribution and structure of pathologically misfolded prion protein, and may lead to new diagnostics and therapeutics for prion diseases.     

Differential expression, induction, and stability of CYP1A4 and CYP1A5 mRNA in chicken and herring gull embryo hepatocytes

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces cytochrome P4501A (CYP1A) catalyzed ethoxyresorufin-O-deethylase (EROD) activity in chickens and other avian species. To investigate mechanisms underlying the effectiveness of EROD activity as a biomarker for exposure to dioxin-like compounds in avian models, we characterized inter-species differences in isoform-specific CYP1A mRNA expression, induction, and stability in chickens (Gallus gallus domesticus) and herring gulls (Larus argentatus). Exposure to 100 nM TCDD significantly increased CYP1A4 and CYP1A5 mRNA expression in chicken and herring gull embryo hepatocyte cultures. Chicken CYP1A4 and CYP1A5 were induced 61-fold and 25-fold respectively. The herring gull isoforms were induced 2.2- and 4.3-fold respectively. In both species, the isoform that was preferentially induced exhibited lower constitutive expression. Half-lives of chicken CYP1A4, chicken CYP1A5, and herring gull CYP1A5 mRNA ranged from 5.0 to 7.0 h in cultured hepatocytes. The half-life of herring gull CYP1A4 mRNA was 2.5 h. Our findings indicate that expression, induction, and stability of CYP1A4 and CYP1A5 mRNA are differentially regulated in chickens and herring gulls. In particular, CYP1A4 is preferentially induced in chickens, while CYP1A5 is preferentially induced in herring gulls. We propose that CYP1A5 mRNA expression may be a sensitive biomarker of exposure to dioxin-like compounds in some avian species.     

Evidence for the extralenticular expression of members of the β-crystallin gene family in the chick and a comparison with δ-crystallin during differentiation and transdifferentiation

The beta-crystallins are major water soluble proteins of vertebrate lens fibre cells and have previously been regarded as lens-specific proteins: however beta B2-and beta A3/A1-crystallin RNAs are transcribed and beta-crystallin polypeptides are detectable in the developing chick retina. The beta-crystallin RNA is transcribed in a subpopulation of retina cells and the number of transcribing cells and the level of beta-crystallin polypeptides increase during the differentiation of the retina. Several tissues express beta-crystallin polypeptides, but individual tissues are characterised by qualitative and quantitative differences in the beta- and delta-crystallin polypeptides expressed. The expression of beta-crystallins appears to be non-random as defined by tissue distribution, cellular localisation and ontogeny, implying a function for extralenticular beta-crystallins and a complex mechanism for the regulation of their expression.     

The effect of positive air ions on reproduction and growth in laboratory rats

The aim of the present investigation was to determine the growth rates, reproductive success and early mortality of laboratory rats maintained at 10,000 positive ions/ml over two generations. These findings were compared with those from animals maintained at ambient ion levels. The present work indicates that positive ions do not have any adverse effects on the reproductive capabilities or the growth of laboratory rats. In contrast it is shown that exposure to elevated levels of positive ions promotes overall growth, particularly in male rats. This action of positive ions increases with each successive generation exposed to the ions. It is suggested that the growth promoting effect of positive ions may be mediated via some modulation of the endocrine system.     

Potent induction of TNF-α during interaction of immune effectors with oral tumors as a potential mechanism for the loss of NK cell viability and function

The inhibitory role of TNF-α on survival of naïve and IL-2 treated NK cells has been demonstrated in the past. However, its effect on the function of these cells against tumor cells, in particular against oral tumors has not been established. We investigated the significance of secreted TNF-α in death and functional loss of splenocytes and NK cells in ex-vivo cultures with oral tumors. Oral tumors trigger potent secretion of TNF-α by human and murine immune effectors. Absence of TNF-α increases the cytotoxic activity and secretion of IFN-γ by IL-2 treated splenocytes and NK cells in co-cultures with MOK L2D1+/p53?/? oral tumor cells. IL-2 treated splenocytes and NK cells from TNF-α ?/? mice survive and proliferate more when compared to cells from TNF-α +/+ mice. Cell death induced by F. nucleatum, an oral bacteria, in TNF-α ?/? splenocytes are considerably lower than that induced in TNF-α +/+ splenocytes where potent release of TNF-α is reproducibly observed. Addition of exogenous rTNF-α to IL-2 treated splenocytes and NK cells decreased survival and function of splenocytes and NK cells obtained from TNF-α ?/? mice against oral tumors. These findings suggest that potent induction of TNF-α during interaction of immune effectors with oral tumors and/or oral bacteria is an important factor in decreasing the function and survival of cytotoxic immune effectors. Strategies to neutralize TNF-α may be beneficial in the treatment of oral cancers.