Two tarantula peptides inhibit activation of multiple sodium channels

Two peptides, ProTx-I and ProTx-II, from the venom of the tarantula Thrixopelma pruriens, have been isolated and characterized. These peptides were purified on the basis of their ability to reversibly inhibit the tetrodotoxin-resistant Na channel, Na(V) 1.8, and are shown to belong to the inhibitory cystine knot (ICK) family of peptide toxins interacting with voltage-gated ion channels. The family has several hallmarks: cystine bridge connectivity, mechanism of channel inhibition, and promiscuity across channels within and across channel families. The cystine bridge connectivity of ProTx-II is very similar to that of other members of this family, i.e., C(2) to C(16), C(9) to C(21), and C(15) to C(25). These peptides are the first high-affinity ligands for tetrodotoxin-resistant peripheral nerve Na(V) channels, but also inhibit other Na(V) channels (IC(50)'s < 100 nM). ProTx-I and ProTx-II shift the voltage dependence of activation of Na(V) 1.5 to more positive voltages, similar to other gating-modifier ICK family members. ProTx-I also shifts the voltage dependence of activation of Ca(V) 3.1 (alpha(1G), T-type, IC(50) = 50 nM) without affecting the voltage dependence of inactivation. To enable further structural and functional studies, synthetic ProTx-II was made; it adopts the same structure and has the same functional properties as the native peptide. Synthetic ProTx-I was also made and exhibits the same potency as the native peptide. Synthetic ProTx-I, but not ProTx-II, also inhibits K(V) 2.1 channels with 10-fold less potency than its potency on Na(V) channels. These peptides represent novel tools for exploring the gating mechanisms of several Na(V) and Ca(V) channels.     

Pathological apoptosis by xanthurenic acid, a tryptophan metabolite: activation of cell caspases but not cytoskeleton breakdown

Background  

A family of aspartate-specific cysteinyl proteases, named caspases, mediates programmed cell death, apoptosis. In this function, caspases are important for physiological processes such as development and maintenance of organ homeostasis. Caspases are, however, also engaged in aging and disease development. The factors inducing age-related caspase activation are not known. Xanthurenic acid, a product of tryptophan degradation, is present in blood and urine, and accumulates in organs with aging.     

Immunohistochemical localization of S100 beta in human nervous system tumors by using monoclonal antibodies with specificity for the S100 beta polypeptide

The immunohistochemical localization of the calcium-binding protein, S100 beta, in human nervous system tumors has been examined by using a monoclonal antibody with specificity for the S100 beta polypeptide. S100 beta-specific immunoreactivity is detected in astrocytoma, glioblastoma, Schwannoma, ependymoma, and craniopharyngioma, whereas no reactivity is seen in oligodendroglioma, meningioma, neuroblastoma, or medulloblastoma. These data suggest that analysis of S100 beta localization with these monoclonal antibodies may be useful for research or diagnostic purposes.     

Site-specific Nitration of Apolipoprotein A-I at Tyrosine 166 Is Both Abundant within Human Atherosclerotic Plaque and Dysfunctional

We reported previously that apolipoprotein A-I (apoA-I) is oxidatively modified in the artery wall at tyrosine 166 (Tyr¹⁶⁶), serving as a preferred site for post-translational modification through nitration. Recent studies, however, question the extent and functional importance of apoA-I Tyr¹⁶⁶ nitration based upon studies of HDL-like particles recovered from atherosclerotic lesions. We developed a monoclonal antibody (mAb 4G11.2) that recognizes, in both free and HDL-bound forms, apoA-I harboring a 3-nitrotyrosine at position 166 apoA-I (NO₂-Tyr¹⁶⁶-apoA-I) to investigate the presence, distribution, and function of this modified apoA-I form in atherosclerotic and normal artery wall. We also developed recombinant apoA-I with site-specific 3-nitrotyrosine incorporation only at position 166 using an evolved orthogonal nitro-Tyr-aminoacyl-tRNA synthetase/tRNA_CUA pair for functional studies. Studies with mAb 4G11.2 showed that NO₂-Tyr¹⁶⁶-apoA-I was easily detected in atherosclerotic human coronary arteries and accounted for ∼8% of total apoA-I within the artery wall but was nearly undetectable (>100-fold less) in normal coronary arteries. Buoyant density ultracentrifugation analyses showed that NO₂-Tyr¹⁶⁶-apoA-I existed as a lipid-poor lipoprotein with <3% recovered within the HDL-like fraction (d = 1.063–1.21). NO₂-Tyr¹⁶⁶-apoA-I in plasma showed a similar distribution. Recovery of NO₂-Tyr¹⁶⁶-apoA-I using immobilized mAb 4G11.2 showed an apoA-I form with 88.1 ± 8.5% reduction in lecithin-cholesterol acyltransferase activity, a finding corroborated using a recombinant apoA-I specifically designed to include the unnatural amino acid exclusively at position 166. Thus, site-specific nitration of apoA-I at Tyr¹⁶⁶ is an abundant modification within the artery wall that results in selective functional impairments. Plasma levels of this modified apoA-I form may provide insights into a pathophysiological process within the diseased artery wall.     

High‐yield recombinant bacterial expression of 13C‐, 15N‐labeled,serine‐16 phosphorylated,murine amelogenin using a modified third generation genetic code expansion protocol

Amelogenin constitutes ~90% of the enamel matrix in the secretory stage of amelogenesis, a still poorly understood process that results in the formation of the hardest and most mineralized tissue in vertebrates—enamel. Most biophysical research with amelogenin uses recombinant protein expressed in Escherichia coli. In addition to providing copious amounts of protein, recombinant expression allows ¹³C‐ and ¹⁵N‐labeling for detailed structural studies using NMR spectroscopy. However, native amelogenin is phosphorylated at one position, Ser‐16 in murine amelogenin, and there is mounting evidence that Ser‐16 phosphorylation is important. Using a modified genetic code expansion protocol we have expressed and purified uniformly ¹³C‐, ¹⁵N‐labeled murine amelogenin (pS16M179) with ~95% of the protein being correctly phosphorylated. Homogeneous phosphorylation was achieved using commercially available, enriched, ¹³C‐, ¹⁵N‐labeled media, and protein expression was induced with isopropyl β‐D‐1‐thiogalactopyranoside at 310 K. Phosphoserine incorporation was verified from one‐dimensional ³¹P NMR spectra, comparison of ¹H‐¹⁵N HSQC spectra, Phos‐tag SDS PAGE, and mass spectrometry. Phosphorus‐31 NMR spectra for pS16M179 under conditions known to trigger amelogenin self‐assembly into nanospheres confirm nanosphere models with buried N‐termini. Lambda phosphatase treatment of these nanospheres results in the dephosphorylation of pS16M179, confirming that smaller oligomers and monomers with exposed N‐termini are in equilibrium with nanospheres. Such ¹³C‐, ¹⁵N‐labeling of amelogenin with accurately encoded phosphoserine incorporation will accelerate biomineralization research to understand amelogenesis and stimulate the expanded use of genetic code expansion protocols to introduce phosphorylated amino acids into proteins.     

Using the Lives Saved Tool (LiST) to Model mHealth Impact on Neonatal Survival in Resource-Limited Settings

While the importance of mHealth scale-up has been broadly emphasized in the mHealth community, it is necessary to guide scale up efforts and investment in ways to help achieve the mortality reduction targets set by global calls to action such as the Millennium Development Goals, not merely to expand programs. We used the Lives Saved Tool (LiST)–an evidence-based modeling software–to identify priority areas for maternal and neonatal health services, by formulating six individual and combined interventions scenarios for two countries, Bangladesh and Uganda. Our findings show that skilled birth attendance and increased facility delivery as targets for mHealth strategies are likely to provide the biggest mortality impact relative to other intervention scenarios. Although further validation of this model is desirable, tools such as LiST can help us leverage the benefit of mHealth by articulating the most appropriate delivery points in the continuum of care to save lives.     

Nucleation and Crystal Growth in a Vitrification Solution Tested for Organ Cryopreservation by Vitrification

Nucleation and crystal growth are investigated for vitrification solution VS41A (dimethyl sulfoxide, formamide, and 1,2-propanediol) in an aqueous carrier solution giving, when added to these three cryoprotectants, a concentration of other solutes in the whole solution the same as that in Euro-Collins, with a 55% (w/v) cryoprotectant concentration. This concentration is assumed to achieve physical properties under 1 atmosphere similar to those of solution VS4 used under 1000 atmospheres. The thermal range and the kinetics of nucleation and crystal growth are investigated by DSC through different thermal treatments. It is found that the nucleation thermal range is below -90 degrees C and that of crystal growth is above -85 degrees C for a relatively long experimental time. The nucleation density is also studied through direct observations by cryomicroscopy and is related to the amount of crystallization calorimetrically recorded. The effect of storage below the glass transition shows the possibility of a slow increase in nucleation below the glass transition, as already observed by other authors for different aqueous solutions. Isothermal crystallization is analyzed within the Johnson-Mehl-Avrami model for temperatures above -75 degrees C. The corresponding samples have been cooled and warmed at the same rate of 40 degrees C/min and calculations give, at constant nuclei numbers, an activation energy of 9.3 +/- 0.3 kcal/mol and the Avrami exponent n = 2.2 +/- 0.05. This shows a two-dimensional crystal growth as observed by cryomicroscopy. The estimated critical warming rate relevant to the preservation of rabbit kidneys by vitrification is 270 degrees C/min with or without an increase in the nucleus density during storage. The present results support the possibility of using VS4 solution for vitrification of rabbit kidneys if pressure is not a limiting factor. Copyright 1993, 1999 Academic Press.     

Hydrogel biopolymer created from the self-assembly of a designed protein containing a four-helix bundle forming motif

A protein hydrogel system based on the assembly of a four-helix bundle motif was proposed and synthesized by genetic engineering methods. This new polypeptide, named GBH1, consists of identical amphipathic helices of 22 residues in length oriented in opposite fashion to one another at each end of a polypeptide with a total length of 227 amino acids. The middle portion of the polypeptide (residues 79-147) is an unstructured random coil. The region between the amphipathic and unstructured segment is an α-helical stretch (23-78 and 148-204) not possessing a sequence compatible with a coiled-coil conformation, but rather possesses regions that have overwinding of the helix. The thermal unfolding of GBH1 shows more than one inflection point (T(m1) = 30.5 °C, T(m2) = 64.6 °C), indicative of a partially unfolded intermediate and, thus, multiple interactions in the folded state. A qualitative assessment of hydrogel formation with varying pH showed that acidic conditions did not support the gel state, indirectly indicating that the proposed four-helix bundle is the major cross-linking structure and not a leucine zipper motif. Scanning electron microscopy reveals a network of interacting protein molecules forming a spongelike matrix with numerous pores that would be occupied with water molecules.