Misfolding of vWF to Pathologically Disordered Conformations Impacts the Severity of von Willebrand Disease

The primary hemostatic von Willebrand factor (vWF) functions to sequester platelets from rheological blood flow and mediates their adhesion to damaged subendothelium at sites of vascular injury. We have surveyed the effect of 16 disease-causing mutations identified in patients diagnosed with the bleeding diathesis disorder, von Willebrand disease (vWD), on the structure and rheology of vWF A1 domain adhesiveness to the platelet GPIbα receptor. These mutations have a dynamic phenotypical range of bleeding from lack of platelet adhesion to severe thrombocytopenia. Using new rheological tools in combination with classical thermodynamic, biophysical, and spectroscopic metrics, we establish a high propensity of the A1 domain to misfold to pathological molten globule conformations that differentially alter the strength of platelet adhesion under shear flow. Rheodynamic analysis establishes a quantitative rank order between shear-rate-dependent platelet-translocation pause times that linearly correlate with clinically reported measures of patient platelet counts and the severity of thrombocytopenia. These results suggest that specific secondary structure elements remaining in these pathological conformations of the A1 domain regulate GPIbα binding and the strength of vWF-platelet interactions, which affects the vWD functional phenotype and the severity of thrombocytopenia.     

Suppressible P-element alleles of the vestigial locus in Drosophila melanogaster

Summary A series of P-element insertion mutations at one site in the vestigial (vg) locus was tested for cytotype dependent effects on vg expression. The mutant phenotypes for four P-element vg alleles were suppressed when the alleles were stabilized in the P-cytotype. The suppression was observed whenever repressor-producing P-elements were present in the genome. Genetic and molecular analysis indicated that the suppression is not due to excision or other irreversible alterations of the inserts. The results are consistent with a model in which somatic P-element repressor binding to the ends of P-element inserts can modify the effects of these inserts on target gene expression.     

Possible interrelationship between vitamins E and b12 in the disturbance in methylmalonate metabolism in vitamin E deficiency.

The presence of three major proteins alpha, beta and gamma in rat ventral prostate was demonstrated by electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. Their regulation by androgens was studied by measuring the rates of synthesis of the proteins in minced prostatic tissue by using L-[35S]methionine. The three proteins account for 30-40% of the proteins synthesized in the gland. After castration, their rates of synthesis rapidly decline to about 1% that of normal animals, and this cannot be accounted for by the accompanying decrease in general protein synthesis. Testosterone reverses these changes in castrated animals, so that after 4 days normal synthesis is restored. The regulation is specific for androgens, since cyproterone acetate, an anti-androgen, is inhibitory and oestradiol-17beta and corticosterone are without effect. Preliminary characterization of the proteins indicates that protein alpha (mol.wt. 22000, pI unknown) is a glycoprotein containing glucose and/or mannose residues and occurs in both the mitochondrial and cytosol fractions. Protein beta (mol.wt. 12000, pI5.4) is also a glycoprotein, but is found exclusively in the cytosol fraction. Protein gamma (mol.wt. 8000, pI5.4) is also a glycoprotein, but is found exclusively in the cytosol fraction. Protein gamma (mol.wt. 8000, pI5.4) is also found exclusively in the cytosol fraction.     

Alterations of Phospholipid Metabolism in Rat Cerebral Cortex Mince Induced by Cationic Amphiphilic Drugs

Abstract Cationic amphiphilic drugs (CADs) of varied clinical use were screened to determine their capacity to alter the pattern of labeling with ³²Pj of cerebral cortex mince phospholipids. The altered phospholipid labeling patterns were qualitatively similar, the prominent features being reduced incorporation into phosphatidylcholine and increased incorporation into phosphatidic acid. Relative potencies were: (±)-propranolol > chlorpromazine = 4,4'-bis(diethylaminoethoxy) α,β -diethyldiphenylethane > desipramine > di-bucaine > pimozide > oxymetazoline = fenfluramine = haloperidol = chloroquine > amphetamine = no drug added. Propranolol was used to study the action of CADs further. Its effect was time- and dose-dependent, but in contrast with pineal gland, no label appeared in phosphatidyl-CMP (CDP-diacylglycerol), nor did dialysis of the mince to reduce diffusible substrates or exogenous addition of substrates cause appearance of liponucleotide. Thus lack of diffusible precursors is not responsible for CAD effects in vitro. Pulse-chase experiments with ³²P₁ and [2-³H]glycerol suggested that inhibition of phosphatidate phosphohydrolase may be partly responsible for the observed alterations in phospholipid labeling in the presence of CADs.     

Morphological and molecular screening of rice germplasm lines for low soil P tolerance

Phosphorus (P) is an essential macronutrient to all crops including rice and it plays a key role in various plant activities and development. Low availability of P in the soils negatively, influences rice crop growth and causes significant yield loss. In the present study, we characterized a set of 56 germplasm lines for their tolerance to low soil P by screening them at low soil P and optimum soil P levels along with low soil P tolerant and sensitive check varieties. These lines were genotyped for the presence/absence of tolerant allele with respect to the major low soil P tolerance QTL, Pup1, using a set of locus specific PCR-based markers, viz., K46-1, K46-2, K52 and K46CG-1. High genetic variability was observed for various traits associated with low soil P tolerance. The yield parameters from normal and low soil P conditions were used to calculate stress tolerance indices and classify the genotypes according to their tolerance level. Out of the total germplasm lines screened, 15 lines were found to be tolerant to low soil P condition based on the yield reduction in comparison to the tolerant check, but most of them harbored the complete or partial Pup1 locus. Interestingly, two tolerant germplasm lines, IC216831 and IC216903 were observed to be completely devoid of Pup1 and hence they can be explored for new loci underlying low soil P tolerance.

     

Tristetraprolin Mediates Radiation-Induced TNF-α Production in Lung Macrophages

The efficacy of radiation therapy for lung cancer is limited by radiation-induced lung toxicity (RILT). Although tumor necrosis factor-alpha (TNF-α) signaling plays a critical role in RILT, the molecular regulators of radiation-induced TNF-α production remain unknown. We investigated the role of a major TNF-α regulator, Tristetraprolin (TTP), in radiation-induced TNF-α production by macrophages. For in vitro studies we irradiated (4 Gy) either a mouse lung macrophage cell line, MH-S or macrophages isolated from TTP knockout mice, and studied the effects of radiation on TTP and TNF-α levels. To study the in vivo relevance, mouse lungs were irradiated with a single dose (15 Gy) and assessed at varying times for TTP alterations. Irradiation of MH-S cells caused TTP to undergo an inhibitory phosphorylation at Ser-178 and proteasome-mediated degradation, which resulted in increased TNF-α mRNA stabilization and secretion. Similarly, MH-S cells treated with TTP siRNA or macrophages isolated from ttp (−/−) mice had higher basal levels of TNF-α, which was increased minimally after irradiation. Conversely, cells overexpressing TTP mutants defective in undergoing phosphorylation released significantly lower levels of TNF-α. Inhibition of p38, a known kinase for TTP, by either siRNA or a small molecule inhibitor abrogated radiation-induced TNF-α release by MH-S cells. Lung irradiation induced TTP^Ser178 phosphorylation and protein degradation and a simultaneous increase in TNF-α production in C57BL/6 mice starting 24 h post-radiation. In conclusion, irradiation of lung macrophages causes TTP inactivation via p38-mediated phosphorylation and proteasome-mediated degradation, leading to TNF-α production. These findings suggest that agents capable of blocking TTP phosphorylation or stabilizing TTP after irradiation could decrease RILT.     

Quantitative characterization of intrinsic disorder in polyglutamine: insights from analysis based on polymer theories

Intrinsically disordered proteins (IDPs) are unfolded under physiological conditions. Here we ask if archetypal IDPs in aqueous milieus are best described as swollen disordered coils in a good solvent or collapsed disordered globules in a poor solvent. To answer this question, we analyzed data from molecular simulations for a 20-residue polyglutamine peptide and concluded, in accord with experimental results, that water is a poor solvent for this system. The relevance of monomeric polyglutamine is twofold: It is an archetypal IDP sequence and its aggregation is associated with nine neurodegenerative diseases. The main advance in this work lies in our ability to make accurate assessments of solvent quality from analysis of simulations for a single, rather than multiple chain lengths. We achieved this through the proper design of simulations and analysis of order parameters that are used to describe conformational equilibria in polymer physics theories. Despite the preference for collapsed structures, we find that polyglutamine is disordered because a heterogeneous ensemble of conformations of equivalent compactness is populated at equilibrium. It is surprising that water is a poor solvent for polar polyglutamine and the question is: why? Our preliminary analysis suggests that intrabackbone interactions provide at least part of the driving force for the collapse of polyglutamine in water. We also show that dynamics for conversion between distinct conformations resemble structural relaxation in disordered, glassy systems, i.e., the energy landscape for monomeric polyglutamine is rugged. We end by discussing generalizations of our methods to quantitative studies of conformational equilibria of other low-complexity IDP sequences.     

Hydrodynamic interaction between erythrocytes and leukocytes affects rheology of blood in microvessels

Hydrodynamic interaction between erythrocytes (RBC) and leukocytes (WBC) in a microvessel of size 20-40 micron, typical of a postcapillary venule, is studied using a two-dimensional computational model. The model is based on immersed boundary method, and it takes into consideration the particulate nature of blood by explicitly modeling individual blood cell, and cell deformation. Due to their highly flexible nature, RBC drift away from the wall and toward the center of a vessel creating a cell-free layer. It is shown here that the lateral motion of RBC is strongly affected in presence of a WBC, and is dependent on whether the WBC is non-adherent or firmly adhered. When the WBC is non-adherent, some RBC, depending on their initial radial locations and vessel size, may be deflected closer toward the wall, resulting in a decrease in the cell-free layer. The apparent viscosity of the whole blood containing both RBC and WBC is computed, and shown to be much higher than that containing RBC only. The increased viscosity cannot be accounted for by the contribution due to WBC only. This observation is in agreement with a previous in vivo measurement. Here we show that the additional flow resistance is due to the decrease in the cell-free layer resulting from the WBC-RBC interaction. It can be accounted for by a two-layer model of blood when the reduced values of the cell-free layer thickness are used. When the WBC is firmly adhered, RBC easily move away from the wall, and the cell-free layer is not significantly changed. In such cases, the major contribution to whole blood viscosity comes from the WBC alone. The hydrodynamic interaction between WBC and RBC, though it exists, does not contribute significantly when WBC are adhered.     

A simple model for polyproline II structure in unfolded states of alanine-based peptides

The striking similarity between observed circular dichroism spectra of nonprolyl homopolymers and that of regular left-handed polyproline II (P_II) helices prompted Tiffany and Krimm to propose in 1968 that unordered peptides and unfolded proteins are built of P_II segments linked by sharp bends. A large body of experimental evidence, accumulated over the past three decades, provides compelling evidence in support of the original hypothesis of Tiffany and Krimm. Of particular interest are the recent experiments of Shi et al. who find significant P_II structure in a short unfolded alanine-based peptide. What is the physical basis for P_II helices in peptide and protein unfolded states? The widely accepted view is that favorable chain-solvent hydrogen bonds lead to a preference for dynamical fluctuations about noncooperative P_II helices in water. Is this preference simply a consequence of hydrogen bonding or is it a manifestation of a more general trend for unfolded states which are appropriately viewed as chains in a good solvent? The prevalence of closely packed interiors in folded proteins suggests that under conditions that favor folding, water—which is a better solvent for itself than for any polypeptide chain—expels the chain from its midst, thereby maximizing chain packing. Implicit in this view is a complementary idea: under conditions that favor unfolding, chain-solvent interactions are preferred and in a so-called good solvent, chain packing density is minimized. In this work we show that minimization of chain packing density leads to preferred fluctuations for short polyalanyl chains around canonical, noncooperative P_II-like conformations. Minimization of chain packing is modeled using a purely repulsive soft-core potential between polypeptide atoms. Details of chain-solvent interactions are ignored. Remarkably, the simple model captures the essential physics behind the preference of short unfolded alanine-based peptides for P_II helices. Our results are based on a detailed analysis of the potential energy landscape which determines the system''s structural and thermodynamic preferences. We use the inherent structure formalism of Stillinger and Weber, according to which the energy landscape is partitioned into basins of attraction around local minima. We find that the landscape for the experimentally studied seven-residue alanine-based peptide is dominated by fluctuations about two noncooperative structures: the left-handed polyproline II helix and its symmetry mate.