Effect of inhibitors on conformational changes in hen lysozyme around thermal transition point in solution and solid state

Carbon-13 NMR spectroscopy has been used to further document the interaction, at low and high temperatures, of N-acetylglucosamine and its short polymers with hen egg-white lysozyme. The results have been compared with the corresponding X-ray crystallographic data. Two domains, the active site and the hydrophobic box, have been found by NMR to undergo conformational rearrangement while X-ray crystallography only detected changes located in the active site. The extent of the modifications induced by inhibitor binding was proportional to the inhibitor size. The two techniques concurred to show that even in the presence of monosaccharide (N-acetylglucosamine), more than one subsite of the enzyme was occupied at high temperature, the binding at the C-site being the best defined. The thermal transition of lysozyme still occurred in solution when inhibitors were bound. However, in the solid state, crystallographic data showed that the transition was hindered.     

Syndecan 4 Is Involved in Mediating HCV Entry through Interaction with Lipoviral Particle-Associated Apolipoprotein E

Hepatitis C virus (HCV) is a major cause of liver disease worldwide and HCV infection represents a major health problem. HCV associates with host lipoproteins forming host/viral hybrid complexes termed lipoviral particles. Apolipoprotein E (apoE) is a lipoprotein component that interacts with heparan sulfate proteoglycans (HSPG) to mediate hepatic lipoprotein uptake, and may likewise mediate HCV entry. We sought to define the functional regions of apoE with an aim to identify critical apoE binding partners involved in HCV infection. Using adenoviral vectors and siRNA to modulate apoE expression we show a direct correlation of apoE expression and HCV infectivity, whereas no correlation exists with viral protein expression. Mutating the HSPG binding domain (HSPG-BD) of apoE revealed key residues that are critical for mediating HCV infection. Furthermore, a novel synthetic peptide that mimics apoE’s HSPG-BD directly and competitively inhibits HCV infection. Genetic knockdown of the HSPG proteins syndecan (SDC) 1 and 4 revealed that SDC4 principally mediates HCV entry. Our data demonstrate that HCV uses apoE-SDC4 interactions to enter hepatoma cells and establish infection. Targeting apoE-SDC interactions could be an alternative strategy for blocking HCV entry, a critical step in maintaining chronic HCV infection.     

Multiple molecular forms of rat superior cervical ganglion acetylcholinesterase: Developmental aspects in primary cell culture and during postnatal maturation in vivo

Four main molecular forms of acetylcholinesterase (AChE) can be solubilized from newborn rat superior cervical ganglia (SCG), homogenized in the presence of a high-ionic-strength, detergent-containing medium. These forms, respectively referred to as 16, 10, 6.5, and 4 S, are characterized by their sedimentation coefficients. Their relative proportions in SCG are notably different in vivo during postnatal maturation, and in culture. The 16-S AChE appears to be mainly neuronal in origin, is maintained in culture independently of original presynaptic in vivo elements, and its cellular pool is not depleted in the presence of tetrodotoxin (TTX).     

Decrease of calcium turnover during the onset of mineralocorticoid hypertension in the rat.

Administration of choline chloride i.p. to rats causes a dose-dependent increase in the brain concentration of the neurotransmitter, acetylcholine (ACh). This increase is maximal (22% after a 60-mg/kg dose) 40 minutes after injection. These observations suggest that precursor availability may influence brain ACh synthesis, just as brain tryptophan and tyrosine levels have previously been shown to control the synthesis of brain serotonin and catecholamines.     

Space use and genetic structure do not maintain color polymorphism in a species with alternative behavioral strategies

Space use including territoriality and spatial arrangement within a population can reveal important information on the nature, dynamics, and evolutionary maintenance of alternative strategies in color polymorphic species. Despite the prevalence of color polymorphic species as model systems in evolutionary biology, the interaction between space use and genetic structuring of morphs within populations has rarely been examined. Here, we assess the spatial and genetic structure of male throat color morphs within a population of the tawny dragon lizard, Ctenophorus decresii. Male color morphs do not differ in morphology but differ in aggressive and antipredator behaviors as well as androgen levels. Despite these behavioral and endocrine differences, we find that color morphs do not differ in territory size, with their spatial arrangement being essentially random with respect to each other. There were no differences in genetic diversity or relatedness between morphs; however, there was significant, albeit weak, genetic differentiation between morphs, which was unrelated to geographic distance between individuals. Our results indicate potential weak barriers to gene flow between some morphs, potentially due to nonrandom pre‐ or postcopulatory mate choice or postzygotic genetic incompatibilities. However, space use, spatial structure, and nonrandom mating do not appear to be primary mechanisms maintaining color polymorphism in this system, highlighting the complexity and variation in alternative strategies associated with color polymorphism.     

Agonists cause endocytosis of nicotinic acetylcholine receptors on cultured myotubes.

Regulated trafficking of neurotransmitter receptors in excitable cells may play an important role in synaptic plasticity. In addition, agonist-induced endocytosis of nicotinic acetylcholine receptors (nAChRs) in particular might be involved in nicotine tolerance and addiction. The existing evidence concerning regulated internalization of cell-surface nAChRs is indirect and equivocal, however. In the present study, radioligand binding and fluorescence microscopy were used to show that agonists cause substantial endocytosis of nAChRs on cultured myotubes. Exposure to carbachol or nicotine caused a decrease in the intensity of fluorescent labeling of clusters of cell-surface nAChRs that was blocked by low temperature. Overall, myotubes exposed to carbachol or nicotine bound 50-70% less [(125)I]-alpha-bungarotoxin on the cell surface than untreated cells. The effect of carbachol was significant within 5 min, increased progressively for at least 4 h, and had a sensitivity of 100 nM or less. Exposure to carbachol caused the appearance or dramatic expansion of an intracellular pool of nAChRs, which were localized to discrete, largely perinuclear structures. A pulse-chase labeling protocol allowed the selective labeling and localization of nAChRs that had been internalized from the cell surface. In untreated cells, very little internalization of nAChRs occurred over a period of 3 h, indicating that constitutive endocytosis of receptors over this period was minimal. Exposure to carbachol, however, caused a dramatic increase in the endocytosis of nAChRs. These results provide direct evidence that agonists, including the tobacco alkaloid nicotine, can cause substantial endocytosis of cell-surface nAChRs.