Use-dependent block of the voltage-gated Na+ channel by tetrodotoxin and saxitoxin: Effect of pore mutations that change ionic selectivity

Voltage-gated Na⁺ channels (NaV channels) are specifically blocked by guanidinium toxins such as tetrodotoxin (TTX) and saxitoxin (STX) with nanomolar to micromolar affinity depending on key amino acid substitutions in the outer vestibule of the channel that vary with NaV gene isoforms. All NaV channels that have been studied exhibit a use-dependent enhancement of TTX/STX affinity when the channel is stimulated with brief repetitive voltage depolarizations from a hyperpolarized starting voltage. Two models have been proposed to explain the mechanism of TTX/STX use dependence: a conformational mechanism and a trapped ion mechanism. In this study, we used selectivity filter mutations (K1237R, K1237A, and K1237H) of the rat muscle NaV1.4 channel that are known to alter ionic selectivity and Ca²⁺ permeability to test the trapped ion mechanism, which attributes use-dependent enhancement of toxin affinity to electrostatic repulsion between the bound toxin and Ca²⁺ or Na⁺ ions trapped inside the channel vestibule in the closed state. Our results indicate that TTX/STX use dependence is not relieved by mutations that enhance Ca²⁺ permeability, suggesting that ion–toxin repulsion is not the primary factor that determines use dependence. Evidence now favors the idea that TTX/STX use dependence arises from conformational coupling of the voltage sensor domain or domains with residues in the toxin-binding site that are also involved in slow inactivation.     

Studies on the structure of yeast phosphofructokinase

In this paper, we describe an efficient procedure for the purification of yeast phosphofructokinase. This procedure eliminates any time delay and enables to obtain an enzyme with minimum proteolytic alterations. The molecular weights of the oligomeric enzyme and of its constitutive subunits were both evaluated by means of several independent methods. However, the accuracy of each measurement was not sufficient to discriminate between an hexameric and an octameric structure of the enzyme oligomer. On the other hand, crosslinking experiments demonstrated the octameric structure of yeast phosphofructokinase. Obviously, some methods of molecular weight determination have led to erroneous results. In particular, our experiments show that the reliability of molecular weight determinations performed by gel filtration of native proteins must be considered with caution.     

Studies on Nucleotide Chemistry 15. Synthesis of Oligodeoxynucleotides Using Amidine Protected Nucleosides

Abstract

An in situ method is described for synthesizing DNA which incorporates a new series of amidine protected deoxy-nucleosides and bis-dialkylaminophosphines as phosphitylating agents. These procedures were used to synthesize d(GGGAATTCCC) which was digested by EcoRI.     

A Host YB-1 Ribonucleoprotein Complex Is Hijacked by Hepatitis C Virus for the Control of NS3-Dependent Particle Production

Hepatitis C virus (HCV) orchestrates the different stages of its life cycle in time and space through the sequential participation of HCV proteins and cellular machineries; hence, these represent tractable molecular host targets for HCV elimination by combination therapies. We recently identified multifunctional Y-box-binding protein 1 (YB-1 or YBX1) as an interacting partner of NS3/4A protein and HCV genomic RNA that negatively regulates the equilibrium between viral translation/replication and particle production. To identify novel host factors that regulate the production of infectious particles, we elucidated the YB-1 interactome in human hepatoma cells by a quantitative mass spectrometry approach. We identified 71 YB-1-associated proteins that included previously reported HCV regulators DDX3, heterogeneous nuclear RNP A1, and ILF2. Of the potential YB-1 interactors, 26 proteins significantly modulated HCV replication in a gene-silencing screening. Following extensive interaction and functional validation, we identified three YB-1 partners, C1QBP, LARP-1, and IGF2BP2, that redistribute to the surface of core-containing lipid droplets in HCV JFH-1-expressing cells, similarly to YB-1 and DDX6. Importantly, knockdown of these proteins stimulated the release and/or egress of HCV particles without affecting virus assembly, suggesting a functional YB-1 protein complex that negatively regulates virus production. Furthermore, a JFH-1 strain with the NS3 Q221L mutation, which promotes virus production, was less sensitive to this negative regulation, suggesting that this HCV-specific YB-1 protein complex modulates an NS3-dependent step in virus production. Overall, our data support a model in which HCV hijacks host cell machinery containing numerous RNA-binding proteins to control the equilibrium between viral RNA replication and NS3-dependent late steps in particle production.     

Size at birth and some sociodemographic factors in gypsies in Hungary

In 10,108 babies born to gypsy parents, birth weight, birth length and gestational age are compared with a national reference sample, taking into account maternal education, age, and birth interval. The gypsy babies tend consistently to be smaller, and this difference is attributable to the circumstances in which the gypsies live as well as the poorer maternal education. There appears to be a vicious circle--poor hygiene and living conditions adversely affect birth size, which in turn puts the child at a disadvantage in terms of survival, health and development.     

Morphological effects of the catechol estrogens on rat epididymal epithelia

The 2-hydroxy and 4-hydroxyestradiols (2-/4-OHE₂) caused marked cytotoxic effects, including vacuolation and nuclear changes, in rat epididymal epithelia, after exposure to very low levels (40 ng/rat/week) for 20 weeks. The effects of the 2-/4-OHE₂ metabolites were more pronounced than that of estradiol-17β(E₂).