Short interfering RNA-mediated inhibition of herpes simplex virus type 1 gene expression and function during infection of human keratinocytes

RNA interference (RNAi) is an antiviral mechanism that is activated when double-stranded RNA is cleaved into fragments, called short interfering RNA (siRNA), that prime an inducible gene silencing enzyme complex. We applied RNAi against a herpes simplex virus type 1 (HSV-1) gene, glycoprotein E, which mediates cell-to-cell spread and immune evasion. In an in vitro model of infection, human keratinocytes were transfected with siRNA specific for glycoprotein E and then infected with wild-type HSV-1. RNAi-mediated gene silencing reproduced the small plaque phenotype of a gE-deletion mutant virus. The specificity of gene targeting was demonstrated by flow cytometry and Northern blot analyses. Exogenous siRNA can suppress HSV-1 glycoprotein E expression and function during active infection in vitro through RNAi. This work establishes RNAi as a genetic tool for the study of HSV and provides a foundation for development of RNAi as a novel antiviral therapy.     

Calcium waves propagate through radial glial cells and modulate proliferation in the developing neocortex

The majority of neurons in the adult neocortex are produced embryonically during a brief but intense period of neuronal proliferation. The radial glial cell, a transient embryonic cell type known for its crucial role in neuronal migration, has recently been shown to function as a neuronal progenitor cell and appears to produce most cortical pyramidal neurons. Radial glial cell modulation could thus affect neuron production, neuronal migration, and overall cortical architecture; however, signaling mechanisms among radial glia have not been studied directly. We demonstrate here that calcium waves propagate through radial glial cells in the proliferative cortical ventricular zone (VZ). Radial glial calcium waves occur spontaneously and require connexin hemichannels, P2Y1 ATP receptors, and intracellular IP3-mediated calcium release. Furthermore, we show that wave disruption decreases VZ proliferation during the peak of embryonic neurogenesis. Taken together, these results demonstrate a radial glial signaling mechanism that may regulate cortical neuronal production.     

A Mutation Associated with CMT2A Neuropathy Causes Defects in Fzo1 GTP Hydrolysis,Ubiquitylation, and Protein Turnover

Charcot-Marie-Tooth disease type 2A (CMT2A) is caused by mutations in the gene MFN2 and is one of the most common inherited peripheral neuropathies. Mfn2 is one of two mammalian mitofusin GTPases that promote mitochondrial fusion and maintain organelle integrity. It is not known how mitofusin mutations cause axonal degeneration and CMT2A disease. We used the conserved yeast mitofusin FZO1 to study the molecular consequences of CMT2A mutations on Fzo1 function in vivo and in vitro. One mutation (analogous to the CMT2A I213T substitution in the GTPase domain of Mfn2) not only abolishes GTP hydrolysis and mitochondrial membrane fusion but also reduces Mdm30-mediated ubiquitylation and degradation of the mutant protein. Importantly, complexes of wild type and the mutant Fzo1 protein are GTPase active and restore ubiquitylation and degradation of the latter. These studies identify diverse and unexpected effects of CMT2A mutations, including a possible role for mitofusin ubiquitylation and degradation in CMT2A pathogenesis, and provide evidence for a novel link between Fzo1 GTP hydrolysis, ubiquitylation, and mitochondrial fusion.     

Analysis of cardiac signals using spatial filling index and time-frequency domain

Background  

Analysis of heart rate variation (HRV) has become a popular noninvasive tool for assessing the activities of the autonomic nervous system (ANS). HRV analysis is based on the concept that fast fluctuations may specifically reflect changes of sympathetic and vagal activity. It shows that the structure generating the signal is not simply linear, but also involves nonlinear contributions. These signals are essentially non-stationary; may contain indicators of current disease, or even warnings about impending diseases. The indicators may be present at all times or may occur at random in the time scale. However, to study and pinpoint abnormalities in voluminous data collected over several hours is strenuous and time consuming.     

A dynamic mathematical model for microbial removal of pyritic sulfur from coal

A dynamic mathematical model has been developed to describe microbial desulfurization of coal by Thiobacillus ferrooxidans. The model considers adsorption and desorption of cells on coal particles and microbial oxidation of pyritic sulfur on particle surfaces. The influence of certain parameters, such as microbial growth rate constants, adsorption-descrption constants, pulp density, coal particle size, initial cell and solid phase substrate concentration on the maximum rate of pyritic sulfur removal, have been elucidated. The maximum rate of pyritic sulfur removal was strongly dependent upon the number of attached cells per coal particle. At sufficiently high initial cell concentrations, the surfaces of coal particles are nearly saturated by the cells and the maximum leaching rate is limited either by total external surface area of coal particles or by the concentration of pyritic sulfur in the coal phase. The maximum volumetric rate of pyritic sulfur removal (mg S/h cm(3) mixture) increases with the pulp density of coal and reaches a saturation level at high pulp densities (e.g. 45%). The maximum rate also increases with decreasing particle diameter in a hyperbolic form. Increases in adsorption coefficient or decreases in the desorption coefficient also result in considerable improvements in this rate. The model can be applied to other systems consisting of suspended solid substrate particles in liquid medium with microbial oxidation occurring on the particle surfaces (e.g., bacterial ore leaching). The results obtained from this model are in good agreement with published experimental data on microbial desulfurization of coal and bacterial ore leaching.     

Dissection and design of yeast prions

Many proteins can misfold into β-sheet-rich, self-seeding polymers (amyloids). Prions are exceptional among such aggregates in that they are also infectious. In fungi, prions are not pathogenic but rather act as epigenetic regulators of cell physiology, providing a powerful model for studying the mechanism of prion replication. We used prion-forming domains from two budding yeast proteins (Sup35p and New1p) to examine the requirements for prion formation and inheritance. In both proteins, a glutamine/asparagine-rich (Q/N-rich) tract mediates sequence-specific aggregation, while an adjacent motif, the oligopeptide repeat, is required for the replication and stable inheritance of these aggregates. Our findings help to explain why although Q/N-rich proteins are relatively common, few form heritable aggregates: prion inheritance requires both an aggregation sequence responsible for self-seeded growth and an element that permits chaperone-dependent replication of the aggregate. Using this knowledge, we have designed novel artificial prions by fusing the replication element of Sup35p to aggregation-prone sequences from other proteins, including pathogenically expanded polyglutamine.     

Isolation of molecules recognized by monoclonal antibodies and antisera: the solid phase immunoisolation technique

Coupling of Ca2+ transport to ATP hydrolysis in isolated sarcoplasmic reticulum vesicles has been studied following pulsed additions of either ATP or Ca2+. ATP was infused as a pulse into medium, whose free Ca2+ concentration was maintained constant at saturating levels by a calciumstat procedure, using either a Ca2+-selective electrode or the spectrophotometric arsenazo III technique as Ca2+ indicators. The low ATP levels virtually exclude contributions by "basal" ATPase activity. Passive leakage of Ca2+, monitored after an ATP pulse, does not contribute more than 5% to subintegral coupling ratios. Pulsed additions of Ca2+ were made into medium. containing saturating concentrations of ATP, whose hydrolysis was monitored by a pH-stat procedure. Ca2+-stimulated hydrolysis continued until all the Ca2+ was transported into the vesicles. Values for the coupling ratio, Ca2+/ATP, of 1.82 +/- 0.12 and 1.79 +/- 0.15 were obtained by the ATP- and Ca2+-pulse methods, respectively.