Single-channel analysis of inactivation-defective rat skeletal muscle sodium channels containing the F1304Q mutation.

The intracellular linker between domains III and IV of the voltage-gated Na channel mediates fast inactivation. Targeted alteration of one or more of a triplet of hydrophobic amino acids within this linker region results in a marked slowing in the decay of ionic current. The mechanism of this defective inactivation was explored in rat skeletal muscle sodium channels (mu 1) containing the F1304Q mutation in Xenopus laevis oocytes with and without coexpression of the rat brain beta 1 subunit. Cell-attached single-channel patch-clamp recordings revealed that the mu 1-F1304Q channel reopens multiple times with open times that are prolonged compared with those of the wild-type channel. Coexpression of the beta 1 subunit stabilized a dominant nonbursting gating mode and accelerated the activation kinetics of mu 1-F1304Q but did not modify mean open time or fast-inactivation kinetics. A Markov gating model incorporating separate fast- and slow-inactivation particles reproduced the results by assuming that the F1304Q mutation specifically influences transitions to and from fast-inactivated states. These effects are independent of interactions of the mutant channel with the beta 1 subunit and do not result from a change in modal gating behavior. These results indicate that F1304Q mutant channels can still enter the inactivated state but do so reversibly and with altered kinetics.     

Trypanosoma cruzi glycoprotein of Mr 56000: characterization and assessment of its potential to protect against fatal parasite infections

A ∼ 56 000 Da membrane glycoprotein purified from epimastigotes of Trypanosoma cruzi was characterized biochemically and tested for its efficacy to induce protection in mice from a lethal challenge with this protozoan parasite. Immunofluorescence assays with live and formalin-fixed epimastigotes and trypomastigotes localized the glycoprotein to the flagellum, the body of the parasite, and the cell membrane. Immunoblotting demonstrated the glyco-protein's presence in nearly equal amounts in all developmental stages of several T. cruzi isolates. Mice immunized with the purified glycoprotein and challenged with 10000 infectious trypomastigote forms of isolate Y survived the controls by up to four days. This significant protection makes this antigen a potential candidate for a multi-subunit vaccine against 7. cruzi.     

Proton Inhibition of Sodium Channels: Mechanism of Gating Shifts and Reduced Conductance

Extracellular acidosis affects both permeation and gating of the expressed rat skeletal muscle Na⁺ channel (μ1). Reduction of the extracellular pH produced a progressive decrease in the maximal whole-cell conductance and a depolarizing shift in the whole-cell current-voltage relationship. A smaller depolarizing shift in the steady-state inactivation curve was observed. The pK of the reduction of maximal conductance was 6.1 over the pH range studied. An upper limit estimate of the pK of the shift of the half-activation voltage was 6.1. The relative reduction in the maximal whole-cell conductance did not change with higher [Na⁺] _o . The conductance of single fenvalerate-modified Na⁺ channels was reduced by extracellular protons. Although the single-channel conductance increased with higher [Na⁺] _o , the maximal conductances at pH 7.6, 7.0 and 6.0 did not converge at [Na⁺] _o up to 280 mm, inconsistent with a simple electrostatic effect. A model incorporating both Na⁺ and H⁺ binding in the pore and cation binding to a Gouy-Chapman surface charge provided a robust fit to the single-channel conductance data with an estimated surface charge density of 1e⁻/439?². Neither surface charge nor proton block alone suffices to explain the effects of extracellular acidosis on Na⁺ channel permeation; both effects play major roles in mediating the response to extracellular pH. Received: 14 May 1996/Revised: 19 September 1996     

Analysis of the initial stages of plant protoplast development using 33258 Hoechst: reactivation of the cell cycle

A microfluorimetric procedure, employing the fluorescent stain 33258 Hoechst, has been developed for the investigation of the process of DNA synthesis during the initial stages of culture of tobacco ( N. tabacum cv. Xanthi) leaf protoplasts.
In this system, the freshly-isolated protoplasts exhibited a unimodal distribution of nuclear DNA content characteristic of the diploid state. The almost immediate onset of DNA synthesis during culture resulted in a doubling of nuclear DNA levels prior to the first mitoses. Although the majority of the protoplasts subsequently entered into synchronous mitosis and cell division, a proportion of the remainder developed into large polyploid cells. Upon further culture, the polyploid cells became subdivided into clusters of small diploid cells. Measurement of total cell protein and cell volumes during culture indicated that a relationship existed between these parameters and the initiation of mitosis. The significance of these observations is discussed.     

Chemoirradiation for Glioblastoma Multiforme: The National Cancer Institute Experience

Purpose

Standard treatment for glioblastoma (GBM) is surgery followed by radiation (RT) and temozolomide (TMZ). While there is variability in survival based on several established prognostic factors, the prognostic utility of other factors such as tumor size and location are not well established.

Experimental Design

The charts of ninety two patients with GBM treated with RT at the National Cancer Institute (NCI) between 1998 and 2012 were retrospectively reviewed. Most patients received RT with concurrent and adjuvant TMZ. Topographic locations were classified using preoperative imaging. Gross tumor volumes were contoured using treatment planning systems utilizing both pre-operative and post-operative MR imaging.

Results

At a median follow-up of 18.7 months, the median overall survival (OS) and progression-free survival (PFS) for all patients was 17.9 and 7.6 months. Patients with the smallest tumors had a median OS of 52.3 months compared to 16.3 months among patients with the largest tumors, P = 0.006. The patients who received bevacizumab after recurrence had a median OS of 23.3 months, compared to 16.3 months in patients who did not receive it, P = 0.0284. The median PFS and OS in patients with periventricular tumors was 5.7 and 17.5 months, versus 8.9 and 23.3 months in patients with non-periventricular tumors, P = 0.005.

Conclusions

Survival in our cohort was comparable to the outcome of the defining EORTC-NCIC trial establishing the use of RT+TMZ. This study also identifies several potential prognostic factors that may be useful in stratifying patients.     

Inducible reporter/driver lines for the Arabidopsis root with intrinsic reporting of activity state

Cell‐, tissue‐ or organ‐specific inducible expression systems are powerful tools for functional analysis of changes to the pattern, level or timing of gene expression. However, plant researchers lack standardised reagents that promote reproducibility across the community. Here, we report the development and functional testing of a Gateway‐based system for quantitatively, spatially and temporally controlling inducible gene expression in Arabidopsis that overcomes several drawbacks of the legacy systems. We used this modular driver/effector system with intrinsic reporting of spatio‐temporal promoter activity to generate 18 well‐characterised homozygous transformed lines showing the expected expression patterns specific for the major cell types of the Arabidopsis root; seed and plasmid vectors are available through the Arabidopsis stock centre. The system's tight regulation was validated by assessing the effects of diphtheria toxin A chain expression. We assessed the utility of Production of Anthocyanin Pigment 1 (PAP1) as an encoded effector mediating cell‐autonomous marks. With this shared resource of characterised reference driver lines, which can be expanded with additional promoters and the use of other fluorescent proteins, we aim to contribute towards enhancing reproducibility of qualitative and quantitative analyses.     

Target cell APOBEC3C can induce limited G-to-A mutation in HIV-1

The evolutionary success of primate lentiviruses reflects their high capacity to mutate and adapt to new host species, immune responses within individual hosts, and, in recent years, antiviral drugs. APOBEC3G (A3G) and APOBEC3F (A3F) are host cell DNA-editing enzymes that induce extensive HIV-1 mutation that severely attenuates viral replication. The HIV-1 virion infectivity factor (Vif), expressed in vivo, counteracts the antiviral activity of A3G and A3F by inducing their degradation. Other APOBECs may contribute more to viral diversity by inducing less extensive mutations allowing viral replication to persist. Here we show that in APOBEC3C (A3C)-expressing cells infected with the patient-derived HIV-1 molecular clones 210WW, 210WM, 210MW, and 210MM, and the lab-adapted molecular clone LAI, viral G-to-A mutations were detected in the presence of Vif expression. Mutations occurred primarily in the GA context and were relatively infrequent, thereby allowing for spreading infection. The mutations were absent in cells lacking A3C but were induced after transient expression of A3C in the infected target cell. Inhibiting endogenous A3C by RNA interference in Magi cells prevented the viral mutations. Thus, A3C is necessary and sufficient for G-to-A mutations in some HIV-1 strains. A3C-induced mutations occur at levels that allow replication to persist and may therefore contribute to viral diversity. Developing drugs that inhibit A3C may be a novel strategy for delaying viral escape from immune or antiretroviral inhibition.