Effect of postnatal development on calcium currents and slow charge movement in mammalian skeletal muscle

Single- (whole-cell patch) and two-electrode voltage-clamp techniques were used to measure transient (Ifast) and sustained (Islow) calcium currents, linear capacitance, and slow, voltage-dependent charge movements in freshly dissociated fibers of the flexor digitorum brevis (FDB) muscle of rats of various postnatal ages. Peak Ifast was largest in FDB fibers of neonatal (1-5 d) rats, having a magnitude in 10 mM external Ca of 1.4 +/- 0.9 pA/pF (mean +/- SD; current normalized by linear fiber capacitance). Peak Ifast was smaller in FDB fibers of older animals, and by approximately 3 wk postnatal, it was so small as to be unmeasurable. By contrast, the magnitudes of Islow and charge movement increased substantially during postnatal development. Peak Islow was 3.6 +/- 2.5 pA/pF in FDB fibers of 1-5-d rats and increased to 16.4 +/- 6.5 pA/pF in 45-50-d-old rats; for these same two age groups, Qmax, the total mobile charge measurable as charge movement, was 6.0 +/- 1.7 and 23.8 +/- 4.0 nC/microF, respectively. As both Islow and charge movement are thought to arise in the transverse-tubular system, linear capacitance normalized by the area of fiber surface was determined as an indirect measure of the membrane area of the t-system relative to that of the fiber surface. This parameter increased from 1.5 +/- 0.2 microF/cm2 in 2-d fibers to 2.9 +/- 0.4 microF/cm2 in 44-d fibers. The increases in peak Islow, Qmax, and normalized linear capacitance all had similar time courses. Although the function of Islow is unknown, the substantial postnatal increase in its magnitude suggests that it plays an important role in the physiology of skeletal muscle.     

Slow charge movement in mammalian skeletal muscle

Voltage-dependent charge movements were measured in the rat omohyoid muscle with the three-microelectrode voltage-clamp technique. Contraction was abolished with hypertonic sucrose. The standard (ON-OFF) protocol for eliciting charge movements was to depolarize the fiber from -90 mV to a variable test potential (V) and then repolarize the fiber to -90 mV. The quantity of charge moved saturated at test potentials of approximately 0 mV. The steady state dependence of the amount of charge that moves as a function of test potential could be well fitted by the Boltzmann relation: Q = Qmax/(1 + exp[-(V - V)/k]), where Qmax is the maximum charge that can be moved, V is the potential at which half the charge moves, and k is a constant. At 15 degrees C, these values were Qmax = 28.5 nC/microF, V = -34.2 mV, and k = 8.7 mV. Qmax, k, and V exhibited little temperature dependence over the range 7-25 degrees C. "Stepped OFF" charge movements were elicited by depolarizing the fiber from -90 mV to a fixed conditioning level that moved nearly all the mobile charge (0 mV), and then repolarizing the fiber to varying test potentials. The sum of the charge that moved when the fiber was depolarized directly from -90 mV to a given test potential and the stepped OFF charge that moved when the fiber was repolarized to the same test potential had at all test potentials a value close to Qmax for that fiber. In nearly all cases, the decay phase of ON, OFF, and stepped OFF charge movements could be well fitted with a single exponential. The time constant, tau decay, for an ON charge movement at a given test potential was comparable to tau decay for a stepped OFF charge movement at the same test potential. Tau decay had a bell-shaped dependence on membrane potential: it was slowest at a potential near V (the midpoint of the steady state charge distribution) and became symmetrically faster on either side of this potential. Raising the temperature from 7 to 15 degrees C caused tau decay to become faster by about the same proportion at all potentials, with a Q10 averaging 2.16. Raising the temperature from 15 to 25 degrees C caused tau decay to become faster at potentials near V, but not at potentials farther away.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.