Distribution of mitochondria within Muller cells – II. Post-natal development of the rabbit retinal periphery in vivo and in vitro: dependence on oxygen supply

The occurrence and localization of mitochondria within glial (Muller) cells and neurons of the peripheral (avascular) rabbit retina was studied electron microscopically and by immunocytochemical demonstration of the mitochondrial enzyme GABA transaminase (GABA-T). Post-natal development in vivo was compared with development of organ cultures from neonatal rabbit retinae, grown over 2 weeks in vitro. The adult pattern of mitochondrial localization (restriction to the sclerad end of the cells) was observed from the beginning of enzyme expression at early post-natal stages. However, when neonatal retinal pieces were grown in vitro with their vitread surface exposed to the air, their Muller cells contained mitochondria along most of their length. When functionally developed retinae from postnatal day 14 were explanted in vitro, they retained their sclerad mitochondrial distribution for almost 24 h but thereafter the inner portions of their cytoplasm became occupied by mitochondria within a few hours. This was achieved mainly by mitochondrial migration rather than by formation of new mitochondria because it was not prevented by cycloheximide-induced inhibition of protein synthesis. These data support the following hypotheses: (1) the mitochondrial distribution in Muller cells is determined by the local cytoplasmic O2 pressure (pO2), (2) existing mitochondria move towards cytoplasmic regions of sufficient pO2 by rather rapid migration and (3) the start of this migration is delayed by almost 24 h due to the action of as yet unknown control mechanisms. In contrast, the mitochondrial content of retinal ganglion and amacrine cells in the vitread retinal layers was virtually independent of the source and level of oxygen supply.     

The Arabidopsis transposable element Tag1 is widely distributed among Arabidopsis ecotypes

Tag1 is an autonomous transposable element (3.3 kb in length) first identified as an insertion in the CHL1 (NRT1) gene of Arabidopsis thaliana. Tag1 has been found in the Landsberg erecta ecotype of A. thaliana but not in Columbia or WS. In this paper, 41 additional ecotypes were examined for the presence of Tag1. Using an internal Tag1 fragment as probe, we found that DNA from 19 of the 41 ecotypes strongly hybridized to Tag1. Almost all of the Tag1-containing ecotypes had only one or two copies of Tag1 per haploid genome, as determined by Southern blot analysis. The only exception, Bf-1 from Bretagny-sur-Orge, France, had four copies. Two ecotypes, Di-G and S96, gave identical Southern blot patterns to that of Landsberg erecta and were subsequently shown to contain Tag1 at the same two positions found in Landsberg erecta (loci designated as Tag1-2 and Tag1-3). Two other ecotypes, Ag-0 and Lo-1, had a Tag1 element located at Tag1-2 but not at Tag1-3. The distance between these two loci was determined to be 0.37 cM. Analysis of DNA from two related species, A. griffithiana and A. pumila, showed that both species contain sequences that hybridize to Tag1 and that could be amplified with an oligonucleotide specific to the terminal inverted repeats of Tag1. These results show that Tag1 and related elements are present, and may be useful for insertional mutagenesis, in many A. thaliana ecotypes and several Arabidopsis species.     

Albumin synthesis after intense intermittent exercise in human subjects

Yang, Roger C., Gary W. Mack, Robert R. Wolfe, and Ethan R. Nadel. Albumin synthesis after intense intermittent exercise inhuman subjects. J. Appl. Physiol.84(2): 584-592, 1998.We measured hepatic albumin synthesis infive volunteers (4 men and 1 woman) at 3 and 6 h after recovery fromintense exercise. A primed-constant infusion of a stable isotopictracer of phenylalanine was used to determine hepatic fractionalsynthetic rate (FSR) and absolute synthetic rate (ASR) of albumin fromthe enrichment of phenylalanine in albumin. The infusion of the stableisotope tracer began 2 h after upright exercise or upright rest.Albumin FSR and ASR were 6.39 ± 0.48%/day and 120 ± 9 mg · kg bodywt¹ · day¹,respectively, 3-6 h after recovery from exercise; the FSR and ASRon the time control study day were 5.94 ± 0.47%/day and 104 ± 9 mg · kg bodywt¹ · day¹,respectively. The 6 and 16% increases(P < 0.05) in FSR and ASR afterexercise were associated with an elevated plasma albumin content at 5 and 6 h of recovery (P < 0.05), anincreased total protein content throughout recovery(P < 0.05), and a negative freewater clearance (P < 0.05) at 2, 3, and 6.5 h of recovery compared with baseline values; these variableswere unchanged from their baselines on the time control study day.Increased albumin content and reduced free water clearance contributeto a retention of fluid within the circulation after intense exercise. The measured increase in albumin synthesis could not account for theentire increase in albumin content at 6 h of recovery from exercise.However, we estimate that if the increased activity was maintained forthe next 18 h, it could account for the expected increase in albumincontent at 24 h of recovery.

     

Nevirapine Concentration in Hair Samples Is a Strong Predictor of Virologic Suppression in a Prospective Cohort of HIV-Infected Patients

Effective antiretroviral (ARV) therapy depends on adequate drug exposure, yet methods to assess ARV exposure are limited. Concentrations of ARV in hair are the product of steady-state pharmacokinetics factors and longitudinal adherence. We investigated nevirapine (NVP) concentrations in hair as a predictor of treatment response in women receiving ARVs. In participants of the Women’s Interagency HIV Study, who reported NVP use for >1 month from 2003–2008, NVP concentrations in hair were measured via liquid-chromatography-tandem mass-spectrometry. The outcome was virologic suppression (plasma HIV RNA below assay threshold) at the time of hair sampling and the primary predictor was nevirapine concentration categorized into quartiles. We controlled for age, race/ethnicity, pre-treatment HIV RNA, CD4 cell count, and self-reported adherence over the 6-month visit interval (categorized ≤ 74%, 75%–94% or ≥ 95%). We also assessed the relation of NVP concentration with changes in hepatic transaminase levels via multivariate random intercept logistic regression and linear regression analyses. 271 women contributed 1089 person-visits to the analysis (median 3 of semi-annual visits). Viral suppression was least frequent in concentration quartile 1 (86/178 (48.3%)) and increased in higher quartiles (to 158/204 (77.5%) for quartile 4). The odds of viral suppression in the highest concentration quartile were 9.17 times (95% CI 3.2–26, P < 0.0001) those in the lowest. African-American race was associated with lower rates of virologic suppression independent of NVP hair concentration. NVP concentration was not significantly associated with patterns of serum transaminases. Concentration of NVP in hair was a strong independent predictor of virologic suppression in women taking NVP, stronger than self-reported adherence, but did not appear to be strongly predictive of hepatotoxicity.     

Characterization of HTT Inclusion Size,Location, and Timing in the zQ175 Mouse Model of Huntington′s Disease: An In Vivo High-Content Imaging Study

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. Major pathological hallmarks of HD include inclusions of mutant huntingtin (mHTT) protein, loss of neurons predominantly in the caudate nucleus, and atrophy of multiple brain regions. However, the early sequence of histological events that manifest in region- and cell-specific manner has not been well characterized. Here we use a high-content histological approach to precisely monitor changes in HTT expression and characterize deposition dynamics of mHTT protein inclusion bodies in the recently characterized zQ175 knock-in mouse line. We carried out an automated multi-parameter quantitative analysis of individual cortical and striatal cells in tissue slices from mice aged 2–12 months and confirmed biochemical reports of an age-associated increase in mHTT inclusions in this model. We also found distinct regional and subregional dynamics for inclusion number, size and distribution with subcellular resolution. We used viral-mediated suppression of total HTT in the striatum of zQ175 mice as an example of a therapeutically-relevant but heterogeneously transducing strategy to demonstrate successful application of this platform to quantitatively assess target engagement and outcome on a cellular basis.     

Do Foliar,Litter, and Root Nitrogen and Phosphorus Concentrations Reflect Nutrient Limitation in a Lowland Tropical Wet Forest?

Understanding nutrient limitation of net primary productivity (NPP) is critical to predict how plant communities will respond to environmental change. Foliar nutrients, especially nitrogen and phosphorus concentrations ([N] and [P]) and their ratio, have been used widely as indicators of plant nutritional status and have been linked directly to nutrient limitation of NPP. In tropical systems, however, a high number of confounding factors can limit the ability to predict nutrient limitation —as defined mechanistically by NPP responses to fertilization— based on the stoichiometric signal of the plant community. We used a long-term full factorial N and P fertilization experiment in a lowland tropical wet forest in Costa Rica to explore how tissue (foliar, litter and root) [N] and [P] changed with fertilization, how different tree size classes and taxa influenced the community response, and how tissue nutrients related to NPP. Consistent with NPP responses to fertilization, there were no changes in community-wide foliar [N] and [P], two years after fertilization. Nevertheless, litterfall [N] increased with N additions and root [P] increased with P additions. The most common tree species (Pentaclethra macroloba) had 9 % higher mean foliar [N] with NP additions and the most common palm species (Socratea exohrriza) had 15% and 19% higher mean foliar [P] with P and NP additions, respectively. Moreover, N:P ratios were not indicative of NPP responses to fertilization, either at the community or at the taxa level. Our study suggests that in these diverse tropical forests, tissue [N] and [P] are driven by the interaction of multiple factors and are not always indicative of the nutritional status of the plant community.     

GRAF1 promotes ferlin-dependent myoblast fusion

Myoblast fusion (a critical process by which muscles grow) occurs in a multi-step fashion that requires actin and membrane remodeling; but important questions remain regarding the spatial/temporal regulation of and interrelationship between these processes. We recently reported that the Rho-GAP, GRAF1, was particularly abundant in muscles undergoing fusion to form multinucleated fibers and that enforced expression of GRAF1 in cultured myoblasts induced robust fusion by a process that required GAP-dependent actin remodeling and BAR domain-dependent membrane sculpting. Herein we developed a novel line of GRAF1-deficient mice to explore a role for this protein in the formation/maturation of myotubes in vivo. Post-natal muscles from GRAF1-depleted mice exhibited a significant and persistent reduction in cross-sectional area, impaired regenerative capacity and a significant decrease in force production indicative of lack of efficient myoblast fusion. A significant fusion defect was recapitulated in isolated myoblasts depleted of GRAF1 or its closely related family member GRAF2. Mechanistically, we show that GRAF1 and 2 facilitate myoblast fusion, at least in part, by promoting vesicle-mediated translocation of fusogenic ferlin proteins to the plasma membrane.     

Hypofractionated radiotherapy has the potential for second cancer reduction

Background and Purpose  

A model for carcinoma and sarcoma induction was used to study the dependence of carcinogenesis after radiotherapy on fractionation.