Posttranslational regulation of I-Ed by affinity for CLIP

Several MHC class II alleles linked with autoimmune diseases form unusually low stability complexes with CLIP, leading us to hypothesize that this is an important feature contributing to autoimmune pathogenesis. To investigate cellular consequences of altering class II/CLIP affinity, we evaluated invariant chain (Ii) mutants with varying CLIP affinity for a mouse class II allele, I-E(d), which has low affinity for wild-type CLIP and is associated with a mouse model of spontaneous, autoimmune joint inflammation. Increasing CLIP affinity for I-E(d) resulted in increased cell surface and total cellular abundance and half-life of I-E(d). This reveals a post-endoplasmic reticulum chaperoning capacity of Ii via its CLIP peptides. Quantitative effects on I-E(d) were less pronounced in DM-expressing cells, suggesting complementary chaperoning effects mediated by Ii and DM, and implying that the impact of allelic variation in CLIP affinity on immune responses will be highest in cells with limited DM activity. Differences in the ability of cell lines expressing wild-type or high-CLIP-affinity mutant Ii to present Ag to T cells suggest a model in which increased CLIP affinity for class II serves to restrict peptide loading to DM-containing compartments, ensuring proper editing of antigenic peptides.     

Effect of fluorocitrate on cerebral oxidation of lactate and glucose in freely moving rats

Glucose is the primary carbon source to enter the adult brain for catabolic and anabolic reactions. Some studies suggest that astrocytes may metabolize glucose to lactate; the latter serving as a preferential substrate for neurons, especially during neuronal activation. The current study utilizes the aconitase inhibitor fluorocitrate to differentially inhibit oxidative metabolism in glial cells in vivo. Oxidative metabolism of 14C-lactate and 14C-glucose was monitored in vivo using microdialysis and quantitating 14CO2 in the microdialysis eluate following pulse labeling of the interstitial glucose or lactate pool. After establishing a baseline oxidation rate, fluorocitrate was added to the perfusate. Neither lactate nor glucose oxidation was affected by 5 micromol/L fluorocitrate. However, 20 and 100 micromol/L fluorocitrate reduced lactate oxidation by 55 +/- 20% and 68 +/- 12%, respectively (p < 0.05 for both). Twenty and 100 micromol/L fluorocitrate reduced 14C-glucose oxidation by 50 +/- 14% (p < 0.05) and 24 +/- 19% (ns), respectively. Addition of non-radioactive lactate to (14)C-glucose plus fluorocitrate decreased 14C-glucose oxidation by an additional 29% and 38%, respectively. These results indicate that astrocytes oxidize about 50% of the interstitial lactate and about 35% of the glucose. By subtraction, neurons metabolize a maximum of 50% of the interstitial lactate and 65% of the interstitial glucose.     

Metabolic rate does not scale with body mass in cultured mammalian cells

The allometric scaling of metabolic rate with organism body mass can be partially accounted for by differences in cellular metabolic rates. For example, hepatocytes isolated from horses consume almost 10-fold less oxygen per unit time as mouse hepatocytes [Porter and Brand, Am J Physiol Regul Integr Comp Physiol 269: R226-R228, 1995]. This could reflect a genetically programmed, species-specific, intrinsic metabolic rate set point, or simply the adaptation of individual cells to their particular in situ environment (i.e., within the organism). We studied cultured cell lines derived from 10 mammalian species with donor body masses ranging from 5 to 600,000 g to determine whether cells propagated in an identical environment (media) exhibited metabolic rate scaling. Neither metabolic rate nor the maximal activities of key enzymes of oxidative or anaerobic metabolism scaled significantly with donor body mass in cultured cells, indicating the absence of intrinsic, species-specific, cellular metabolic rate set points. Furthermore, we suggest that changes in the metabolic rates of isolated cells probably occur within 24 h and involve a reduction of cellular metabolism toward values observed in lower metabolic rate organisms. The rate of oxygen delivery has been proposed to limit cellular metabolic rates in larger organisms. To examine the effect of oxygen on steady-state cellular respiration rates, we grew cells under a variety of physiologically relevant oxygen regimens. Long-term exposure to higher medium oxygen levels increased respiration rates of all cells, consistent with the hypothesis that higher rates of oxygen delivery in smaller mammals might increase cellular metabolic rates.     

Expanding our view of genomic diversity in Candidatus Accumulibacter clades

Enhanced biological phosphorus removal (EBPR) is an important industrial wastewater treatment process mediated by polyphosphate‐accumulating organisms (PAOs). Members of the genus Candidatus Accumulibacter are one of the most extensively studied PAO as they are commonly enriched in lab‐scale EBPR reactors. Members of different Accumulibacter clades are often enriched through changes in reactor process conditions; however, the two currently sequenced Accumulibacter genomes show extensive metabolic similarity. Here, we expand our understanding of Accumulibacter genomic diversity through recovery of eight population genomes using deep metagenomics, including seven from phylogenetic clades with no previously sequenced representative. Comparative genomic analysis revealed a core of shared genes involved primarily in carbon and phosphorus metabolism; however, each Accumulibacter genome also encoded a substantial number of unique genes (> 700 genes). A major difference between the Accumulibacter clades was the type of nitrate reductase encoded and the capacity to perform subsequent steps in denitrification. The Accumulibacter clade IIF genomes also contained acetaldehyde dehydrogenase that may allow ethanol to be used as carbon source. These differences in metabolism between Accumulibacter genomes provide a molecular basis for niche differentiation observed in lab‐scale reactors and may offer new opportunities for process optimization.     

Wetland development in a previously mined landscape of East Texas,USA

We studied wetland development in a chronosequence of created wetlands in a reclaimed landscape in east Texas seasonally for 1 year. The purpose of the study was to identify features (i.e., indicators) that best reflected changes in wetland ecosystem state through time and could serve as indicators of “maturity” for bond-release. Features considered included surface water nutrients, soil nutrients, soil redox potential, vegetative biomass and diversity, and benthic invertebrate biomass and diversity. Our sampling focused on nine wetlands representing three different-age classes (n = 3 for each) as a surrogate for time. All wetland sites were created with the same homogenized mine spoil and had similar hydrology and climate. Age-specific changes in all parameters were observed, except for surface water nutrients. The oldest wetlands (i.e., “mature”) exhibited highest soil concentrations of N, C, K, P, and Ca. Soil redox potential was significantly lower in the mature wetlands, in addition to within-wetland (lowest in deepest sampling zones) and intra-annual variability (i.e., lowest during the summer). Mature created wetlands supported the highest vegetative biomass and species richness and highest densities of invertebrates; however, taxa richness was similar across all age groups. Of all parameters we measured, vegetation metrics were among the simplest and most cost-effective measures used to track the early development of mitigated wetlands. This study provides the basis from which to track the development of these reclaimed ecosystems in a more rigorous and easily replicated manner. With further validation, select use of these parameter sets in east Texas and other similar landscapes could aid both in determining compliance for regulatory purposes as well as tracking success of ecological mitigation.     

Stochastic exit from mitosis in budding yeast: Model predictions and experimental observations

Unlike many mutants that are completely viable or inviable, the CLB2-dbΔ clb5Δ mutant of Saccharomyces cerevisiae is inviable in glucose but partially viable on slower growth media such as raffinose. On raffinose, the mutant cells can bud and divide but in each cycle there is a chance that a cell will fail to divide (telophase arrest), causing it to exit the cell cycle. This effect gives rise to a stochastic phenotype that cannot be explained by a deterministic model. We measure the interbud times of wild-type and mutant cells growing on raffinose and compute statistics and distributions to characterize the mutant''s behavior. We convert a detailed deterministic model of the budding yeast cell cycle to a stochastic model and determine the extent to which it captures the stochastic phenotype of the mutant strain. Predictions of the mathematical model are in reasonable agreement with our experimental data and suggest directions for improving the model. Ultimately, the ability to accurately model stochastic phenotypes may prove critical to understanding disease and therapeutic interventions in higher eukaryotes.Key words: stochastic phenotype, mitotic exit, non-genetic variability, cell cycle modeling, computational biology, stochastic modeling, deterministic modeling     

Genetic diversity, social structure, and conservation value of the elephants of the Nakai Plateau, Lao PDR, based on non-invasive sampling

The Lao People??s Democratic Republic (PDR) may have the largest Asian elephant population in Indochina. However, elephants on Lao PDR??s Nakai Plateau are potentially threatened by the construction of a hydropower dam that will flood important habitat. We conducted a non-invasive genetic study of elephants in this region to provide baseline data on genetic diversity and social structure prior to dam construction. For the 102 elephants we detected, values of observed heterozygosity (0.711) and allelic diversity (8.0 alleles/locus) at microsatellite loci were higher than those found in elephant populations in India and Vietnam, while mitochondrial diversity (haplotype diversity 0.741; nucleotide diversity 0.011) was similar to that reported for the Lao/Vietnam region. Six mitochondrial haplotypes were detected, representing both major clades previously reported in this species. Relatedness estimates between females and young detected near each other are consistent with familial relationships, and relatedness estimates between adult males and females suggest male locational dispersal. Since family group structure appears to be intact in the Nakai region, these elephants will likely move as relatively large family groups in response to habitat disturbance. These results have positive implications for the viability of the elephant population in this region, demonstrate its conservation significance, and will be valuable for predicting and monitoring the effects of the hydropower dam over time.     

The two domains of centrin have distinct basal body functions in Tetrahymena

The basal body is a microtubule-organizing center responsible for organizing the cilium, a structure important for cell locomotion and sensing of the surrounding environment. A widely conserved basal body component is the Ca(2+)-binding protein centrin. Analyses of centrin function suggest a role in basal body assembly and stability; however, its molecular mechanisms remain unclear. Here we describe a mutagenic strategy to study the function and essential nature of the various structural features of Cen1 in the ciliate Tetrahymena. We find that the two domains of Cen1 are both essential, and examination of strains containing mutant CEN1 alleles indicates that there are two predominant basal body phenotypes: misorientation of newly assembled basal bodies and stability defects. The results also show that the two domains of Cen1 are able to bind Ca(2+) and that perturbation of Ca(2+) binding affects Cen1 function. In all, the data suggest that the two domains of Cen1 have distinct functions.     

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons^1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser⁵. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response^1,3,7.We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.