Aberrant Mitochondria in a Bethlem Myopathy Patient with a Homozygous Amino Acid Substitution That Destabilizes the Collagen VI α2(VI) Chain

Bethlem myopathy and Ullrich congenital muscular dystrophy (UCMD) sit at opposite ends of a clinical spectrum caused by mutations in the extracellular matrix protein collagen VI. Bethlem myopathy is relatively mild, and patients remain ambulant in adulthood while many UCMD patients lose ambulation by their teenage years and require respiratory interventions. Dominant and recessive mutations are found across the entire clinical spectrum; however, recessive Bethlem myopathy is rare, and our understanding of the molecular pathology is limited. We studied a patient with Bethlem myopathy. Electron microscopy of his muscle biopsy revealed abnormal mitochondria. We identified a homozygous COL6A2 p.D871N amino acid substitution in the C-terminal C2 A-domain. Mutant α2(VI) chains are unable to associate with α1(VI) and α3(VI) and are degraded by the proteasomal pathway. Some collagen VI is assembled, albeit more slowly than normal, and is secreted. These molecules contain the minor α2(VI) C2a splice form that has an alternative C terminus that does include the mutation. Collagen VI tetramers containing the α2(VI) C2a chain do not assemble efficiently into microfibrils and there is a severe collagen VI deficiency in the extracellular matrix. We expressed wild-type and mutant α2(VI) C2 domains in mammalian cells and showed that while wild-type C2 domains are efficiently secreted, the mutant p.D871N domain is retained in the cell. These studies shed new light on the protein domains important for intracellular and extracellular collagen VI assembly and emphasize the importance of molecular investigations for families with collagen VI disorders to ensure accurate diagnosis and genetic counseling.     

Quantifying the importance of geographic replication and representativeness when estimating demographic rates,using a coastal species as a case study

Demographic rates are rarely estimated over an entire species range, limiting empirical tests of ecological patterns and theories, and raising questions about the representativeness of studies that use data from a small part of a range. The uncertainty that results from using demographic rates from just a few sites is especially pervasive in population projections, which are critical for a wide range of questions in ecology and conservation. We developed a simple simulation to quantify how this lack of geographic representativeness can affect inferences about the global mean and variance of growth rates, which has implications for the robust design of a wide range of population studies. Using a coastal songbird, saltmarsh sparrow Ammodramus caudacutus, as a case study, we first estimated survival, fecundity, and population growth rates at 21 sites distributed across much of their breeding range. We then subsampled this large, representative dataset according to five sampling scenarios in order to simulate a variety of geographic biases in study design. We found spatial variation in demographic rates, but no large systematic patterns. Estimating the global mean and variance of growth rates using subsets of the data suggested that at least 10–15 sites were required for reasonably unbiased estimates, highlighting how relying on demographic data from just a few sites can lead to biased results when extrapolating across a species range. Sampling at the full 21 sites, however, offered diminishing returns, raising the possibility that for some species accepting some geographical bias in sampling can still allow for robust range‐wide inferences. The subsampling approach presented here, while conceptually simple, could be used with both new and existing data to encourage efficiency in the design of long‐term or large‐scale ecological studies.     

Long‐term increases in tropical flowering activity across growth forms in response to rising CO2 and climate change

Mounting evidence suggests that anthropogenic global change is altering plant species composition in tropical forests. Fewer studies, however, have focused on long‐term trends in reproductive activity, in part because of the lack of data from tropical sites. Here, we analyze a 28‐year record of tropical flower phenology in response to anthropogenic climate and atmospheric change. We show that a multidecadal increase in flower activity is most strongly associated with rising atmospheric CO₂ concentrations using yearly aggregated data. Compared to significant climatic factors, CO₂ had on average an approximately three‐, four‐, or fivefold stronger effect than rainfall, solar radiation, and the Multivariate ENSO Index, respectively. Peaks in flower activity were associated with greater solar radiation and lower rainfall during El Niño years. The effect of atmospheric CO₂ on flowering has diminished over the most recent decade for lianas and canopy trees, whereas flowering of midstory trees and shrub species continued to increase with rising CO₂. Increases in flowering were accompanied by a lengthening of flowering duration for canopy and midstory trees. Understory treelets did not show increases in flowering but did show increases in duration. Given that atmospheric CO₂ will likely continue to climb over the next century, a long‐term increase in flowering activity may persist in some growth forms until checked by nutrient limitation or by climate change through rising temperatures, increasing drought frequency and/or increasing cloudiness and reduced insolation.     

The PGK epitope of human thyroglobulin: A molecular marker of alternatively spliced thyroglobulin molecules?

Summary Human thyroglobulin (hTg) is the 2748 aa precursor of the thyroid hormones T3 and T4. In autoimmune thyroid diseases, autoantibodies to hTg appeared which showed a restricted epitope specificity for the central region of the molecule (residues 1149–1251). Our hypothesis to explain why this particular region becomes autoantigenic is presented, which involves the existence of truncated, alternatively spliced forms of hTg in the bloodstream. To try to prove this hypothesis, we have undertaken the identification of the peptide epitopes recognized by monoclonal antibodies on the thyroglobulin molecule by multiple peptide synthesis methods; we report here on the identification of the three-residue epitope, Pro-Gly-Lys in position 1282–1284 of the hTg sequence which is recognized by monoclonal antibodies Tg2 and Tg8. Due to their sequence specificity, these antibodies could provide a means to tag the region of the hTg sequence which is suggested to be the site of an alternative processing phenomenon. Our results are discussed in terms of both the specificity of anti-hTg monoclonal antibodies and of the mechanism of appearance of autoantibodies recognizing the central region of hTg.     

Amino acid biosynthesis and metabolic flux profiling of Pichia pastoris.

Amino acid biosynthesis and central carbon metabolism of Pichia pastoris were studied using biosynthetically directed fractional (13)C labeling. Cells were grown aerobically in a chemostat culture fed at two dilution rates (0.05 h(-1), 0.16 h(-1)) with glycerol as the sole carbon source. For investigation of amino acid biosynthesis and comparison with glycerol cultivations, cells were also grown at 0.16 h(-1) on glucose. Our results show that, firstly, amino acids are synthesized as in Saccharomyces cerevisiae. Secondly, biosynthesis of mitochondrial pyruvate via the malic enzyme is not registered for any of the three cultivations. Thirdly, transfer of oxaloacetate across the mitochondrial membrane appears bidirectional, with a smaller fraction of cytosolic oxaloacetate stemming from the mitochondrial pool at the higher dilution rate of 0.16 h(-1) (for glucose or glycerol cultivation) when compared to the glycerol cultivation at 0.05 h(-1). Fourthly, the fraction of anaplerotic synthesis of oxaloacetate increases from 33% to 48% when increasing the dilution rate for glycerol supply, while 38% is detected when glucose is fed. Finally, the cultivation on glucose also allowed qualitative comparison with the flux ratio profile previously published for Pichia stipitis and S. cerevisiae grown on glucose in a chemostat culture at a dilution rate of 0.1 h(-1). This provided a first indication that regulation of central carbon metabolism in P. pastoris and S. cerevisiae might be more similar to each other than to P. stipitis.     

Functional Changes in Rat Nigral GABAA Receptors Induced by Degeneration of the Striatonigral GABAergic Pathway: An Electrophysiological Study of Receptors Incorporated into Xenopus Oocytes

Abstract: Expression of rat brain γ-aminobutyric acid type A (GABA_A) receptors in Xenopus laevis oocytes can be achieved by injection of the oocytes with synaptosomes. This approach has now been applied to evaluate changes in the function of nigral GABA_A receptors after degeneration of the striatonigral GABAergic pathway induced by the unilateral infusion of kainic acid into the rat striatum. Ten days after striatal injection, synaptosomal membranes were prepared from the substantia nigra and introduced into oocytes. Nigral GABA_A receptors incorporated into the oocyte cell membrane were then characterized electrophysiologically under voltage-clamp conditions. The maximal amplitude of GABA-induced Cl^? currents in oocytes injected with synaptosomes from denervated substantia nigra was twice that observed in oocytes injected with synaptosomes from control substantia nigra. The concentration of GABA required for the half-maximal response did not differ between the two groups of oocytes. In addition, the potentiation of GABA-induced currents by the benzodiazepine diazepam (1 µM) and the steroid derivative allopregnanolone (3 µM) was increased by ～65 and 60%, respectively, in oocytes injected with synaptosomes from denervated substantia nigra compared with those injected with control synaptosomes. The concentrations of diazepam and allopregnanolone giving half-maximal responses were not affected by denervation. In contrast, the inhibitory effects of the benzodiazepine receptor inverse agonists FG 7142 (10 µM) and 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylic acid ethyl ester (1 µM) were reduced by 48 and 38%, respectively, after denervation. These results indicate that the up-regulation of nigral GABA_A receptors induced by degeneration of the striatonigral GABAergic pathway is associated with an increased efficacy of positive allosteric modulators, such as benzodiazepines and steroids, and with a reduced efficacy of negative allosteric modulators such as β-carbolines.