Regulation of development of leucine uptake activity by glutamine in the scutellum of germinating barley grain

Scutella from ungerminated grains of barley (Hordeum vulgare L. cv Pirkka) take up leucine at a slow rate, which increases rapidly during germination. When endosperms were removed from the grains after imbibition for 4 hours or after germination for 12 or 72 hours, the increase in the rate of leucine uptake was greatly accelerated during subsequent incubation of the embryos or scutella. These increases were rapidly inhibited by cordycepin and cycloheximide, suggesting that protein synthesis, probably synthesis of the carrier protein, was required for the development of the uptake activity.

In separated embryos or scutella, the increases in the leucine uptake activity were inhibited by glutamine. The inhibitions caused by glutamine and cycloheximide were not additive, suggesting that glutamine did not interfere with the function of the carrier but repressed its synthesis. Glutamine did not inhibit the simultaneous increase in peptide uptake; in this respect, its effect was specific for leucine uptake, which appears to be due to a general amino acid uptake system.

Some other protein amino acids also inhibited the increase in leucine uptake without inhibiting the increase in peptide uptake. However, these effects were smaller than that of glutamine.

These results suggest that the transfer of leucine (and other amino acids) from the endosperm to the seedling in a germinating barley grain is regulated at the uptake step by repression of the synthesis of the amino acid carrier protein by glutamine and—possibly to a lesser extent—by some other amino acids taken up from the endosperm.

     

Determination of short-chain fatty acids in serum by hollow fiber supported liquid membrane extraction coupled with gas chromatography

A method based on hollow fiber supported liquid membrane extraction coupled with a gas chromatograph equipped with flame ionization detector (GC-FID) was developed for the determination of six short-chain fatty acids including acetic acid, propionic acid, i-butyric acid, n-butyric acid, i-valeric acid and n-valeric acid in serum. Hollow fiber supported liquid membrane extraction was employed for preconcentration and clean-up of the samples. The fatty acids were extracted from the acidic donor (diluted serum) into a liquid membrane formed in the wall of the hollow fiber with 10% tri-n-octylphoshphine oxide (TOPO) in di-n-hexyl ether, and then extracted back into a basic acceptor solution filled in the lumen of the hollow fiber. After being acidified with HCl, the acceptor was directly analyzed by GC-FID. The acceptor concentration, donor pH, membrane liquid and extracting time were optimized giving an enrichment factor up to 155 times. The good linearity (r(2)>0.980), reasonable recovery (87.2-121%), and satisfactory intra-assay (8.2-11.5%) and inter-assay (6.1-11.6%) precision illustrated the good performance of the present method. Limits of detection (LOD) ranged from 0.04 to 0.24 microM and limits of quantification (LOQ) varied from 0.13 to 0.80 microM.     

Heterogeneous response to biostimulation for U(VI) reduction in replicated sediment microcosms

A field-scale experiment to assess biostimulation of uranium reduction is underway at the Natural and Accelerated Bioremediation Research Field Research Center (FRC) in Oak Ridge, Tennessee. To simulate the field experiment, we established replicate batch microcosms containing well-mixed contaminated sediment from a well within the FRC treatment zone, and we added an inoculum from a pilot-scale fluidized bed reactor representing the inoculum in the field experiment. After reduction of nitrate, both sulfate and soluble U(VI) concentration decreased. X-ray absorption near edge structure (XANES) spectroscopy confirmed formation of U(IV) in sediment from biostimulated microcosms, but did not detect reduction of solid-phase Fe(III). Two to three fragments dominated terminal restriction fragment length polymorphism (T-RFLP) profiles of the 16S rDNA gene. Comparison to a clone library indicated these fragments represented denitrifying organisms related to Acidovorax, and Acidovorax isolates from the inoculum were subsequently shown to reduce U(VI). Investigation using the T-RFLP Analysis Program (TAP T-RFLP) and chemical analyses detected the presence and activity of fermenting and sulfate-reducing bacteria after 2 weeks. These organisms likely contributed to uranium reduction. In some microcosms, soluble U(VI) concentration leveled off or rebounded, indicating microbial and/or mineralogical heterogeneity among samples. Sulfate, acetate, and ethanol were depleted only in those microcosms exhibiting a rebound in soluble U(VI). This suggests that rates of U(VI) desorption can exceed rates of U(VI) reduction when sulfate-reducing bacteria become substrate-limited. These observations underscore the importance of effective chemical delivery and the role of serial and parallel processes in uranium reduction.     

The structure of the PP2A regulatory subunit B56 gamma: the remaining piece of the PP2A jigsaw puzzle

The PP2A serine/threonine phosphatase regulates a plethora of cellular processes. In the cell the predominant form of the enzyme is a heterotrimer, formed by a core dimer composed of a catalytic and a scaffolding subunit, which assemble together with one of a range of different regulatory B subunits. Here, we present the first structure of a free non-complexed B subunit, B56 gamma. Comparison with the recent structures of a heterotrimeric complex and the core dimer reveals several significant conformational changes in the interface region between the B56 gamma and the core dimer. These allow for an assembly scheme of the PP2A holoenzyme to be put forth where B56 gamma first complexes with the scaffolding subunit and subsequently binds to the catalytic subunit and this induces the formation of a binding site for the invariant C-terminus of the catalytic subunit that locks in the complex as a last step of assembly.     

Quantitative measures of femoral fracture repair in rats derived by micro-computed tomography

Although fracture healing is frequently studied in pre-clinical models of long bone fractures using rodents, there is a dearth of objective quantitative techniques to assess successful healing. Biomechanical testing is possibly the most quantitative and relevant to a successful clinical outcome, but it is a destructive technique providing little insight into the cellular mechanisms associated with healing. The advent of X-ray computed tomography (CT) has provided the opportunity to quantitatively and non-destructively assess bone structure and density, but it is unknown how measurements derived using this technology relate to successful healing. To examine possible relationships, we used a pre-clinical model to test for statistically significant correlations between quantitative characteristics of the callus by micro-CT (μCT) and the bending strength, stiffness, and energy-to-failure of the callus as assessed by three-point bending of excised bones. A closed, transverse fracture was generated in the mid-shaft of rat femurs by impact loading. Shortly thereafter, the rats received a one-time, local injection of either the vehicle or one of four doses of lovastatin. Following sacrifice after 4 weeks of healing, fractured femurs were extracted for μCT analysis and then three-point bending. Setting the region of interest to be 3.2 mm above and below the fracture line, we acquired standard and new μCT-derived measurements. The mineralized callus volume and the mineral density of the callus correlated positively with callus strength (r_xy=?0.315, p=0.016 and r_xy=0.444, p<0.0005, respectively) and stiffness (r_xy=?0.271, p=0.040 and r_xy=0.325, p=0.013, respectively), but the fraction of the callus that mineralized and the moment of inertia of the callus did not. This fraction did correlate with energy-to-failure (r_xy=?0.343, p=0.0085). Of the μCT-derived measurements, quantifying defects within the outer bridging cortices of the callus produced the strongest correlation with both callus strength (r_xy=0.557, p<0.0001) and stiffness (r_xy=0.468, p=0.0002). By both reducing structural defects and increasing mineralization, lovastatin appears to increase the callus strength.     

Metabolism during anaesthesia and recovery in colic and healthy horses: a microdialysis study

Background  

Muscle metabolism in horses has been studied mainly by analysis of substances in blood or plasma and muscle biopsy specimens. By using microdialysis, real-time monitoring of the metabolic events in local tissue with a minimum of trauma is possible. There is limited information about muscle metabolism in the early recovery period after anaesthesia in horses and especially in the colic horse. The aims were to evaluate the microdialysis technique as a complement to plasma analysis and to study the concentration changes in lactate, pyruvate, glucose, glycerol, and urea during anaesthesia and in the recovery period in colic horses undergoing abdominal surgery and in healthy horses not subjected to surgery.     

To speciate,or not to speciate? Resource heterogeneity,the subjectivity of similarity,and the macroevolutionary consequences of niche‐width shifts in plant‐feeding insects

Coevolutionary studies on plants and plant‐feeding insects have significantly improved our understanding of the role of niche shifts in the generation of new species. Evolving plant lineages essentially constitute moving islands and archipelagoes in resource space, and host shifts by insects are usually preceded by colonizations of novel resources. Critical to hypotheses concerning ecological speciation is what happens immediately before and after colonization attempts: if an available plant is too similar to the current host(s), it simply will be incorporated into the existing diet, but if it is too different, it will not be colonized in the first place. It thus seems that the probability of speciation is maximized when alternative hosts are at an ‘intermediate’ distance in resource space. In this review, I wish to highlight the possibility that resource similarity and, thus, the definition of ‘intermediate’, are subjective concepts that depend on the herbivore lineage's tolerance to dietary variation. This subjectivity of similarity means that changes in tolerance can either decrease or increase speciation probabilities depending on the distribution of plants in resource space: insect lineages with narrow tolerances are likely to speciate by ‘island‐hopping’ on young, species‐rich plant groups, whereas more generalized lineages could speciate by shifting among resource archipelagoes formed by higher plant taxa. Repeated and convergent origins of traits known to broaden or to restrict host‐plant use in multiple different insect groups provide opportunities for studying how tolerance and resource heterogeneity may interact to determine speciation rates.     

Pripper: prediction of caspase cleavage sites from whole proteomes

Background  

Caspases are a family of proteases that have central functions in programmed cell death (apoptosis) and inflammation. Caspases mediate their effects through aspartate-specific cleavage of their target proteins, and at present almost 400 caspase substrates are known. There are several methods developed to predict caspase cleavage sites from individual proteins, but currently none of them can be used to predict caspase cleavage sites from multiple proteins or entire proteomes, or to use several classifiers in combination. The possibility to create a database from predicted caspase cleavage products for the whole genome could significantly aid in identifying novel caspase targets from tandem mass spectrometry based proteomic experiments.     

Hormone,vitamin and contaminant status during the moulting/fasting period in ringed seals (Pusa [Phoca] hispida) from Svalbard

This study investigates the potential effects of moulting, and the concomitant period of fasting undertaken by ringed seals, on hormone, vitamin and contaminant status in adult animals in a population from Svalbard, Norway, which has relatively low contaminant levels. Concentrations of circulating total and free thyroxine and triiodothyronine, circulating and hepatic vitamin A, hepatic persistent organic pollutants and their circulating hydroxyl metabolites were higher in moulting seals compared to pre-moulting seals. The opposite trend was observed for body condition, circulating calcitriol levels and hepatic mRNA expression of thyroid hormone receptor β. No differences were observed for circulating or hepatic vitamin E levels or hepatic mRNA expressions for deioidinase 1 or 2, or retinoic acid receptor α between the two seal groups. The observed differences are likely the result of increased metabolic rates required during moulting to maintain thermal balance and replace the pelage, in combination with mobilization of lipid soluble compounds from blubber stores during the fasting period that is associated with moulting. The present study shows that contaminant levels and their relationships with physiological or endogenous variables can be highly confounded by moulting/fasting status. Thus, moulting status and body condition should be taken into consideration when using variables related to thyroid, calcium or vitamin A homeostasis as biomarkers for contaminant effects.     

Structural and functional studies of the human phosphoribosyltransferase domain containing protein 1

Human hypoxanthine-guanine phosphoribosyltransferase (HPRT) (EC 2.4.2.8) catalyzes the conversion of hypoxanthine and guanine to their respective nucleoside monophosphates. Human HPRT deficiency as a result of genetic mutations is linked to both Lesch-Nyhan disease and gout. In the present study, we have characterized phosphoribosyltransferase domain containing protein 1 (PRTFDC1), a human HPRT homolog of unknown function. The PRTFDC1 structure has been determined at 1.7 ? resolution with bound GMP. The overall structure and GMP binding mode are very similar to that observed for HPRT. Using a thermal-melt assay, a nucleotide metabolome library was screened against PRTFDC1 and revealed that hypoxanthine and guanine specifically interacted with the enzyme. It was subsequently confirmed that PRTFDC1 could convert these two bases into their corresponding nucleoside monophosphate. However, the catalytic efficiency (k(cat)/K(m)) of PRTFDC1 towards hypoxanthine and guanine was only 0.26% and 0.09%, respectively, of that of HPRT. This low activity could be explained by the fact that PRTFDC1 has a Gly in the position of the proposed catalytic Asp of HPRT. In PRTFDC1, a water molecule at the position of the aspartic acid side chain position in HPRT might be responsible for the low activity observed by acting as a weak base. The data obtained in the present study indicate that PRTFDC1 does not have a direct catalytic role in the nucleotide salvage pathway.