6-Phosphofructo-2-kinase and D-fructose-2,6-bisphosphatase activities in mung bean seedlings

6-Phosphofructo-2-kinase (ATP: D-fructose-6-phosphate-2-phosphotransferase) and D-fructose-2,6-bisphosphatase activities have been found in extracts prepared from etiolated mung bean seedlings. The activity of 6-phosphofructo-2-kinase exhibits a sigmoidal shape in response to changes in concentrations of both substrates, D-fructose 6-phosphate and ATP (S_0.5 values of 1.8 and 1.2 mM, respectively). Inorganic orthophosphate (P_i) has a strong stimulating effect on the 2-kinase activity (A_0.5 at about 2 mM), moderately increasing the V_max and modifying the response into hyperbolic curves with K_m values of 0.4 and 0.2 mM for fructose 6-phosphate and ATP, respectively. 3-Phosphoglycerate (I_0.5 about 0.15 mM) partially inhibited the kinase activity by counteracting the P_i activation. In contrast, the activity of D-fructose-2,6-bisphosphatase (K_m 0.38 mM) is strongly inhibited by P_i (I_0.5 0.8 mM) lowering its affinity to fructose-2,6-P₂ (K_m 1.4 mM). 3-Phosphoglycerate activites the enzyme (A_0.5 at about 0.3 mM) without causing a significant change in its K_m for fructose-2,6-P₂. The activities of both of these enzymes in relationship to the metabolic role of D-fructose 2,6-bisphosphate in the germinating seed is discussed.     

Platelet activating factor synthesis and metabolism in intestinal ischaemia-reperfusion injury

The object of this study was to characterize the synthesis and metabolism of platelet activating factor (PAF) by intestinal mucosa subjected to ischaemia-reperfusion injury. Canine intestinal mucosa produced 16:0-PAF, 18:0-PAF, and high levels of the corresponding lyso- PAF metabolites. Three h of intestinal ischaemia and ischaemia followed by 1 h of reperfusion did not affect the synthesis or metabolism of PAF by intestinal mucosa. Intestinal mucosa elaborated a factor that rapidly hydrolyzes PAF to lyso-PAF. The observed hydrolysis rate was not altered by ischaemia or ischaemia and reperfusion. In conclusion, this study suggests that intestinal mucosa produces PAF and rapidly hydrolyzes PAF. The PAF synthesis and metabolism rates of intestinal mucosa is not altered by ischaemia reperfusion in this model under the imposed conditions.     

Influence of molecular configuration on the passage of macromolecules across the glomerular capillary wall

The influence of molecular configuration on the filtration of macromolecules across glomerular capillary walls was examined by comparing fractional clearances of two uncharged polysaccharides of distinctly different molecular configuration in the Munich-Wistar rat. The macromolecules employed were dextran, a slightly branched polymer of glucopyranose, and ficoll, a highly cross-linked copolymer of sucrose and epichlorohydrin. Differences in effective shape between these two polymers were determined from measurements of several physical properties of aqueous solutions containing either dextran or ficoll. It was found that dextran is best represented as a prolate ellipsoid with axial ratios of 4, 9, and 16 for molecules with Stokes-Einstein radii of 22, 32, and 40 A, respectively. On the other hand, ficoll is more closely approximated as spherical since the axial ratio was found to be between 1 and 2 for all molecular sizes. Fractional clearances of dextran and ficoll ranging in effective radius from 18 to 44 A were determined in each of seven Munich-Wistar rats. Fractional clearances of dextran were found to be greater than those of ficoll, the difference being significant for molecular radii ranging from 24 to 44 A. In addition, as shown previously for dextran, ficoll was found to be neither secreted nor reabsorbed by the renal tubules. These results, therefore, suggest that in addition to molecular size and charge, molecular configuration is also a determinant of the filtration of macromolecules across the glomerular capillary wall.     

Coupling of M3 Acetylcholine Receptor to Gq16 Activates a Natriuretic Peptide Receptor Guanylyl Cyclase

Muscarinic activation of tracheal smooth muscle (TSM) involves a M₃AChR/heterotrimeric-G protein/NPR-GC coupling mechanism. G protein activators Mastoparan (MAS) and Mastoparan-7 stimulated 4- and 10-fold the NPR-GC respectively, being insensitive to PTX and antibodies against Gα_i/o subfamily. Muscarinic and MAS stimulation of NPR-GC was blocked by antibodies against C-terminal of Gα_q16, whose expression was confirmed by RT-PCR. However, synthetic peptides from C-terminal of Gα_q15/16 stimulated the NPR-GC. Coupling of α_q16 to M₃AChR is supported by MAS decreased [³H]QNB binding, being abolished after M₃AChR-4-DAMP-alkylation. Anti-i₃M₃AChR antibodies blocked the muscarinic activation of NPR-GC, and synthetic peptide from i₃M₃AChR (M₃P) was more potent than MAS increasing GTPγ [³⁵S] and decreasing the [³H]QNB activities. Coupling between NPR-GC and Gα_q16 was evaluated by using trypsin-solubilized-fraction from TSM membranes, which displayed a MAS-sensitive-NPR-GC activity, being immunoprecipitated with anti-Gα_q16, also showing an immunoreactive heterotrimeric-G-β -subunit. These data support the existence of a novel transducing cascade, involving Gα_q16β γ coupling M₃AChR to NPR-GC.     

FireStem2D – A Two-Dimensional Heat Transfer Model for Simulating Tree Stem Injury in Fires

FireStem2D, a software tool for predicting tree stem heating and injury in forest fires, is a physically-based, two-dimensional model of stem thermodynamics that results from heating at the bark surface. It builds on an earlier one-dimensional model (FireStem) and provides improved capabilities for predicting fire-induced mortality and injury before a fire occurs by resolving stem moisture loss, temperatures through the stem, degree of bark charring, and necrotic depth around the stem. We present the results of numerical parameterization and model evaluation experiments for FireStem2D that simulate laboratory stem-heating experiments of 52 tree sections from 25 trees. We also conducted a set of virtual sensitivity analysis experiments to test the effects of unevenness of heating around the stem and with aboveground height using data from two studies: a low-intensity surface fire and a more intense crown fire. The model allows for improved understanding and prediction of the effects of wildland fire on injury and mortality of trees of different species and sizes.     

A holistic high-throughput screening framework for biofuel feedstock assessment that characterises variations in soluble sugars and cell wall composition in <Emphasis Type="Italic">Sorghum bicolor</Emphasis>

Background

A major hindrance to the development of high yielding biofuel feedstocks is the ability to rapidly assess large populations for fermentable sugar yields. Whilst recent advances have outlined methods for the rapid assessment of biomass saccharification efficiency, none take into account the total biomass, or the soluble sugar fraction of the plant. Here we present a holistic high-throughput methodology for assessing sweet Sorghum bicolor feedstocks at 10 days post-anthesis for total fermentable sugar yields including stalk biomass, soluble sugar concentrations, and cell wall saccharification efficiency.

Results

A mathematical method for assessing whole S. bicolor stalks using the fourth internode from the base of the plant proved to be an effective high-throughput strategy for assessing stalk biomass, soluble sugar concentrations, and cell wall composition and allowed calculation of total stalk fermentable sugars. A high-throughput method for measuring soluble sucrose, glucose, and fructose using partial least squares (PLS) modelling of juice Fourier transform infrared (FTIR) spectra was developed. The PLS prediction was shown to be highly accurate with each sugar attaining a coefficient of determination (R ² ) of 0.99 with a root mean squared error of prediction (RMSEP) of 11.93, 5.52, and 3.23 mM for sucrose, glucose, and fructose, respectively, which constitutes an error of <4% in each case. The sugar PLS model correlated well with gas chromatography–mass spectrometry (GC-MS) and brix measures. Similarly, a high-throughput method for predicting enzymatic cell wall digestibility using PLS modelling of FTIR spectra obtained from S. bicolor bagasse was developed. The PLS prediction was shown to be accurate with an R ² of 0.94 and RMSEP of 0.64 μg.mgDW^-1.h^-1.

Conclusions

This methodology has been demonstrated as an efficient and effective way to screen large biofuel feedstock populations for biomass, soluble sugar concentrations, and cell wall digestibility simultaneously allowing a total fermentable yield calculation. It unifies and simplifies previous screening methodologies to produce a holistic assessment of biofuel feedstock potential.

     

The two rat alpha 2,6-sialyltransferase (ST6Gal I) isoforms: evaluation of catalytic activity and intra-Golgi localization

alpha2,6-Sialyltransferase (ST6Gal I) functions in the Golgi to terminally sialylate the N-linked oligosaccharides of glycoproteins. Interestingly, rat ST6Gal I is expressed as two isoforms, STtyr and STcys, that differ by a single amino acid in their catalytic domains. In this article, our goal was to evaluate more carefully possible differences in the catalytic activity and intra-Golgi localization of the two isoforms that had been suggested by earlier work. Using soluble recombinant STtyr and STcys enzymes and three asialoglycoprotein substrates for in vitro analysis, we found that the STcys isoform was somewhat more active than the STtyr isoform. However, we found no differences in isoform substrate choice when these proteins were expressed in Chinese hamster ovary cells, and sialylated substrates were detected by lectin blotting. Immuno-fluorescence and immunoelectron microscopy revealed differences in the relative levels of the isoforms found in the endoplasmic reticulum (ER) and Golgi of transiently expressing cells but similar intra-Golgi localization. STtyr was restricted to the Golgi in most cells, and STcys was found in both the ER and Golgi. The ER localization of STcys was especially pronounced with a C-terminal V5 epitope tag. Ultrastructural and deconvolution studies of immunostained HeLa cells expressing STtyr or STcys showed that within the Golgi both isoforms are found in medial-trans regions. The similar catalytic activities and intra-Golgi localization of the two ST6Gal I isoforms suggest that the particular isoform expressed in specific cells and tissues is not likely to have significant functional consequences.     

Development, characterization, and comparative analysis of polymorphism at common bean SSR loci isolated from genic and genomic sources.

Microsatellites or SSRs (single sequence repeats) have been used to construct and integrate genetic maps in crop species, including Phaseolus vulgaris. In the present study, 3 cDNA libraries generated by the Bean EST project (http://lgm.esalq.usp.br/BEST/), comprising a unigene collection of 3126 sequences and a genomic microsatellite-enriched library, were analyzed for the presence of SSRs. A total of 219 expressed sequence tags (ESTs) were found to carry 240 SSRs (named EST-SSR), whereas 714 genomic sequences contained 471 SSRs (named genomic-SSR). A subset of 80 SSRs, 40 EST-SSRs, and 40 genomic-SSRs were evaluated for molecular polymorphism in 23 genotypes of cultivated beans from the Mesoamerican and Andean genetic pools, including Brazilian cultivars and 2 related species. Of the common bean genotypes, 31 EST-SSR loci were polymorphic, yielding 2-12 alleles as compared with 26 polymorphic genomic-SSRs, accounting for 2-7 alleles. Cluster analysis from data using both genic and genomic-SSR revealed a clear separation between Andean and Mesoamerican beans. The usefulness of these loci for distinguishing bean genotypes and genetic mapping is discussed.     

Impact of QTL properties on the accuracy of multi-breed genomic prediction

Background

Although simulation studies show that combining multiple breeds in one reference population increases accuracy of genomic prediction, this is not always confirmed in empirical studies. This discrepancy might be due to the assumptions on quantitative trait loci (QTL) properties applied in simulation studies, including number of QTL, spectrum of QTL allele frequencies across breeds, and distribution of allele substitution effects. We investigated the effects of QTL properties and of including a random across- and within-breed animal effect in a genomic best linear unbiased prediction (GBLUP) model on accuracy of multi-breed genomic prediction using genotypes of Holstein-Friesian and Jersey cows.

Methods

Genotypes of three classes of variants obtained from whole-genome sequence data, with moderately low, very low or extremely low average minor allele frequencies (MAF), were imputed in 3000 Holstein-Friesian and 3000 Jersey cows that had real high-density genotypes. Phenotypes of traits controlled by QTL with different properties were simulated by sampling 100 or 1000 QTL from one class of variants and their allele substitution effects either randomly from a gamma distribution, or computed such that each QTL explained the same variance, i.e. rare alleles had a large effect. Genomic breeding values for 1000 selection candidates per breed were estimated using GBLUP modelsincluding a random across- and a within-breed animal effect.

Results

For all three classes of QTL allele frequency spectra, accuracies of genomic prediction were not affected by the addition of 2000 individuals of the other breed to a reference population of the same breed as the selection candidates. Accuracies of both single- and multi-breed genomic prediction decreased as MAF of QTL decreased, especially when rare alleles had a large effect. Accuracies of genomic prediction were similar for the models with and without a random within-breed animal effect, probably because of insufficient power to separate across- and within-breed animal effects.

Conclusions

Accuracy of both single- and multi-breed genomic prediction depends on the properties of the QTL that underlie the trait. As QTL MAF decreased, accuracy decreased, especially when rare alleles had a large effect. This demonstrates that QTL properties are key parameters that determine the accuracy of genomic prediction.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-015-0124-6) contains supplementary material, which is available to authorized users.