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.     

The first vitamin B6 zinc complex, pyridoxinato-zinc acetate: A 1D coordination polymer with polar packing through strong inter-chain hydrogen bonding

The biologically important pyridoxinato(1−) ligand (anionic vitamin B₆) shows the rare phenolate-hydroxymethyl chelation plus bridging mode through the pyridine-nitrogen atom towards zinc(II) to give the one-dimensional (1D) coordination polymer {(acetato-κO)-aqua-μ-[2-methyl-3-oxy-4,5-di(hydroxymethyl)pyridine-κN:O,O′]zinc(II)}·monohydrate with polar packing of adjacent chains along the polar c axis (in space group Pc) through strong inter-chain hydrogen bonding.     

Iridoid glucosides in fouquieriaceae

From three Fouquieria sp. 12 iridoid glucosides were isolated and identified. Eight of these were structurally related to galioside (monotropein methylester), while four were hydroxy substitution products of deoxyloganin. In three cases the glucoside occurred together with the corresponding 10-O-acetate.     

Clinical adaptation of a high-performance liquid chromatographic method for the assay of pyridoxal 5′-phosphate in human plasma

Vitamin B6, measured as pyridoxal 5′-phosphate (PLP), is a co-enzyme in the transsulfuration pathway of homocysteine metabolism. Since depletion of PLP has been suggested as an independent risk factor for coronary artery disease, PLP is frequently measured to guide patient care. By a change and utilization of an Aquasil C18 column and the addition of an acetonitrile clean-up gradient to the potassium phosphate, with sodium perchlorate and bisulfite buffer between samples we report the modification of a previously described method for analysis of PLP. The result is a more practical, efficient, reliable and robust method for daily clinical use. We also determined and report that it is critical to protect freshly prepared standard PLP samples from light exposure during assay preparation.     

Microtubules promote the non-cell autonomous action of microRNAs by inhibiting their cytoplasmic loading onto ARGONAUTE1 in Arabidopsis

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Towards Molecular Understanding of the pH Dependence Characterizing NhaA of Which Structural Fold is Shared by Other Transporters

Na⁺/H⁺ antiporters comprise a super-family (CPA) of membrane proteins that are found in all kingdoms of life and are essential in cellular homeostasis of pH, Na⁺ and volume. Their activity is strictly dependent on pH, a property that underpins their role in pH homeostasis. While several human homologues have long been drug targets, NhaA of Escherichia coli has become the paradigm for this class of secondary active transporters as NhaA crystal structure provided insight into the architecture of this molecular machine. However, the mechanism of the strict pH dependence of NhaA is missing. Here, as a follow up of a recent evolutionary analysis that identified a ‘CPA motif’, we rationally designed three E. coli NhaA mutants: D133S, I134T, and the double mutant D133S-I134T. Exploring growth phenotype, transport activity and Li⁺-binding of the mutants, we revealed that Asp133 does not participate directly in proton binding, nor does it directly dictate the pH-dependent transport of NhaA. Strikingly, the variant I134T lost some of the pH control, and the D133S-Il134T double mutant retained Li⁺ binding in a pH independent fashion. Concurrent to loss of pH control, these mutants bound Li⁺ more strongly than the WT. Both positions are in close vicinity to the ion-binding site of the antiporter, attributing the results to electrostatic interaction between these residues and Asp164 of the ion-binding site. This is consistent with pH sensing resulting from direct coupling between cation binding and deprotonation in Asp164, which applies also to other CPA antiporters that are involved in human diseases.     

Prediction of the halothane (Hal) genotypes of pigs by deducing Hal, Phi, Po2, Pgd haplotypes of parents and offspring: results from a large-scale practice in Swedish breeds

Results from a large-scale study, comprising 75 different breeding herds, are reported on predicting the halothane ( Hal ) genotypes of individual pigs by making use of the known close linkage between Hal and three C blood marker loci ( Phi, Po2, Pgd ). The parents haplotypes (involving Hal and marker loci) were determined from the HAL phenotypes (halothane test results) and marker loci phenotypes of their offspring in the first one or two litters studied. In subsequent litters of the Hal -marker loci haplotyped parents, the offspring's expected Hal genotypes could be predicted on the basis of the marker loci haplotypes inherited by them. By comparing the expected and observed HAL phenotypes of offspring in subsequent litters, the predicted Hal genotype was found to be correct in 90–95 % of the 4000 offspring (from Nn × Nn and Nn × nn matings) of Swedish Landrace and Yorkshire breeds studied.
The order of the three marker loci was confirmed as Phi-Po2-Pgd but the position of Hal with regards to Phi could not be resolved. The recombination frequencies between the most distant loci in this region, viz. Hal-Pgd and Phi-Pgd , were estimated to be 3–4.5 % and 4–6 % , respectively. The easy and rapid electrophoretic techniques described in the study to phenotype PHI, PO2, PGD, also allowed phe-notyping of six other polymorphic protein systems on the same gels. Thus Hal genotyping and effective parentage control can be conducted simultaneously.     

A comparative Proteomics Analysis Identified Differentially Expressed Proteins in Pancreatic Cancer–Associated Stellate Cell Small Extracellular Vesicles

Human pancreatic stellate cells (HPSCs) are an essential stromal component and mediators of pancreatic ductal adenocarcinoma (PDAC) progression. Small extracellular vesicles (sEVs) are membrane-enclosed nanoparticles involved in cell-to-cell communications and are released from stromal cells within PDAC. A detailed comparison of sEVs from normal pancreatic stellate cells (HPaStec) and from PDAC-associated stellate cells (HPSCs) remains a gap in our current knowledge regarding stellate cells and PDAC. We hypothesized there would be differences in sEVs secretion and protein expression that might contribute to PDAC biology. To test this hypothesis, we isolated sEVs using ultracentrifugation followed by characterization by electron microscopy and Nanoparticle Tracking Analysis. We report here our initial observations. First, HPSC cells derived from PDAC tumors secrete a higher volume of sEVs when compared to normal pancreatic stellate cells (HPaStec). Although our data revealed that both normal and tumor-derived sEVs demonstrated no significant biological effect on cancer cells, we observed efficient uptake of sEVs by both normal and cancer epithelial cells. Additionally, intact membrane-associated proteins on sEVs were essential for efficient uptake. We then compared sEV proteins isolated from HPSCs and HPaStecs cells using liquid chromatography–tandem mass spectrometry. Most of the 1481 protein groups identified were shared with the exosome database, ExoCarta. Eighty-seven protein groups were differentially expressed (selected by 2-fold difference and adjusted p value ≤0.05) between HPSC and HPaStec sEVs. Of note, HPSC sEVs contained dramatically more CSE1L (chromosome segregation 1–like protein), a described marker of poor prognosis in patients with pancreatic cancer. Based on our results, we have demonstrated unique populations of sEVs originating from stromal cells with PDAC and suggest that these are significant to cancer biology. Further studies should be undertaken to gain a deeper understanding that could drive novel therapy.     

Dynamic and Reversible Aggregation of the Human CAP Superfamily Member GAPR-1 in Protein Inclusions in Saccharomyces cerevisiae

Many proteins that can assemble into higher order structures termed amyloids can also concentrate into cytoplasmic inclusions via liquid–liquid phase separation. Here, we study the assembly of human Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1), an amyloidogenic protein of the Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 proteins (CAP) protein superfamily, into cytosolic inclusions in Saccharomyces cerevisiae. Overexpression of GAPR-1-GFP results in the formation GAPR-1 oligomers and fluorescent inclusions in yeast cytosol. These cytosolic inclusions are dynamic and reversible organelles that gradually increase during time of overexpression and decrease after promoter shut-off. Inclusion formation is, however, a regulated process that is influenced by factors other than protein expression levels. We identified N-myristoylation of GAPR-1 as an important determinant at early stages of inclusion formation. In addition, mutations in the conserved metal-binding site (His54 and His103) enhanced inclusion formation, suggesting that these residues prevent uncontrolled protein sequestration. In agreement with this, we find that addition of Zn²⁺ metal ions enhances inclusion formation. Furthermore, Zn²⁺ reduces GAPR-1 protein degradation, which indicates stabilization of GAPR-1 in inclusions. We propose that the properties underlying both the amyloidogenic properties and the reversible sequestration of GAPR-1 into inclusions play a role in the biological function of GAPR-1 and other CAP family members.