The glycine max xylem sap and apoplast proteome

Molecular signaling interactions in the plant apoplast are important for defense and developmental responses. We examined the soybean proteome of the apoplastic conduit of root-to-shoot communication, the xylem stream, using gel electrophoresis combined with two types of tandem mass spectrometry. We examined soybeans for the presence of a Bradyrhizobium japonicum-induced, long distance developmental signal that controls autoregulation of nodulation (AON) to determine if xylem proteins (XPs) were involved directly or indirectly in AON. The xylem and apoplast fluids collected in hypocotyl, epicotyl, and stem tissue contained a highly similar set of secreted proteins. The XPs were different from those secreted from imbibing seed implying they play important basic roles in xylem function. The XPs of wild-type and nts1007 plants were indistinguishable irrespective of plant age, inoculation status, or time after inoculation suggesting that none was directly involved in AON. XPs were continuously loaded into the xylem stream, as they were present even 28 h after shoot decapitation. These results were consistent with semiquantitative RT-PCR studies that examined the expression of genes corresponding to the XPs under inoculated or uninoculated conditions. Monitoring the expression of XP genes by RT-PCR showed that four possessed root biased expression. This suggested that the corresponding protein products could be produced in roots and travel long distances to shoots. Of these, a species of lipid transfer protein is a candidate for a water-soluble, long-distance signal-carrier due to the presence of hydrophobic clefts that bind known plant signals in vitro. Two soybean XPs identified in this study, lipid transfer protein and Kunitz trypsin inhibitor (KTI), have known roles in plant signaling.     

Alchemilla viridiflora Rothm.: the potent natural inhibitor of angiotensin I-converting enzyme

Molecular and Cellular Biochemistry - Alchemilla viridiflora Rothm., Rosaceae is a herbaceous plant widespread in central Greece, Bulgaria, North Macedonia and Serbia with Kosovo. Liquid...     

Effects of short-term magnesium supplementation on ionized,total magnesium and other relevant electrolytes levels

BioMetals - This study aimed to investigate the short-term effects of three magnesium (Mg) dietary supplements containing mineral immediately available for absorption on Mg biochemical status...     

In Vitro Reassembly of the Ribose ATP-binding Cassette Transporter Reveals a Distinct Set of Transport Complexes

Bacterial ATP-binding cassette (ABC) importers are primary active transporters that are critical for nutrient uptake. Based on structural and functional studies, ABC importers can be divided into two distinct classes, type I and type II. Type I importers follow a strict alternating access mechanism that is driven by the presence of the substrate. Type II importers accept substrates in a nucleotide-free state, with hydrolysis driving an inward facing conformation. The ribose transporter in Escherichia coli is a tripartite complex consisting of a cytoplasmic ATP-binding cassette protein, RbsA, with fused nucleotide binding domains; a transmembrane domain homodimer, RbsC₂; and a periplasmic substrate binding protein, RbsB. To investigate the transport mechanism of the complex RbsABC₂, we probed intersubunit interactions by varying the presence of the substrate ribose and the hydrolysis cofactors, ATP/ADP and Mg²⁺. We were able to purify a full complex, RbsABC₂, in the presence of stable, transition state mimics (ATP, Mg²⁺, and VO₄); a RbsAC complex in the presence of ADP and Mg²⁺; and a heretofore unobserved RbsBC complex in the absence of cofactors. The presence of excess ribose also destabilized complex formation between RbsB and RbsC. These observations suggest that RbsABC₂ shares functional traits with both type I and type II importers, as well as possessing unique features, and employs a distinct mechanism relative to other ABC transporters.     

An Investigation into the Influence of Experimental Conditions on <Emphasis Type="Italic">In Vitro</Emphasis> Drug Release from Immediate-Release Tablets of Levothyroxine Sodium and Its Relation to Oral Bioavailability

The aim of this study was to investigate the influence of experimental conditions on levothyroxine sodium release from two immediate-release tablet formulations which narrowly passed the standard requirements for bioequivalence studies. The in vivo study was conducted as randomised, single-dose, two-way cross-over pharmacokinetic study in 24 healthy subjects. The in vitro study was performed using various dissolution media, and obtained dissolution profiles were compared using the similarity factor value. Drug solubility in different media was also determined. The in vivo results showed narrowly passing bioequivalence. Considering that levothyroxine sodium is classified as Class III drug according to the Biopharmaceutics Classification System, drug bioavailability will be less sensitive to the variation in its dissolution characteristics and it can be assumed that the differences observed in vitro in some of investigated media probably do not have significant influence on the absorption process, as long as rapid and complete dissolution exists. The study results indicate that the current regulatory criteria for the value of similarity factor in comparative dissolution testing, as well as request for very rapid dissolution (more than 85% of drug dissolved in 15 min), are very restricted for immediate-release dosage forms containing highly soluble drug substance and need further investigation. The obtained results also add to the existing debate on the appropriateness of the current bioequivalence standards for levothyroxine sodium products.     

Deficiency of a Niemann-Pick, type C1-related protein in toxoplasma is associated with multiple lipidoses and increased pathogenicity

Several proteins that play key roles in cholesterol synthesis, regulation, trafficking and signaling are united by sharing the phylogenetically conserved 'sterol-sensing domain' (SSD). The intracellular parasite Toxoplasma possesses at least one gene coding for a protein containing the canonical SSD. We investigated the role of this protein to provide information on lipid regulatory mechanisms in the parasite. The protein sequence predicts an uncharacterized Niemann-Pick, type C1-related protein (NPC1) with significant identity to human NPC1, and it contains many residues implicated in human NPC disease. We named this NPC1-related protein, TgNCR1. Mammalian NPC1 localizes to endo-lysosomes and promotes the movement of sterols and sphingolipids across the membranes of these organelles. Miscoding patient mutations in NPC1 cause overloading of these lipids in endo-lysosomes. TgNCR1, however, lacks endosomal targeting signals, and localizes to flattened vesicles beneath the plasma membrane of Toxoplasma. When expressed in mammalian NPC1 mutant cells and properly addressed to endo-lysosomes, TgNCR1 restores cholesterol and GM1 clearance from these organelles. To clarify the role of TgNCR1 in the parasite, we genetically disrupted NCR1; mutant parasites were viable. Quantitative lipidomic analyses on the ΔNCR1 strain reveal normal cholesterol levels but an overaccumulation of several species of cholesteryl esters, sphingomyelins and ceramides. ΔNCR1 parasites are also characterized by abundant storage lipid bodies and long membranous tubules derived from their parasitophorous vacuoles. Interestingly, these mutants can generate multiple daughters per single mother cell at high frequencies, allowing fast replication in vitro, and they are slightly more virulent in mice than the parental strain. These data suggest that the ΔNCR1 strain has lost the ability to control the intracellular levels of several lipids, which subsequently results in the stimulation of lipid storage, membrane biosynthesis and parasite division. Based on these observations, we ascribe a role for TgNCR1 in lipid homeostasis in Toxoplasma.     

Oxidative stress biomarker monitoring in elite women volleyball athletes during a 6-week training period

The objectives of this study were to determine (a) if reactive oxygen metabolites (ROMs) are a reliable parameter for monitoring oxidative stress in athletes alone or in association with other parameters of oxidative stress and depending on whether antioxidant supplements are taken or not; (b) the level of oxidative stress in athletes before the competition season; and (c) if oxidative status could be improved in volleyball athletes. Sixteen women athletes (supplemented group) received an antioxidant cocktail containing vitamin E, vitamin C, zinc gluconate, and selenium as a dietary supplement during a 6-week training period, whereas 12 of them (control group) received no dietary supplement. Blood samples were taken before and after the training period. The following parameters were measured: ROMs, superoxide anion (O2??), malondialdehyde (MDA), advanced oxidation protein products (AOPP), lipid hydroperoxide (LOOH), biological antioxidative potential (BAP), paraoxonase activity toward paraoxon (POase) and diazoxon (DZOase), superoxide dismutase(SOD), total sulfydryl group concentration (SH groups), and lipid status. Reactive oxygen metabolites were taken as the dependent variable and MDA, O2??, AOPP, and LOOH as independent variables. In the group of athletes who have received supplementation, linear regression analysis revealed that the implemented model had a lower influence on dROMs (70.4 vs. 27.9%) after the training period. The general linear model showed significant differences between parameters before and after training/supplementation (Wilks' lambda = 0.074, F = 11.76, p < 0.01). At the partial level, significant increases in ROM levels (p <0.05, 95% confidence interval [CI]: 286-337), SOD activity (CI: 113-144), and BAP (CI: 2,388-2,580) (p < 0.01) were observed. The association between ROMs and other parameters of oxidative stress was reduced in athletes who received supplements. During the precompetition training period, treatment with dietary supplements prevented the depletion of antioxidative defense in volleyball athletes.     

Kinesin molecular motor Eg5 functions during polypeptide synthesis

The kinesin-related molecular motor Eg5 plays roles in cell division, promoting spindle assembly. We show that during interphase Eg5 is associated with ribosomes and is required for optimal nascent polypeptide synthesis. When Eg5 was inhibited, ribosomes no longer bound to microtubules in vitro, ribosome transit rates slowed, and polysomes accumulated in intact cells, suggesting defects in elongation or termination during polypeptide synthesis. These results demonstrate that the molecular motor Eg5 associates with ribosomes and enhances the efficiency of translation.