Over-expression of PHO1 in Arabidopsis leaves reveals its role in mediating phosphate efflux

Inorganic phosphate (Pi) homeostasis in multi-cellular eukaryotes depends not only on Pi influx into cells, but also on Pi efflux. Examples in plants for which Pi efflux is crucial are transfer of Pi into the xylem of roots and release of Pi at the peri-arbuscular interface of mycorrhizal roots. Despite its importance, no protein has been identified that specifically mediates phosphate efflux either in animals or plants. The Arabidopsis thaliana PHO1 gene is expressed in roots, and was previously shown to be involved in long-distance transfer of Pi from the root to the shoot. Here we show that PHO1 over-expression in the shoot of A. thaliana led to a two- to threefold increase in shoot Pi content and a severe reduction in shoot growth. (31) P-NMR in vivo showed a normal initial distribution of intracellular Pi between the cytoplasm and the vacuole in leaves over-expressing PHO1, followed by a large efflux of Pi into the infiltration medium, leading to a rapid reduction of the vacuolar Pi pool. Furthermore, the Pi concentration in leaf xylem exudates from intact plants was more than 100-fold higher in PHO1 over-expressing plants compared to wild-type. Together, these results show that PHO1 over-expression in leaves leads to a dramatic efflux of Pi out of cells and into the xylem vessel, revealing a crucial role for PHO1 in Pi efflux.     

T cells recognizing a peptide contaminant undetectable by mass spectrometry

Synthetic peptides are widely used in immunological research as epitopes to stimulate their cognate T cells. These preparations are never completely pure, but trace contaminants are commonly revealed by mass spectrometry quality controls. In an effort to characterize novel major histocompatibility complex (MHC) Class I-restricted β-cell epitopes in non-obese diabetic (NOD) mice, we identified islet-infiltrating CD8+ T cells recognizing a contaminating peptide. The amount of this contaminant was so small to be undetectable by direct mass spectrometry. Only after concentration by liquid chromatography, we observed a mass peak corresponding to an immunodominant islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)(206-214) epitope described in the literature. Generation of CD8+ T-cell clones recognizing IGRP(206-214) using a novel method confirmed the identity of the contaminant, further underlining the immunodominance of IGRP(206-214). If left undetected, minute impurities in synthetic peptide preparations may thus give spurious results.     

Vaginal delivery and continuous epidural analgesia: should we change our clinical approach?

The aim of the study was to investigate the effects of continuous epidural analgesia (EA) on the course of vaginal delivery with an emphasis on duration of labor and instrumental interventions. In a prospective 2-year trial, the study group included singleton vaginal births between 35 and 41 gestational weeks with a vertex fetus, in which continuous EA with bupivacaine or chirocaine in concentration of 0.125% combined with 2-4 microg of fentanyl or 0.5 microg of sufenta was used. The control group was created randomly from laboring patients with singleton pregnancies but without EA. The groups were adjusted for epidemiological characteristics and compared regarding the obstetric data and perinatal outcome. Student t-test and Mann-Whitney U-test were performed for normally and non-normally distributed results, respectively. Out of 1284 patients, 551 pregnant women were included in the study group and 733 in the control group. The statistically significant differences between the groups related to duration of the first and second stage of labor, frequency of premature rupture of membranes, intrapartal complications, and incidence of operative deliveries were found. Both stages of labor were significantly protracted and the incidence of operative deliveries was higher in the study group of patients compared with controls. There is a need for an active obstetric approach and management of vaginal deliveries of women who receive continuous EA, particularly if it is medically indicated.     

Non-functional Fas ligand increases the formation of cartilage early in the endochondral bone induction by rhBMP-2

It has previously been shown that mice with a defect in Fas ligand-mediated apoptosis have an enhancement of ectopic bone formation. We investigated the expression of bone-related markers--alkaline phosphatase, collagen, bone sialoprotein, osteocalcin, osteopontin, and bone morphogenetic proteins (BMP) -2, -4, and -7; and cytokines interleukin-1alpha (IL-1), IL-1beta, and tumor necrosis factor-alpha (TNF-alpha) in ectopic new bone induced by recombinant human (rh) BMP-2 in mice without functional Fas-ligand (gld mice). At day 6 after rhBMP-2 implantation, gld mice formed more cartilage and mesenchyme compared with their wild type littermates. At later stages, gld mice did not differ from the control mice in the volume of newly formed tissue, expressing higher level of BMP genes and lower levels of genes involved in osteoblast maturation--bone sialoprotein and osteopontin. Differences in the levels of expression of IL-1alpha and TNF-alpha were observed only at day 12 after rhBMP-2 implantation. These results suggest that gld mice have an increased recruitment of cells of mesenchymal origin and an abnormal pattern of differentiation and maturation of the newly formed mesenchymal tissues.     

Inhibitory effect of Torilis anthriscus on growth of microorganisms

Antibacterial and antifungal activities of aqueous, ethanol and ethyl acetate extract of Torilis anthriscus (L.) Gmel. (Apiaceae) were tested in vitro against ten species of bacteria and five of fungi. Antimicrobial properties were determined by disk diffusion and broth tube dilution method. In the minimum inhibitory concentrations (MICs), the ethanol extract showed the highest activity, followed by the ethyl acetate extract and the aqueous extract against bacterial species, while the extracts were inactive against the tested fungi species. The most active extract was chosen to examine the effects of its combinations with commercial antibiotics by checkerboard method. The obtained results showed that the interactions between ethanol extract/streptomycin and ethanol extract/chloramphenicol were additive and indifferent against the tested human-pathogenic bacteria. Synergism and antagonism were not observed.     

5' stem-loop of collagen alpha 1(I) mRNA inhibits translation in vitro but is required for triple helical collagen synthesis in vivo

The 5' stem-loop is a conserved sequence element found around the translation initiation site of three collagen mRNAs, alpha1(I), alpha2(I), and alpha1(III). We show here that the 5' stem-loop of collagen alpha1(I) mRNA is inhibitory to translation in vitro. The sequence 5' to the translation initiation codon, as a part of the 5' stem-loop, is also not efficient in initiating translation under competitive conditions. This suggests that collagen alpha1(I) mRNA may not be a good substrate for translation. Since the 5' stem-loop binds protein factors in collagen-producing cells, this binding may regulate its translation in vivo. We studied in vivo translation of collagen alpha1(I) mRNA after transfecting collagen alpha1(I) genes with and without the 5' stem-loop into Mov 13 fibroblasts. The mRNA with the alpha1(I) 5' stem-loop was translated into pepsin-resistant collagen, which was secreted into the cellular medium. This mRNA also produced more disulfide-bonded high molecular weight collagen found intracellularly. The mRNA in which the 5' stem-loop was mutated, but without affecting the coding region of the gene, was translated into pepsin-sensitive collagen and produced only trace amounts of disulfide-bonded collagen. This suggests that the 5' stem-loop is required for proper folding or stabilization of the collagen triple helix. To our knowledge this is the first example that an RNA element located in the 5'-untranslated region is involved in synthesis of a secreted multisubunit protein. We suggest that 5' stem-loop, with its cognate binding proteins, targets collagen mRNAs for coordinate translation and couples translation apparatus to the rest of the collagen biosynthetic pathway.     

A deoxyribozyme-based molecular automaton

We describe a molecular automaton, called MAYA, which encodes a version of the game of tic-tac-toe and interactively competes against a human opponent. The automaton is a Boolean network of deoxyribozymes that incorporates 23 molecular-scale logic gates and one constitutively active deoxyribozyme arrayed in nine wells (3x3) corresponding to the game board. To make a move, MAYA carries out an analysis of the input oligonucleotide keyed to a particular move by the human opponent and indicates a move by fluorescence signaling in a response well. The cycle of human player input and automaton response continues until there is a draw or a victory for the automaton. The automaton cannot be defeated because it implements a perfect strategy.     

Associations between parental and child overweight and obesity

Results of the analysis showed that parents and children overweight/obesity were significantly correlated. The sample includes 318 pairs of mothers and children, and 336 pairs of fathers and children at the age 11.3 +/- 0.4 years in Trogir, Croatia. Child overweight and obesity were defined according to body mass index (BMI) 25 and 30 equivalents (kg/m2). The prevalence of total overweight in girls was 25.6% and among boys was 20.5%. Mother's weight (p = 0.003) and BMI (p = 0.006) were greater in obese than in other groups of children. Overweight/obese children were more often found among overweight/obese mothers (p = 0.009) and fathers (p = 0.039). Correlation between overweight/obese children and their father (odds ratio 3.2, 95% CI 1.5-6.8) was stronger than between overweight/obese children and their mothers (odds ratio 2.2, 95% CI 1.2-3.9). Associations with mothers' and daughters' overweight/obesity were stronger (p = 0.017) than mothers' and sons'(p = 0.12). Correlations between children's BMI and fathers' BMI (r = 0.265, p < 0.0001) and between children's BMI and mothers' BMI (r = 0.173, p = 0.002) were significant. Children whose parents are overweight/obese look for greater attention in future preventive programme.