A Paecilomyces fumosoroseus mutant over-producing chitinase displays enhanced virulence against Bemisia tabaci

A Paecilomyces fumosoroseus strain was mutagenized by u.v. Among 200 colonies, one mutant (M84), showed a large and stable chitin hydrolysis-halo. Glucose consumption and biomass production were similar for M84 and the parental strain. Chitinase was inducible by chitin and repressed by glucose in both strains but, when they were grown on minimal medium plus colloidal chitin as sole carbon source, the parental and M84 strains yielded 198 and 690 mol N-acetylglucosamine, respectively. This results indicate that the mutant strain synthesized a chitinase with a higher activity. Bioassays against Bemisia tabaci nymph, showed that M84 incited a 2-fold higher incidence of disease compared to the parental strain.     

Plant Virus Cell-to-Cell Movement Is Not Dependent on the Transmembrane Disposition of Its Movement Protein

The cell-to-cell transport of plant viruses depends on one or more virus-encoded movement proteins (MPs). Some MPs are integral membrane proteins that interact with the membrane of the endoplasmic reticulum, but a detailed understanding of the interaction between MPs and biological membranes has been lacking. The cell-to-cell movement of the Prunus necrotic ringspot virus (PNRSV) is facilitated by a single MP of the 30K superfamily. Here, using a myriad of biochemical and biophysical approaches, we show that the PNRSV MP contains only one hydrophobic region (HR) that interacts with the membrane interface, as opposed to being a transmembrane protein. We also show that a proline residue located in the middle of the HR constrains the structural conformation of this region at the membrane interface, and its replacement precludes virus movement.Plant viruses encode movement proteins (MPs) that mediate the intra- and intercellular spread of the viral genome via plasmodesmata, membranous channels that traverse the walls of plant cells and enable intercellular transport and communication. There is a range of diversity in the number and type of viral proteins required for viral movement (21). Research on tobacco mosaic virus (TMV) has played a leading role in understanding MP activity (2). The genome of TMV encodes a single 30-kDa multidomain protein, the namesake of the 30K superfamily (7). Viral RNA is associated with the membrane of the endoplasmic reticulum (ER) and microtubules in the presence of this MP (23, 30).A large number of plant viruses have 30K MPs, which share common abilities, including binding nucleic acids, localizing and increasing the size exclusion limit of plasmodesmata, and interacting with the ER membrane. A topological model has been proposed in which the TMV MP has two putative transmembrane (TM) helices, both the N and C termini oriented toward the cytoplasm, and a short loop exposed in the ER lumen (4). There is less experimental information for other 30K MPs, but they are likely to have some membrane interaction.Direct experimental evidence of the integration of MPs into the membrane has been obtained only for small hydrophobic MPs that do not belong to the 30K superfamily. There are two TM segments in the p9 protein of carnation mottle virus (41), whereas the p6 protein of beet yellow virus (29) and the p7B protein of melon necrotic spot virus (22) have a single TM segment. In viruses with genomes that include three partially overlapping open reading frames, termed the triple-gene block (TGB), all three TGB proteins are required for movement where the two smaller proteins, TGBp2 and TGBp3, are also TM proteins (24). Furthermore, cross-linking experiments with carnation mottle virus p9 protein demonstrated that its membrane insertion occurs cotranslationally in a signal recognition particle-dependent manner and throughout the cellular membrane integration components, the translocon (33, 34).Prunus necrotic ringspot virus (PNRSV) is a tripartite, positive-strand RNA virus in the genus Ilarvirus of the family Bromoviridae. RNAs 1 and 2 encode the polymerase proteins P1 and P2, respectively. RNA 3 is translated into a single 30K-type MP. The coat protein is translated from a subgenomic RNA 4 produced during virus replication.The present study tackled the association of the PNRSV MP with biological membranes. The in vitro translation of model integral membrane protein constructs in the presence of microsomal membranes demonstrated that the hydrophobic region (HR) of the PNRSV MP did not span the membranes. Different biochemical and biophysical experiments suggested that the protein is tightly associated with, but does not traverse, the membrane, leaving both its N- and C-terminal hydrophilic regions facing the cytosol. Finally, a mutational analysis of the HR revealed that both the helicity and hydrophobicity of the region are essential for viral cell-to-cell movement.     

Crystal structures of the G139A, G139A–NO and G143H mutants of human heme oxygenase-1. A finely tuned hydrogen-bonding network controls oxygenase versus peroxidase activity

Conserved glycines, Gly139 and Gly143, in the distal helix of human heme oxygenase-1 (HO-1) provide the flexibility required for the opening and closing of the heme active site for substrate binding and product dissociation during HO-1 catalysis. Earlier mutagenesis work on human HO-1 showed that replacement of either Gly139 or Gly143 suppresses heme oxygenase activity and, in the case of the Gly139 mutants, increases peroxidase activity (Liu et al. in J. Biol. Chem. 275:34501, 2000). To further investigate the role of the conserved distal helix glycines, we have determined the crystal structures of the human HO-1 G139A mutant, the G139A mutant in a complex with NO, and the G143H mutant at 1.88, 2.18 and 2.08 Å, respectively. The results confirm that fine tuning of the previously noted active-site hydrogen-bonding network is critical in determining whether heme oxygenase or peroxidase activity is observed.     

Microsatellite Genotyping of Plasmodium vivax Isolates from Pregnant Women in Four Malaria Endemic Countries

Plasmodium vivax is the most widely distributed human parasite and the main cause of human malaria outside the African continent. However, the knowledge about the genetic variability of P. vivax is limited when compared to the information available for P. falciparum. We present the results of a study aimed at characterizing the genetic structure of P. vivax populations obtained from pregnant women from different malaria endemic settings. Between June 2008 and October 2011 nearly 2000 pregnant women were recruited during routine antenatal care at each site and followed up until delivery. A capillary blood sample from the study participants was collected for genotyping at different time points. Seven P. vivax microsatellite markers were used for genotypic characterization on a total of 229 P. vivax isolates obtained from Brazil, Colombia, India and Papua New Guinea. In each population, the number of alleles per locus, the expected heterozygosity and the levels of multilocus linkage disequilibrium were assessed. The extent of genetic differentiation among populations was also estimated. Six microsatellite loci on 137 P. falciparum isolates from three countries were screened for comparison. The mean value of expected heterozygosity per country ranged from 0.839 to 0.874 for P. vivax and from 0.578 to 0.758 for P. falciparum. P. vivax populations were more diverse than those of P. falciparum. In some of the studied countries, the diversity of P. vivax population was very high compared to the respective level of endemicity. The level of inter-population differentiation was moderate to high in all P. vivax and P. falciparum populations studied.     

Local adaptation versus historical isolation as sources of melanin‐based coloration in the white‐throated thrush Turdus assimilis

Local adaptation seems to be one of the causes of variation in melanin‐based colors in bird plumages, related mainly to the heterogeneity of the environmental conditions along the distribution of a species. Based on comparisons of genetic (mtDNA sequences), ecological (niche models), and quantitative colorimetric data, we explored variation in plumage coloration of the white‐throated thrush Turdus assimilis, a Mesoamerican species whose dorsal color varies from brown (northern and central Mexico) to dark gray (southern Mexico and Central America). Our results suggest the existence of two major patterns of coloration in this bird, which are congruent with the genetic structure, and comparisons of ecological niche models showed that population's niches were more similar than expected by chance, suggesting that color variation in plumage of T. assimilis is not consequence of local adaptation to different environmental conditions. Our results also showed that a greater geographic distance between populations is correlated with greater colorimetric differences, suggesting that color variation in T. assimilis may be consequence of historical isolation.     

Transport of thiamine in human intestine: mechanism and regulation in intestinal epithelial cell model Caco-2

The present studyexamined the intestinal uptake of thiamine (vitaminB₁) using the human-derivedintestinal epithelial cells Caco-2 as an in vitro model system.Thiamine uptake was found to be 1)temperature and energy dependent and occurred with minimal metabolicalteration; 2) pH sensitive;3)Na⁺ independent;4) saturable as a function ofconcentration with an apparent Michaelis-Menten constant of 3.18 ± 0.56 µM and maximal velocity of 13.37 ± 0.94 pmol · mgprotein¹ · 3 min¹;5) inhibited by the thiaminestructural analogs amprolium and oxythiamine, but not by unrelatedorganic cations tetraethylammonium, N-methylnicotinamide, and choline; and6) inhibited in a competitive mannerby amiloride with an inhibition constant of 0.2 mM. The role ofspecific protein kinase-mediated pathways in the regulation of thiamineuptake by Caco-2 cells was also examined using specific modulators ofthese pathways. The results showed possible involvement of aCa²⁺/calmodulin (CaM)-mediatedpathway in the regulation of thiamine uptake. No role for proteinkinase C- and protein tyrosine kinase-mediated pathways in theregulation of thiamine uptake was evident. These results demonstratethe involvement of a carrier-mediated system for thiamine uptake byCaco-2 intestinal epithelial cells. This system isNa⁺ independent and is differentfrom the transport systems of organic cations. Furthermore, aCaM-mediated pathway appears to play a role in regulating thiamineuptake in these cells.

     

Human Peroxin PEX3 Is Co‐translationally Integrated into the ER and Exits the ER in Budding Vesicles

The long‐standing paradigm that all peroxisomal proteins are imported post‐translationally into pre‐existing peroxisomes has been challenged by the detection of peroxisomal membrane proteins (PMPs) inside the endoplasmic reticulum (ER). In mammals, the mechanisms of ER entry and exit of PMPs are completely unknown. We show that the human PMP PEX3 inserts co‐translationally into the mammalian ER via the Sec61 translocon. Photocrosslinking and fluorescence spectroscopy studies demonstrate that the N‐terminal transmembrane segment (TMS) of ribosome‐bound PEX3 is recognized by the signal recognition particle (SRP). Binding to SRP is a prerequisite for targeting of the PEX3‐containing ribosome?nascent chain complex (RNC) to the translocon, where an ordered multistep pathway integrates the nascent chain into the membrane adjacent to translocon proteins Sec61α and TRAM. This insertion of PEX3 into the ER is physiologically relevant because PEX3 then exits the ER via budding vesicles in an ATP‐dependent process. This study identifies early steps in human peroxisomal biogenesis by demonstrating sequential stages of PMP passage through the mammalian ER.