Genetic diversity of Couratari multiflora and Couratari guianensis (Lecythidaceae): consequences of two types of rarity in central Amazonia

We quantified the within-population genetic variation of Couratari multiflora and C. guianensis, two tree species found in terra firme forests of central Amazonia. Both species have some ecological features in common, but they differ in population abundance across their geographic ranges. While C. multiflora has been found only in low-density populations in all sites studied to date, C. guianensis is relatively common in some sites and very scarce in others. In a 400-ha plot, we found 41 and 29 adults of C. multiflora and C. guianensis, respectively. Twenty-two saplings of C. guianensis and 103 seedlings of C. multiflora were also examined. The mean expected heterozygosities (H_em) of seedlings and adults of C. multiflora were 0.431 and 0.436, and the mean fixation indices (F_m), 0.114 and 0.176, respectively. For C. guianensis, saplings and adults presented H_em equal to 0.425 and 0.429, and the F_m were 0.393 and 0.527, respectively. These low-density populations of two congeneric species did not differ in terms of genetic diversity, but rather they differed in terms of mean observed heterozygosity (H_om), and therefore F_m. The species with variable population density had lower H_om and greater F_m relative to the species that is always found in low-density.     

Discovery of novel cyclic peptide inhibitors of dengue virus NS2B-NS3 protease with antiviral activity

NS2B-NS3 protease is an essential enzyme for the replication of dengue virus (DENV), which continues to be a serious threat to worldwide public health. We designed and synthesized a series of cyclic peptides mimicking the substrates of this enzyme, and assayed their activity against the DENV-2 NS2B-NS3 protease. The introduction of aromatic residues at the appropriate positions and conformational restriction generated the most promising cyclic peptide with an IC₅₀ of 0.95 μM against NS2B-NS3 protease. Cyclic peptides with proper positioning of additional arginines and aromatic residues exhibited antiviral activity against DENV. Furthermore, replacing the C-terminal amide bond of the polybasic amino acid sequence with an amino methylene moiety stabilized the cyclic peptides against hydrolysis by NS2B-NS3 protease, while maintaining their enzyme inhibitory activity and antiviral activity.     

Niemann-Pick C1 protein transports copper to the secretory compartment from late endosomes where ATP7B resides

Wilson disease is a genetic disorder characterized by the accumulation of copper in the body by defective biliary copper excretion. Wilson disease gene product (ATP7B) functions in copper incorporation to ceruloplasmin (Cp) and biliary copper excretion. However, copper metabolism in hepatocytes has been still unclear. Niemann-Pick disease type C (NPC) is a lipid storage disorder and the most commonly mutated gene is NPC1 and its gene product NPC1 is a late endosome protein and regulates intracellular vesicle traffic. In the present study, we induced NPC phenotype and examined the localization of ATP7B and secretion of holo-Cp, a copper-binding mature form of Cp. The vesicle traffic was modulated using U18666A, which induces NPC phenotype, and knock down of NPC1 by RNA interference. ATP7B colocalized with the late endosome markers, but not with the trans-Golgi network markers. U18666A and NPC1 knock down decreased holo-Cp secretion to culture medium, but did not affect the secretion of other secretory proteins. Copper accumulated in the cells after the treatment with U18666A. These findings suggest that ATP7B localizes in the late endosomes and that copper in the late endosomes is transported to the secretory compartment via NPC1-dependent pathway and incorporated into apo-Cp to form holo-Cp.     

High levels of genetic differentiation and selfing in the Brazilian cerrado fruit tree Dipteryx alata Vog. (Fabaceae)

Dipteryx alata is a native fruit tree species of the cerrado (Brazilian savanna) that has great economic potential because of its multiple uses. Knowledge of how the genetic variability of this species is organized within and among populations would be useful for genetic conservation and breeding programs. We used nine simple sequence repeat (SSR) primers developed for Dipteryx odorata to evaluate the genetic structure of three populations of D. alata located in central Brazil based on a leaf sample analysis from 101 adults. The outcrossing rate was evaluated using 300 open-pollinated offspring from 25 seed-trees. Pollen dispersal was measured by parentage analysis. We used spatial genetic structure (SGS) to test the minimal distance for harvesting seeds in conservation and breeding programs. Our data indicate that the populations studied had a high degree of genetic diversity and population structure, as suggested by the high level of divergence among populations . The estimated outcrossing rate suggested a mixed mating system, and the intrapopulation fixation index was influenced by SGS. We conclude that seed harvesting for genetic conservation and breeding programs requires a minimum distance between trees of 196 m to avoid collecting seeds from related seed-trees.     

Novel Strain of Spiroplasma Found in Flower Bugs of the Genus Orius (Hemiptera: Anthocoridae): Transovarial Transmission,Coexistence with Wolbachia and Varied Population Density

Spiroplasma, a group of small, wall-less, helical, and motile bacteria belonging to the Mollicutes, contains species with diverse life histories. To date, all the Spiroplasma strains that are known to be transmitted vertically in arthropod lineages belong to either the Spiroplasma ixodetis group or the Spiroplasma poulsonii group. Here, we found that a unique strain of Spiroplasma vertically transmitted in predatory flower bugs of the genus Orius belongs to the Spiroplasma insolitum group, which is a group of bacteria phylogenetically closely related to S. insolitum derived from the tickseed sunflower, Bidens sp. (Asterales: Asteraceae). The infection frequencies in natural populations were16.0 % in Orius sauteri (n?=?75), 40.5 % in Orius nagaii (n?=?37), and 8.0 % in Orius minutus (n?=?87). Orius strigicollis was not infected with Spiroplasma (n?=?147). In the early stage of oogenesis (i.e., within the germarium), a large number of bacteria with the typical morphology of Spiroplasma existed, keeping a distance from Wolbachia bacteria. The Spiroplasma population seemed to increase during host development but Wolbachia population did not.     

Detection of elevated antibody against calreticulin by ELISA in aged cynomolgus monkey plasma

Calreticulin (Crt) is a molecular chaperone ubiquitously present in the endoplasmic reticulum. In non-human primates, age-related occurrence of anti-Crt antibody has not been reported. We developed an ELISA assay for an anti-Crt antibody and determined the age-related increase in the levels of anti-Crt antibody in three groups of cynomolgus monkeys: juvenile (1.5 yr), young adults (5-10 yr) and aged adults (20-34 yr). Mean ± SD auto-antibody levels at 450 nm in juvenile, young adults and aged groups were 0.23 ± 0.18, 0.30 ± 0.28, and 0.55 ± 0.33, respectively. Statistically significant differences were noted in the autoantibody levels to Crt among the aged group and juvenile or young adults. This is the first report to demonstrate the expression of anti-Crt autoantibody in aged monkeys and indicates that cynomologous monkeys may serve as an appropriate nonhuman primate model for studies of age-related alteration of immune function in elderly humans. Though preliminary, this finding merits further investigation to determine the relationship between immunosenescence and expression of antibodies to Crt.     

Impairment of ubiquitin-proteasome system by E334K cMyBPC modifies channel proteins, leading to electrophysiological dysfunction

Cardiac arrhythmogenesis is regulated by channel proteins whose protein levels are in turn regulated by the ubiquitin-proteasome system (UPS). We have previously reported on UPS impairment induced by E334K cardiac myosin-binding protein C (cMyBPC), which causes hypertrophic cardiomyopathy (HCM) accompanied by arrhythmia. We hypothesized that UPS impairment induced by E334K cMyBPC causes accumulation of cardiac channel proteins, leading to electrophysiological dysfunction. Wild-type or E334K cMyBPC was overexpressed in HL-1 cells and primary cultured neonatal rat cardiac myocytes. The expression of E334K cMyBPC suppressed cellular proteasome activities. The protein levels of K_v1.5, Na_v1.5, Hcn4, Ca_v3.2, Ca_v1.2, Serca, RyR2, and Ncx1 were significantly higher in cells expressing E334K cMyBPC than in wild type. They further increased in cells pretreated with MG132 and had longer protein decays. The channel proteins retained the correct localization. Cells expressing E334K cMyBPC exhibited higher Ca²⁺ transients and longer action potential durations (APDs), accompanied by afterdepolarizations and higher apoptosis. Those augments of APD and Ca²⁺ transients were recapitulated by a simulation model. Although a Ca²⁺ antagonist, azelnidipine, neither protected E334K cMyBPC from degradation nor affected E334K cMyBPC incorporation into the sarcomere, it normalized the APD and Ca²⁺ transients and partially reversed the levels of those proteins regulating apoptosis, thereby attenuating apoptosis. In conclusion, UPS impairment caused by E334K cMyBPC may modify the levels of channel proteins, leading to electrophysiological dysfunction. Therefore, UPS impairment due to a mutant cMyBPC may partly contribute to the observed clinical arrhythmias in HCM patients.