Mechanism of nucleic acid unwinding by SARS-CoV helicase

The non-structural protein 13 (nsp13) of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is a helicase that separates double-stranded RNA (dsRNA) or DNA (dsDNA) with a 5' → 3' polarity, using the energy of nucleotide hydrolysis. We determined the minimal mechanism of helicase function by nsp13. We showed a clear unwinding lag with increasing length of the double-stranded region of the nucleic acid, suggesting the presence of intermediates in the unwinding process. To elucidate the nature of the intermediates we carried out transient kinetic analysis of the nsp13 helicase activity. We demonstrated that the enzyme unwinds nucleic acid in discrete steps of 9.3 base-pairs (bp) each, with a catalytic rate of 30 steps per second. Therefore the net unwinding rate is ~280 base-pairs per second. We also showed that nsp12, the SARS-CoV RNA-dependent RNA polymerase (RdRp), enhances (2-fold) the catalytic efficiency of nsp13 by increasing the step size of nucleic acid (RNA/RNA or DNA/DNA) unwinding. This effect is specific for SARS-CoV nsp12, as no change in nsp13 activity was observed when foot-and-mouth-disease virus RdRp was used in place of nsp12. Our data provide experimental evidence that nsp13 and nsp12 can function in a concerted manner to improve the efficiency of viral replication and enhance our understanding of nsp13 function during SARS-CoV RNA synthesis.     

Chemodiversity of Nonacosan‐10‐ol and n‐Alkanes in the Needle Wax of Pinus heldreichii

This is the first report of individual variability and population diversity of the contents of nonacosan‐10‐ol and n‐alkanes in the needle cuticular waxes of Bosnian pines originated from Montenegro, regarded as Pinus heldreichii var. leucodermis, and from Serbia, regarded as P. heldreichii var. pan?i?i. The amount of nonacosan‐10‐ol varied individually from 27.4 to 73.2% (55.5% in average), but differences between the four investigated populations were not statistically confirmed. The size of the n‐alkanes ranged from C₁₈ to C₃₃. The most abundant n‐alkanes were C₂₃, C₂₇, and C₂₅ (12.2, 11.2, and 10.8% in average, resp.). The carbon preference index (CPI) of the n‐alkanes ranged from 0.8 to 3.1 (1.6 in average), while the average chain length (ACL) ranged from 20.9 to 26.5 (24.4 in average). Long‐chain and mid‐chain n‐alkanes prevailed (49.6 and 37.9% in average, resp.). It was also found that the populations of P. heldreichii var. leucodermis had predominantly a narrower range of n‐alkanes (C₁₈? C₃₁) than the trees of the variety pan?i?i (C₁₈? C₃₃). Differences between the varieties were also significant for most of the other characteristics of the n‐alkane pattern (e.g., most abundant n‐alkanes, CPI, ACL, and relative proportion of short‐, mid‐, and long‐chain n‐alkanes). The principle component and cluster analyses of eleven n‐alkanes confirmed the significant diversity of these two varieties.     

Composition of n‐Alkanes in Natural Populations of Pinus nigra from Serbia – Chemotaxonomic Implications

This is the first report on the composition and variability of the needle‐wax n‐alkanes in natural populations of Pinus nigra in Serbia. Samples of 195 trees from seven populations belonging to several infraspecific taxa (ssp. nigra, var. gocensis, ssp. pallasiana, and var. banatica) were analyzed. In general, the size of the n‐alkanes ranged from C₁₆ to C₃₃, with the exception of ssp. nigra, for which it ranged from C₁₈ to C₃₃. The most abundant were C₂₃‐, C₂₅‐, C₂₇‐, and C₂₉‐alkanes. The needle waxes of Populations I–III and V were characterized by a higher content of C₂₃‐, C₂₅‐, and C₂₇‐alkanes and a lower content of C₂₄‐, C₂₆‐, C₂₈‐, and C₃₀‐alkanes, compared to the other populations, and the trees of these populations could be assigned to ssp. nigra. The samples of Population VI were characterized by higher amounts of C₂₂‐, C₂₄‐, C₃₀‐, and C₃₂‐alkanes and lower amounts of C₂₅‐ and C₂₇‐alkanes, and the trees could be considered as ssp. pallasiana. The samples of Population VII, consisting of trees belonging to var. banatica, were richer in C₂₉‐, C₃₁‐, and C₃₃‐alkanes. The wax compositions of Populations IV and V, both composed of trees previously determined as P. nigra var. gocensis, showed a tendency of splitting. Indeed, the alkane composition of Population IV was closer to that of ssp. pallasiana pines, while that of Population V was more similar to that of ssp. nigra pines. From the results presented here, it is obvious that in the central part of the Balkan Peninsula, significant diversification and differentiation of the populations of black pine exists, and these populations could be defined as different intraspecific taxa. Our results also indicate the validity of n‐alkanes as chemotaxonomic characters within this aggregate.     

Optimization of extraction of high-ester pectin from passion fruit peel (Passiflora edulis flavicarpa) with citric acid by using response surface methodology

A central composite design was employed to optimize the extraction of pectin with citric acid. The independent variables were citric acid concentration (0.086–2.91% w/v) and extraction time (17–102 min). The combined effect of these variables on the degree of esterification was investigated. Results have shown that the generated regression models adequately explained the data variation and significantly represented the actual relationship between the independent variables and the responses. Besides that, the citric acid concentration was the most important factor to affect the degree of esterification, as it exerted a significant influence on the dependent variable. Lower citric acid concentration increased the pectin degree of esterification. The surface response showed the relationships between the independent variables, and thus responses were generated. Through this surface, the satisfactory condition of 0.086% w/v citric acid for 60 min was established for extraction of high-ester yellow passion fruit pectin.     

A flow fluorimetric analysis of the cell cycle during growth and differentiation inDictyostelium discoideum

Summary We report a flow fluorimetric analysis of the DNA content of cells and nuclei from vegetative populations and various developmental stages of the cellular slime mouldDictyostelium discoideum using the dyes Hoechst 33258 and mithramycin. Nuclei from all of these populations showed an identical single DNA-content peak, indicating that most vegetative cells and most cells in all developmental stages are in one phase of the cell cycle. Our own data and findings in the literature indicate that this phase is G₂. On the other hand, we also found that various stages, subpopulations of cells at early stages and the different differentiated cell types in the slug stage differ in DNA content per cell. Any particular population typically has one major peak of DNA content, with a modal value that is characteristic for the cell type and for the developmental stage. These differences presumably reflect differences in mitochondrial DNA content per cell.     

Fungi associated with black pepper plants in Quang Tri province (Vietnam), and interaction between Meloidogyne incognita and Fusarium solani

During a survey of nurseries and plantations of black pepper plants in Quang Tri province in Vietnam during the rainy season of 2007, nine fungal taxa were isolated from the roots of the black pepper plants. Fusarium solani was found in about one out of four black pepper root samples examined but not in the nurseries and also not from black pepper plants younger than five years growing in plantations. Since in these nurseries about one out of two black pepper plants examined had yellow leaves, this observation suggests that another pathogen must be the initial cause of the yellowing of the leaves. A likely pathogenic candidate is M. incognita which was extracted from every single black pepper plant examined in the nurseries. During the same survey, we also observed that F. solani was not isolated from the roots of black pepper plants that did not had yellow leaves and that the percentage of black pepper plants with yellow leaves increased with increased frequency of occurrence of F. solani. This observation indicates that F. solani plays a role in the yellowing of the leaves of black pepper plants in a later stage of the development of the plants. The results of a greenhouse experiment showed the negative effects inoculation with M. incognita alone or in combination with F. solani may have on the percentage of black pepper plants with yellow leaves and on plant growth. No effect of inoculation with F. solani before, at the same time, or two weeks after inoculation with M. incognita on root galling and nematode reproduction was observed.     

Spatial and environmental correlates of intraspecific morphological variation in three species of passerine birds from the Purus–Madeira interfluvium,Central Amazonia

Biogeographic studies in Amazonia typically describe biodiversity across interfluvia, rarely within them, where geographic variability in morphological traits might be observed. We tested for intraspecific phenotypic variation in three bird species within the Purus–Madeira interfluvium (Central Amazon) and whether phenotypes were correlated with environmental heterogeneity or geographic distance among sites. We compared coloration indexes derived from reflectance spectra and morphometrics of up to five adult individuals of each sex among 11 sites within the interfluvium and contrasted them with proxies for geographic distance and environmental variation (tree basal area and bird community). Environmental heterogeneity was minimally spatially autocorrelated, and there were no obvious geographical barriers to dispersal in the study region. The null hypothesis was that we would see either no phenotypic variation or random variation that was not explained by the tested variables. Half of the cases analyzed showed intraspecific morphological variation. Coloration varied more frequently than morphometrics, and color was better explained by environmental heterogeneity, particularly in males, whereas brightness also varied with geographic distance. Geographic distance explained the only case of variation in morphometrics. Our results indicate that coloration, particularly plumage brightness, is more labile than morphometric traits and that plumage color might be under stronger effects of local adaptation than brightness, which also seems to be under effects of neutral drift and gene flow among populations. Higher frequencies of association between male coloration and the environment suggest a role of non-arbitrary mechanisms of sexual selection on the expression of male phenotypes, whereas arbitrary intersexual selection might explain the randomly distributed variation that is not explained by environmental heterogeneity or geographic distance. We revealed intraspecific phenotypic variation in a spatial extent usually not considered in biogeographic studies in the Amazon and demonstrate that both local adaptation and neutral drift are important to explain intraspecific trait diversification at this geographical scale.     

Orientation and spectral properties of two stilbazolium merocyanine dyes in stretched and unstretched polyvinyl alcohol films

Spectral properties (anisotropy coefficients calculated for absorption, emission and fluorescence decay time) of two stilbazolium merocyanine dyes have been determined to evaluate the applicability of these dyes as sensitizers in photodynamic therapy. The dyes were embedded in an anisotropic polymer matrix. Analysis of the emission decay components measured in polarized light provides information on the interactions of the dye molecules with the polymer matrix being a model of an anisotropic biological system. Different values of the emission anisotropies obtained from various polarized components of fluorescence decays have shown that the orientations of the dye molecules influence their interactions with the polymer. This means that differently oriented dye molecules located in biological systems should exhibit different interactions with membranes. The chain length and type of side groups attached as well as the salt form of the dye molecule were shown to influence the dye-polymer interactions and should be taken into account before the application of merocyanine dyes in medicine. These dyes seem to be promising optical sensors with spectral properties, including the calculated anisotropy coefficients, sensitive to the molecular environment, useful to study orientation and interaction with neighbouring molecules in biological membranes.