Genetic variability of introduced and local Spanish peach cultivars determined by SSR markers

A set of 94 peach cultivars including Spanish native peach and foreign commercial cultivars were analyzed using 15 SSR markers, selected for their high level of polymorphism. The number of alleles obtained varied from two to 11 with an average of 6.73 giving 185 different genotypes. All the cultivars showed a unique genetic profile, each one using different genotypic combination of all loci. BPPCT001 was the most informative locus showing also the highest discrimination power. Only six loci allowed the unambiguous separation of all the Spanish native cultivars studied, and the genotypic combination of only eight loci permitted the total differentiation of the 94 peach cultivars analyzed. The six selected loci (BPPCT001, BPPCT006, BPPCT008, PS9f8, UDP98-022, and UDP98-412) seem to be very useful for future Spanish peach identification works, and they will help to establish a molecular data base for native peach cultivars. UPGMA analysis was performed from the genetic distance matrix, and allowed the arrangement of all genotypes according to their genetic diversity. The genetic diversity among cultivars, observed in this work, led to their separation according to their regional origin, their morphological characteristics, and especially according to their fruit traits. Analysis of molecular variance was performed for seven populations from different regions of Spain and USA to examine the distribution of genetic variation of the studied accessions, showing that the major variation occurred within populations in each geographic site. The results reveal the existence of two diversity regions in Spain for peach germplasm.     

Serine/threonine protein kinase PrkA of the human pathogen Listeria monocytogenes: biochemical characterization and identification of interacting partners through proteomic approaches

Listeria monocytogenes is the causative agent of listeriosis, a very serious food-borne human disease. The analysis of the proteins coded by the L. monocytogenes genome reveals the presence of two eukaryotic-type Ser/Thr-kinases (lmo1820 and lmo0618) and a Ser/Thr-phosphatase (lmo1821). Protein phosphorylation regulates enzyme activities and protein interactions participating in physiological and pathophysiological processes in bacterial diseases. However in the case of L. monocytogenes there is scarce information about biochemical properties of these enzymes, as well as the physiological processes that they modulate. In the present work the catalytic domain of the protein coded by lmo1820 was produced as a functional His(6)-tagged Ser/Thr-kinase, and was denominated PrkA. PrkA was able to autophosphorylate specific Thr residues within its activation loop sequence. A similar autophosphorylation pattern was previously reported for Ser/Thr-kinases from related prokaryotes, whose role in kinase activity and substrate recruitment was demonstrated. We studied the kinase interactome using affinity chromatography and proteomic approaches. We identified 62 proteins that interact, either directly or indirectly, with the catalytic domain of PrkA, including proteins that participate in carbohydrates metabolism, cell wall metabolism and protein synthesis. Our results suggest that PrkA could be involved in the regulation of a variety of fundamental biological processes.     

No evidence of extensive non-CpG methylation in mtDNA

While most research suggests mitochondrial DNA (mtDNA) harbors low or no methylation, a few studies claim to report evidence of high-level methylation in the mtDNA. The reasons behind these contradictory results are likely to be methodological but remain largely unexplored. Here, we critically reanalyzed a recent study by Patil et al. (2019) reporting extensive methylation in human mtDNA in a non-CpG context. Our analyses refute the original findings and show that these do not reflect the biology of the tested samples, but rather stem from a combination of methodological and technical pitfalls. The authors employ an oversimplified model that defines as methylated all reference positions with methylation proportions above an arbitrary cutoff of 9%. This substantially exacerbates the overestimation of methylated cytosines due to the selective degradation of unmethylated cytosine-rich regions. Additional limitations are the small sample sizes and lack of sample-specific controls for bisulfite conversion efficiency. In conclusion, using the same dataset employed in the original study by Patil et al., we find no evidence supporting the existence of extensive non-CpG methylation in the human mtDNA.     

Using tunable diode laser spectroscopy to measure carbon isotope discrimination and mesophyll conductance to CO₂ diffusion dynamically at different CO₂ concentrations

In C₃ leaves, the mesophyll conductance to CO₂ diffusion, g_m, determines the drawdown in CO₂ concentration from intercellular airspace to the chloroplast stroma. Both g_m and stomatal conductance limit photosynthetic rate and vary in response to the environment. We investigated the response of g_m to changes in CO₂ in two Arabidopsis genotypes (including a mutant with open stomata, ost1), tobacco and wheat. We combined measurements of gas exchange with carbon isotope discrimination using tunable diode laser absorption spectroscopy with a CO₂ calibration system specially designed for a range of CO₂ and O₂ concentrations. CO₂ was initially increased from 200 to 1000 ppm and then decreased stepwise to 200 ppm and increased stepwise back to 1000 ppm, or the sequence was reversed. In 2% O₂ a step increase from 200 to 1000 ppm significantly decreased g_m by 26–40% in all three species, whereas following a step decrease from 1000 to 200 ppm, the 26–38% increase in g_m was not statistically significant. The response of g_m to CO₂ was less in 21% O₂. Comparing wild type against the ost1 revealed that mesophyll and stomatal conductance varied independently in response to CO₂. We discuss the effects of isotope fractionation factors on estimating g_m.     

Metabolon disruption: a mechanism that regulates bicarbonate transport

Carbonic anhydrases (CA) catalyze the reversible conversion of CO2 to HCO3-. Some bicarbonate transporters bind CA, forming a complex called a transport metabolon, to maximize the coupled catalytic/transport flux. SLC26A6, a plasma membrane Cl-/HCO3- exchanger with a suggested role in pancreatic HCO3- secretion, was found to bind the cytoplasmic enzyme CAII. Mutation of the identified CAII binding (CAB) site greatly reduced SLC26A6 activity, demonstrating the importance of the interaction. Regulation of SLC26A6 bicarbonate transport by protein kinase C (PKC) was investigated. Angiotensin II (AngII), which activates PKC, decreased Cl-/HCO3- exchange in cells coexpressing SLC26A6 and AT1a-AngII receptor. Activation of PKC reduced SLC26A6/CAII association in immunoprecipitates. Similarly, PKC activation displaced CAII from the plasma membrane, as monitored by immunofluorescence. Finally, mutation of a PKC site adjacent to the SLC26A6 CAB site rendered the transporter unresponsive to PKC. PKC therefore reduces CAII/SLC26A6 interaction, reducing bicarbonate transport rate. Taken together, our data support a mechanism for acute regulation of membrane transport: metabolon disruption.     

A truncated isoform of pyroglutamyl aminopeptidase II produced by exon extension has dominant-negative activity

Thyrotropin-releasing hormone is inactivated in the extracellular space by a membrane-bound peptidase, pyroglutamyl aminopeptidase II (PPII), a member of the M1 family of zinc metallopeptidases. The functional significance of multiple PPII RNA species expression is unknown. We detected, in rat tissues, a RNA species derived from an alternative processing at the exon 14-intron 14 boundary. The alternatively processed RNA encoded a shorter version of PPII (PPII*), lacking part of the C-terminal domain. PPII* was expressed in COS-7 (or C6 glioma) cells but it did not exhibit any PPII activity. Co-transfection of PPII and increasing amounts of PPII* expression vectors resulted in a dose-dependent reduction in PPII activity and the formation of covalent PPII-PPII* heterodimers. PPII* is therefore a powerful dominant-negative isoform of PPII, and heterodimerization may be its mechanism of action. Natural expression of shortened versions of M1 aminopeptidases may constitute a new mode of regulation of their activity.     

Monitoring of resistance to the pyrethroid cypermethrin in Brazilian Aedes aegypti (Diptera: Culicidae) populations collected between 2001 and 2003

Resistance to cypermethrin of different Aedes aegypti Brazilian populations, collected at two successive periods (2001 and 2002/2003), was monitored using the insecticide-coated bottles bioassay. Slight modifications were included in the method to discriminate between mortality and the knock down effect. Although this pyrethroid was recently started to be used in the country to control the dengue vector, a decrease in susceptibility was noted between both periods analyzed, particularly in the city of Rio de Janeiro. The results indicate that resistance is due at least in part to a target site alteration.     

The genus Cyphophthalmus (Arachnida, Opiliones, Cyphophthalmi) in Europe: a phylogenetic approach to Balkan Peninsula biogeography

In this study, we present phylogenetic data to characterize the relationships among sironids centered in the Balkan region, and use these results to discuss biogeographical aspects of sironid evolution. Analysis of ca. 4.5 kb of sequence data from three nuclear and two mitochondrial genes reveals monophyly of a Balkan clade for which we resurrect the name Cyphophthalmus, considered a junior synonym of Siro for over a century. This clade diversified into several groups, and at least three of them--the duricorius group, the serbicus group, and the minutus group--are well corroborated by the data as monophyletic lineages. The members of the different groups, mostly living in troglobitic environments, have diversified in overlapping geographic regions, with evidence of an eastern origin for the group. Our data also suggest that mitochondrial and nuclear genes are all contributing towards the final resolution of the combined analysis of the data.