A new role for PGRP-S (Tag7) in immune defense: lymphocyte migration is induced by a chemoattractant complex of Tag7 with Mts1

PGRP-S (Tag7) is an innate immunity protein involved in the antimicrobial defense systems, both in insects and in mammals. We have previously shown that Tag7 specifically interacts with several proteins, including Hsp70 and the calcium binding protein S100A4 (Mts1), providing a number of novel cellular functions. Here we show that Tag7–Mts1 complex causes chemotactic migration of lymphocytes, with NK cells being a preferred target. Cells of either innate immunity (neutrophils and monocytes) or acquired immunity (CD4⁺ and CD8⁺ lymphocytes) can produce this complex, which confirms the close connection between components of the 2 branches of immune response.     

Regulation of human Cdc25A stability by Serine 75 phosphorylation is not sufficient to activate a S phase checkpoint

Degradation of Cdc25A phosphatase is an ubiquitous feature of stress. There are some discrepancies in the reported roles for different phosphorylation sites in the regulation of Cdc25A stability. Using a panel of doxycycline-inducible phosphorylation mutants we show that the stability of human Cdc25A protein is dependent upon phosphorylation at S75. In non-stressed conditions and in non-mitotic cells, Cdc25A is unstable and its stability is regulated in a Chk1-dependent manner. During mitosis, Cdc25A becomes stable and does not undergo degradation after DNA damage. We further show that Chk1 kinase regulates Cdc25A stability after UV irradiation. Similar to Chk1 kinase, p38 MAPK controls Cdc25A protein level after osmotic stress. Using phospho-specific antibodies, we find that both kinases can phosphorylate S75 and S123 in vitro. Inactivation of either Chk1 after UV-irradiation or p38 MAPK after osmotic stress prevents activation of a S phase checkpoint and S75 and S123 phosphorylation. However, introduction of stable Cdc25A (S75A or S75/123A) proteins is not sufficient to overcome this checkpoint. We propose that regulation of human Cdc25A stability by its phosphorylation at S75 may contribute to S phase checkpoint activation only in cooperation with other regulatory mechanisms.     

Identification of signature and primers specific to genus <Emphasis Type="Italic">Pseudomonas</Emphasis> using mismatched patterns of 16S rDNA sequences

Background

Pseudomonas, a soil bacterium, has been observed as a dominant genus that survives in different habitats with wide hostile conditions. We had a basic assumption that the species level variation in 16S rDNA sequences of a bacterial genus is mainly due to substitutions rather than insertion or deletion of bases. Keeping this in view, the aim was to identify a region of 16S rDNA sequence and within that focus on substitution prone stretches indicating species level variation and to derive patterns from these stretches that are specific to the genus.

Results

Repeating elements that are highly conserved across different species of Pseudomonas were considered as guiding markers to locate a region within the 16S gene. Four repeating patterns showing more than 80% consistency across fifty different species of Pseudomonas were identified. The sub-sequences between the repeating patterns yielded a continuous region of 495 bases. The sub-sequences after alignment and using Shanon's entropy measure yielded a consensus pattern. A stretch of 24 base positions in this region, showing maximum variations across the sampled sequences was focused for possible genus specific patterns. Nine patterns in this stretch showed nearly 70% specificity to the target genus. These patterns were further used to obtain a signature that is highly specific to Pseudomonas. The signature region was used to design PCR primers, which yielded a PCR product of 150 bp whose specificity was validated through a sample experiment.

Conclusions

The developed approach was successfully applied to genus Pseudomonas. It could be tried in other bacterial genera to obtain respective signature patterns and thereby PCR primers, for their rapid tracking in the environmental samples.

     

Meiotic analysis of four cross-pollinated generations in a synthetic autotetraploid population of husk tomato (<i>Physalis ixocarpa</i>)

The cultivated husk tomato (Physalis ixocarpa) (2n = 2x = 24) is native from Mexico and Central America and shows a wide genetic variation. Presently, it is the fourth horticultural crop in cultivation surface in Mexico. The working team of this research previously developed an autotetraploid population by using colchicine. The objectives of the present work were to analyze the ploidy level and meiotic behavior of the subsequent generations (C3, C4, C5, C6) from the original (C2) composed only by plants with the duplicated genome from the Rendidora cultivar, and to determine pollen viability. As a diploid control the cultivar Rendidora of P. ixocarpa was used. Ploidy level was determined by flow citometry and meiotic analysis. For the meiotic study, the microsporocytes were prepared by the squash method, stained with carmin and analyzed in diakinesis. Pollen viability was evaluated through 0.01% Buffalo Black staining. The tetraploid condition prevailed through four cross-pollinating generations, maintaining a constant chromosome number 2n = 4x = 48. In diakinesis, the chromosomes of the diploid cultivar were associated into bivalents, whereas in tetraploid plants the chromosomes associated into univalents, bivalents and trivalents. Highly significant differences in bivalent pairing were detected between autotetraploid plants and between generations. Pollen viability did not show significant differences between generations and allowed reproduction. These results indicate that it is possible to develop an autotetraploid cultivar, because the polyploid state is naturally maintained and the plants are fertile. Furthermore, given the differences in bivalent pairing between plants and generations, a response to selection toward meiotic stability is expected.     

Molecular and genetic analysis of NS gene from high pathogenic strains of the avian influenza (H5N1) virus isolated in Kazakhstan

The high pathogenic strains of the avian influenza H5N1 virus isolated in Kazakhstan have NS of different genotypes. The influenza virus strains isolated in 2005 is of NS1E Qinghai genotype. A/swan/Mangystau/3/2006 strain is of NS2A genotype that is typical for Gs/Gd-like strains. The results of the analysis allow assuming that A/swan/Mangystau/3/2006 strain has been brought onto the territory of Kazakhstan from the European part of the continent along the Black Sea-Mediterranean flyway.     

Two RecA protein types that mediate different modes of hyperrecombination

RecAX53 is a chimeric variant of the Escherichia coli RecA protein (RecAEc) that contains a part of the central domain of Pseudomonas aeruginosa RecA (RecAPa), encompassing a region that differs from RecAEc at 12 amino acid positions. Like RecAPa, this chimera exhibits hyperrecombination activity in E. coli cells, increasing the frequency of recombination exchanges per DNA unit length (FRE). RecAX53 confers the largest increase in FRE observed to date. The contrasting properties of RecAX53 and RecAPa are manifested by in vivo differences in the dependence of the FRE value on the integrity of the mutS gene and thus in the ratio of conversion and crossover events observed among their hyperrecombination products. In strains expressing the RecAPa or RecAEc protein, crossovers are the main mode of hyperrecombination. In contrast, conversions are the primary result of reactions promoted by RecAX53. The biochemical activities of RecAX53 and its ancestors, RecAEc and RecAPa, have been compared. Whereas RecAPa generates a RecA presynaptic complex (PC) that is more stable than that of RecAEc, RecAX53 produces a more dynamic PC (relative to both RecAEc and RecAPa). The properties of RecAX53 result in a more rapid initiation of the three-strand exchange reaction but an inability to complete the four-strand transfer. This indicates that RecAX53 can form heteroduplexes rapidly but is unable to convert them into crossover configurations. A more dynamic RecA activity thus translates into an increase in conversion events relative to crossovers.     

An asymmetric approach to preserve common intervals while sorting by reversals

Background  

The reversal distance and optimal sequences of reversals to transform a genome into another are useful tools to analyse evolutionary scenarios. However, the number of sequences is huge and some additional criteria should be used to obtain a more accurate analysis. One strategy is searching for sequences that respect constraints, such as the common intervals (clusters of co-localised genes). Another approach is to explore the whole space of sorting sequences, eventually grouping them into classes of equivalence. Recently both strategies started to be put together, to restrain the space to the sequences that respect constraints. In particular an algorithm has been proposed to list classes whose sorting sequences do not break the common intervals detected between the two inital genomes A and B. This approach may reduce the space of sequences and is symmetric (the result of the analysis sorting A into B can be obtained from the analysis sorting B into A).     

Study of the mitotic and meiotic chromosomes of sotol (<i>Dasylirion cedrosanum</i> Trel.)

The genus Dasylirion is a group of plants typically present in the Chihuahuan Desert, perennial, with a dioecious sexual behavior and commonly called sotoles. This genus has been little studied from the biological point of view, and the bases of its reproductive response remain unknown. In this work we studied the chromosome number and meiotic response of Dasylirion cedrosanum in the county of Saltillo, Coahuila, located at the North East of Mexico. For the preparation of mitotic chromosomes, we used a technique based on enzymatic treatment with pectolyase and cellulase, as well as staining with acetocarmin dye. For the study of meiosis, male flower buds were collected, fixed and stained for analysis with the same dye. As a result, the gametic (n = x = 19) and somatic chromosome (2n = 38) numbers of D. cedrosanum are reported for the first time, being consistent with previous findings in other Dasylirion species, which points to a constant ploidy level across the genus. Variation was observed in the morphology and size of the somatic chromosomes, with types ranging from submetacentric to subtelocentric, and sizes oscillating in a range of 4.43 µm, with an average total length of 112.38 µm for the diploid chromosome complement. This shows that the chromosome complement of D. cedrosanom would belong to a 3B classification of Stebins, with a medium variation between chromosome lengths and low chromosome asymmetry. This variation indicates the feasibility of constructing a chromosome ideotype for this species. The meiotic chromosome pairing showed a chromosome behavior consistent with a disomic inheritance characteristic of a diploid species, with prevalence of ring and chain bivalents, typically without pairing abnormalities. Bivalent configurations in all cases were symmetrical.The normal and symmetrical meiotic pairing indicates a balanced production of gametes, and suggests the absence of heteromorphic sex determination.     

L29M] substitution in the interface of subunit-subunit interactions enhances Escherichia coli RecA protein properties important for its recombinogenic activity.

Genetic analysis of RecA protein chimeras and their ancestors, RecAEc (from Escherichia coli) and RecAPa (Pseudomonas aeruginosa) had allowed us to place these proteins with respect to their recombinogenic activities in the following order: RecAPa>RecAX21>RecAX20=RecAEc. While RecAX20 differs from RecAEc in five amino acid residues with two substitutions ([S25A] and [I26V]) at the interface of subunit interactions in the RecA polymer, RecAX20 and RecAX21 differ only by a single substitution [L29M] present at the interface. Here, we present an analysis of the biochemical properties considered important for the recombinogenic activity of all four RecA proteins. While RecAX20 was very similar to RecAEc by all activities analysed, RecAX21 differed from RecAEc in several respects. These differences included an increased affinity for double-stranded DNA, a more active displacement of SSB protein from single-stranded DNA (ssDNA), a decreased end-dependent RecAX21 protein dissociation from a presynaptic complex, and a greater accumulation of intermediate products relative to the final product in the strand-exchange reaction. RecAPa was more tolerant than RecAX21 only to the end-dependent RecA protein dissociation. In addition, RecAPa was more resistant to temperature and salt concentrations in its ability to form a presynaptic RecAPa::ATP::ssDNA filament. Calculations of conformational energy revealed that the [L29M] substitution in RecAX21 polymer caused an increase in its flexibility. This led us to conclude that even a small change in the flexibility of the RecA presynaptic complex could profoundly affect its recombinogenic properties.     

Molecular Analysis of the Microbial Community Developing in Continuous Culture of Methylococcus sp. Concept-8 on Natural Gas

Microbiology - The composition of the microbial community formed in the course of long-term (over 60 days) continuous cultivation of an obligate methanotroph Methylococcus sp. Concept-8 on natural...