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.     

Some Properties of New DNA-Specific Bisbenzimidazole Fluorochromes without a Piperazine Ring

Three new bisbenzimidazole (BBI) compounds, which differ from Hoechst 33258 mainly by substitution of a N-dimethylaminopro-pylcarboxamide group in place of the N-methyl-piperazine ring, were studied for their DNA- and AT-base pair specificity as well as for their ability to be quenched by incorporated 5-bromodeoxy-uridine (BrdU). Each of them had DNA binding specificity comparable to or greater than that of Hoechst 33258 and each had a greater specificity for AT-rich regions than did Hoechst 33258. The dependence of fluorescence of new dyes on the BrdU-incorporation into DNA is different from that of Hoechst 33258 and related compounds with piperazine ring. The quenching effect is much weaker, and two of the new compounds (BBI-1 and BBI-2) even show somewhat enhanced binding (fluorescence) at lower concentrations. Certain BBI dyes without piperazine ring may have some advantage over Hoechst for accurate DNA [AT-specific] measurements. The piperazine ring appears to play an important role in the yet unknown mechanism of Hoechst quenching by incorporated BrdU.     

Genome elimination in diploid and triploid Rana esculenta males: cytological evidence from DNA flow cytometry

Cytological aspects of hemiclonal (meroclonal) inheritance in diploid and triploid males of the hybridogenetic frog Rana esculenta (Rana ridibunda x Rana lessonae) have been studied by DNA flow cytometry. The fact that the R. ridibunda genome contains 16% more DNA than the R. lessonae genome provides the ability to discern cells containing genomes of any species from the water-frog complex under study. Data are presented showing that elimination of the R. ridibunda genome occurs in hybridogenetic males from certain populations. In triploid males, the cytogenetic mechanism of hemiclonal inheritance is simpler than in diploids: after the elimination of a genome (always the genome in the minority in the triploid set; "homogenizing elimination"), no compensatory duplication of the remaining genetic material is necessary, as it is in diploids. The process of elimination can be visualized in triploid males by using DNA flow cytometry to identify cells in the special phase of the spermatogonial cell cycle that we termed the E phase.     

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.

     

Optional Endoreplication and Selective Elimination of Parental Genomes during Oogenesis in Diploid and Triploid Hybrid European Water Frogs

Incompatibilities between parental genomes decrease viability of interspecific hybrids; however, deviations from canonical gametogenesis such as genome endoreplication and elimination can rescue hybrid organisms. To evaluate frequency and regularity of genome elimination and endoreplication during gametogenesis in hybrid animals with different ploidy, we examined genome composition in oocytes of di- and triploid hybrid frogs of the Pelophylax esculentus complex. Obtained results allowed us to suggest that during oogenesis the endoreplication involves all genomes occurring before the selective genome elimination. We accepted the hypothesis that only elimination of one copied genome occurs premeiotically in most of triploid hybrid females. At the same time, we rejected the hypothesis stating that the genome of parental species hybrid frogs co-exist with is always eliminated during oogenesis in diploid hybrids. Diploid hybrid frogs demonstrate an enlarged frequency of deviations in oogenesis comparatively to triploid hybrids. Typical for hybrid frogs deviations in gametogenesis increase variability of produced gametes and provide a mechanism for appearance of different forms of hybrids.     

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.     

Loosening of cell cycle controls of human lymphocytes under the action of tumour promoter TPA

The effect of tumour promoter TPA (12-O-tetradecanoylphorbol-13-acetate) on the cell cycle of human peripheral blood lymphocytes stimulated by phytohaemagglutinin (PHA) in vitro was studied and it was found that TPA caused cells to accumulate in all the cell cycle phases. This accumulation took place preferentially at later culture passages, when lymphocytes stimulated by PHA alone stopped mainly in G0/G1 phases. Other effects of TPA were cell induction to enter higher DNA ploidy and to survive and even synthesize DNA under colchicine block of mitosis or under cytochalasin block of cytokinesis. In addition, in experiments in which a transitory block through the G1 phase of cell cycle was applied with use of aminopterin, we could show that a fraction of TPA-treated cells still entered the active phase of DNA synthesis. These findings suggest that TPA causes cell cycle controls to become loose, thereby enhancing adaptability of human lymphocytes to various hindrances in the course of cell cycle and eventually causing them to acquire characteristics known to be common for tumour cells.     

Gamete production patterns and mating systems in water frogs of the hybridogenetic Pelophylax esculentus complex in north‐eastern Ukraine

Hybridization and polyploidy play an important role in animal speciation. European water frogs of the Pelophylax esculentus complex demonstrate unusual genetic phenomena associated with hybridization, clonality and polyploidy which presumably indicate an initial stage of reticulate speciation. The Seversky Donets River drainage in north‐eastern Ukraine is inhabited by both sexes of the diploid and triploid hybrid P. esculentus and only one parental species Pelophylax ridibundus. Based on the presence of various types of hybrids, all populations studied can be divided into three geographical groups: I) P. ridibundus–P. esculentus without triploids; II) P. ridibundus–P. esculentus without diploid hybrids; and III) P. ridibundus–P. esculentus with a mixture of diploids and triploids. A study of gametogenesis revealed that diploid P. esculentus in populations of the first type usually produced haploid gametes of P. ridibundus and a mixture of haploid gametes that carried one or another parental genome (hybrid amphispermy). In populations of the second type, hybrids are derived from crosses of P. ridibundus males with triploid hybrid females producing haploid eggs with a genome of P. lessonae. Therefore, we suggest that clonal genome duplication in these eggs might be the result of suppression of second polar body formation or extra precleavage endoreduplication. In populations of the third type, some diploid females can produce diploid gametes. Fertilization of these eggs with haploid sperm can result in triploid hybrids. Other hybrids here produce haploid gametes with one or another parental genome or their mixture giving rise to new diploid hybrids.