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.     

Cleavage of cytoskeletal proteins by caspases during ovarian cell death: evidence that cell-free systems do not always mimic apoptotic events in intact cells

Several lines of evidence support a role for protease activation during apoptosis. Herein, we investigated the involvement of several members of the CASP (cysteine aspartic acid-specific protease; CED-3- or ICE-like protease) gene family in fodrin and actin cleavage using mouse ovarian cells and HeLa cells combined with immunoblot analysis. Hormone deprivation-induced apo-ptosis in granulosa cells of mouse antral follicles incubated for 24 h was attenuated by two specific peptide inhibitors of caspases, zVAD-FMK and zDEVD-FMK (50-500 microM), confirming that these enzymes are involved in this paradigm of cell death. Proteolysis of actin was not observed in follicles incubated in vitro while fodrin was cleaved to the 120 kDa fragment that accompanies apoptosis. Fodrin, but not actin, cleavage was also detected in HeLa cells treated with various apoptotic stimuli. These findings suggest that, in contrast to recent data, proteolysis of cytoplasmic actin may not be a component of the cell death cascade. To confirm and extend these data, total cell proteins collected from mouse ovaries or non-apoptotic HeLa cells were incubated without and with recombinant caspase-1 (ICE), caspase-2 (ICH-1) or caspase-3 (CPP32). Immunoblot analysis revealed that caspase-3, but not caspase-1 nor caspase-2, cleaved fodrin to a 120 kDa fragment, wheres both caspases-1 and -3 (but not caspase-2) cleaved actin. We conclude that CASP gene family members participate in granulosa cell apoptosis during ovarian follicular atresia, and that collapse of the granulosa cell cytoskeleton may result from caspase-3-catalyzed fodrin proteolysis. However, the discrepancy in the data obtained using intact cells (actin not cleaved) versus the cell-free extract assays (actin cleaved) raises concern over previous conclusions drawn related to the role of actin cleavage in apoptosis.     

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.     

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.     

Caspase-2 deficiency prevents programmed germ cell death resulting from cytokine insufficiency but not meiotic defects caused by loss of ataxia telangiectasia-mutated (Atm) gene function

It is well established that programmed cell death claims up to two-thirds of the oocytes produced during gametogenesis in the developing fetal ovaries. However, the mechanisms underlying prenatal germ cell loss in females remain poorly understood. Herein we report that caspase-11 null female mice are born with a reduced number of oocyte-containing primordial follicles. This phenotype is likely due to failed cytokine processing known to occur in caspase-11 mutants since neonatal female mice lacking both interleukin (IL)-1alpha and IL-1beta also exhibit a reduced endowment of primordial follicles. In addition, germ cell death in wild-type fetal ovaries cultured ex vivo is suppressed by either cytokine, likely via ligand activation of type 1 IL-1 receptors expressed in fetal germ cells. Normal oocyte endowment can be restored in caspase-11 null female mice by simultaneous inactivation of the gene encoding the cell death executioner enzyme, caspase-2. However, caspase-2 deficiency cannot overcome gametogenic failure resulting from meiotic recombination defects in ataxia telangiectasia-mutated (Atm) null female mice. Thus, genetically distinct mechanisms exist for developmental deletion of oocytes via programmed cell death, one of which probably functions as a meiotic quality-control checkpoint that cannot be overridden.     

The timing of alpha-gustducin expression during cell renewal in rat vallate taste buds

The G protein subunit alpha-gustducin is expressed in a subset of light (Type II) but not in dark (Type I) cells in rat vallate taste buds. The thymidine analogue 5-bromo-2'-deoxyuridine (BrdU) is incorporated into DNA during the S-phase of the cell cycle and can be used to determine the time of origin of a cell. In this study, 31 rats were injected with BrdU (50 mg/kg i.p.) and perfused at various times, from 2.5 to 10.5 days, following BrdU administration. Vallate papillae were embedded in polyester wax, cut into 4 microm transverse sections, and characterized with antibodies to BrdU and alpha-gustducin. Sections were processed for indirect immunofluorescence or with an immunoperoxidase procedure. From immunoperoxidase material on 21 rats, counts of alpha-gustducin- and BrdU-labeled cells were obtained from 300-800 taste bud profiles at each survival time; a total of 4122 taste bud profiles were examined. Cells with nuclei immunoreactive for BrdU occurred within the taste buds at 2.5 days and double-labeled cells were clearly evident at 3.5 days; a small number of double-labeled cells were seen as early as 2.5 days. Double-labeled cells reached a peak at 6.5 days and did not decline significantly by 10.5 days. Cells labeled for BrdU but not alpha-gustducin peaked at 5.5 days and showed a significant decline by 8.5 days. These latter cells included light cells not expressing alpha- gustducin and dark cells, which have previously been shown to have a shorter life span than light cells. These data suggest that expression of alpha-gustducin appears very early in a cell's life span and that these cells are longer lived than many of the cells that do not express this G protein.      

Tonic GABAergic inhibition of taste-responsive neurons in the nucleus of the solitary tract

The effects of gamma-aminobutyric acid (GABA) and the GABAA receptor antagonist bicuculline methiodide (BICM) on the activity of taste- responsive neurons in the nucleus of the solitary tract (NST) were examined electrophysiologically in urethane-anesthetized hamsters. Single neurons in the NST were recorded extracellularly and drugs (21 nl) were microinjected into the vicinity of the cell via a multibarrel pipette. The response of each cell was recorded to lingual stimulation with 0.032 M NaCl, 0.032 M sucrose, 0.0032 M citric acid and 0.032 M quinine hydrochloride (QHCl). Forty-six neurons were tested for the effects of GABA; the activity of 29 cells (63%) was inhibited by 5 mM GABA. Whether activity was elicited in these cells by repetitive anodal current stimulation (25 microA, 0.5 s, 0.1 Hz) of the tongue (n = 13 cells) or the cells were spontaneously active (n = 13 cells), GABA produced a dose-dependent (1, 2 and 5 mM) decrement in activity. Forty- seven NST neurons were tested for the effects of BICM on their responses to chemical stimulation of the tongue; the responses of 28 cells (60%) were enhanced by 10 mM BICM. The gustatory responses of 26 of these cells were tested with three concentrations (0.2, 2 and 10 mM) of BICM, which produced a dose-dependent increase in both spontaneous activity and taste-evoked responses. Nine of these neurons were sucrose- best, seven were NaCl-best, eight were acid-best and two responded best to QHCl. The responses to all four tastants were enhanced, with no difference among neuron types. For 18 cells that were tested with two or more gustatory stimuli, BICM increased their breadth of responsiveness to their two most effective stimuli. These data show that approximately 60% of the taste-responsive neurons in the rostral NST are inhibited by GABA and/or subject to a tonic inhibitory influence, which is mediated by GABAA receptors. The modulation of these cells by GABA provides a mechanism by which the breadth of tuning of the cell can be sharpened. Modulation of gustatory activity following a number of physiological changes could be mediated by such a GABAergic circuit.      

Targeted expression of Bcl-2 in mouse oocytes inhibits ovarian follicle atresia and prevents spontaneous and chemotherapy-induced oocyte apoptosis in vitro.

Members of the Bcl-2 family serve as central checkpoints for cell death regulation, and overexpression of Bcl-2 is known to inhibit apoptosis in many cell types. To determine whether targeted expression of Bcl-2 could be used to protect female germ cells from apoptosis, we generated transgenic mice expressing fully functional human Bcl-2 protein only in oocytes. Transgenic mice were produced using a previously characterized 480-bp fragment of the mouse zona pellucida protein-3 (ZP3) gene 5'-flanking region to direct oocyte-specific expression of a human bcl-2 complementary DNA. Immunohistochemical analyses using a human Bcl-2-specific antibody showed that transgene expression was restricted to growing oocytes and was not observed in the surrounding ovarian somatic cells or in any other nonovarian tissues. Histomorphometric analyses revealed that ovaries collected from transgenic female mice possessed significantly fewer atretic small preantral follicles compared with wild-type sisters, resulting in a larger population of healthy maturing follicles per ovary. However, the number of oocytes ovulated in response to exogenous gonadotropin priming and the number of pups per litter were not significantly different among wild-type vs. transgenic female mice. Nonetheless, oocytes obtained from transgenic mice and cultured in vitro were found to be resistant to spontaneous and anticancer drug-induced apoptosis. We conclude that targeted expression of Bcl-2 only in oocytes can be achieved as a means to convey resistance of the female germ line to naturally occurring and chemotherapy-induced apoptosis.