Sublethal damage during cryopreservation of rainbow trout sperm

Although cellular damage during cryopreservation of freshwater fish spermatozoa has been reported in several studies, there is a lack of correlation between this damage and the fertility rates of eggs using postthawed milt. The apparent lack of such correlation may be due to other undetected sublethal cryodamage, which could affect the cell functionality and viability. This may be extremely important for freshwater fish spermatozoa whose ability to fertilize the egg requires dilution in water or hypoosmotic solutions, an hazardous environment for the cells. This study tested the change in cell permeability during cryopreservation, using Hoechst 33258 to assess cell permeability. The permeability of spermatozoa at different times after dilution in several hypoosmotic media were investigated. In the first trial, fresh semen, sperm diluted in freezing media (CPT), and freeze/thawed semen were studied. Three CPT were tested (Me2SO, DMA, and methanol). In the second trial, the addition of egg yolk as a membrane stabilizer was investigated. Samples were frozen at -20 degreesC/min in a programmable cooler and thawed in a 25 degreesC water bath. Dilution in the CPTs slightly increased the susceptibility of cells to damage but freezing/thawing caused a dramatic increase in the fragility of cells, which were killed in a few seconds after their contact with the hypoosmotic solutions. Egg yolk provided a significant protection to the membrane, allowing the cells a greater and more prolonged survival in the fertilization media. Samples frozen with Me2SO displayed the best results. These results are consistent with the achieved fertility rates that demonstrated sublethal cryodamage in the function of the sperm membrane that was not detected by standard procedures. Copyright 1998 Academic Press.     

Slipped-strand mispairing in a plastid gene: rpoC2 in grasses (Poaceae)

An exception to the generally conservative nature of plastid gene evolution is the gene coding for the beta" subunit of RNA polymerase, rpoC2. Previous work by others has shown that maize and rice have an insertion in the coding region of rpoC2, relative to spinach and tobacco. To assess the distribution of this extra coding sequence, we surveyed a broad phylogenetic sample comprising 55 species from 17 angiosperm families by using Southern hybridization. The extra coding sequence is restricted to the grasses (Poaceae). DNA sequence analysis of 11 species from all five subfamilies within the grass family demonstrates that the extra sequence in the coding region of rpoC2 is a repetitive array that exhibits more than a twofold increase in nucleotide substitution, as well as a large number of insertion/deletion events, relative to the adjacent flanking sequences. The structure of the array suggests that slipped-strand mispairing causes the repeated motifs and adds to the mechanisms through which the coding sequence of plastid genes are known to evolve. Phylogenetic analyses based on the sequence data from grass species support several relationships previously suggested by morphological work, but they are ambiguous about broad relationships within the family.      

Recombination at subtelomeres is regulated by physical distance,double‐strand break resection and chromatin status

Homologous recombination (HR) is a conserved mechanism that repairs broken chromosomes via intact homologous sequences. How different genomic, chromatin and subnuclear contexts influence HR efficiency and outcome is poorly understood. We developed an assay to assess HR outcome by gene conversion (GC) and break‐induced replication (BIR), and discovered that subtelomeric double‐stranded breaks (DSBs) are preferentially repaired by BIR despite the presence of flanking homologous sequences. Overexpression of a silencing‐deficient SIR3 mutant led to active grouping of telomeres and specifically increased the GC efficiency between subtelomeres. Thus, physical distance limits GC at subtelomeres. However, the repair efficiency between reciprocal intrachromosomal and subtelomeric sequences varies up to 15‐fold, depending on the location of the DSB, indicating that spatial proximity is not the only limiting factor for HR. EXO1 deletion limited the resection at subtelomeric DSBs and improved GC efficiency. The presence of repressive chromatin at subtelomeric DSBs also favoured recombination, by counteracting EXO1‐mediated resection. Thus, repressive chromatin promotes HR at subtelomeric DSBs by limiting DSB resection and protecting against genetic information loss.     

Analysis of silver binding nucleolar organizer regions in exfoliative cytology smears of potentially malignant and malignant oral lesions

Nucleolar organizer regions are nucleolar components that contain proteins that are stained selectively by silver methods; they can be identified as black dots throughout the nucleolus and are known as silver binding nucleolar organizer regions (AgNOR). The number of AgNOR is related to the cell cycle and the proliferative activity of the cells. We investigated AgNOR using exfoliative cytology smears of potentially malignant oral lesions. Eighty individuals were divided into four equal groups: healthy controls, oral leukoplakia, oral submucous fibrosis and oral squamous cell carcinoma. The mean number of AgNOR in each study group gradually increased from control to oral leukoplakia to oral submucous fibrosis to oral squamous cell carcinoma. The proliferative index was increased in the oral premalignant and malignant patients compared to normal subjects. The mean AgNOR size gradually increased from control to oral leukoplakia to oral submucous fibrosis to oral squamous cell carcinoma. Spherical shaped AgNOR were most common in controls, whereas large, clustered and kidney shapes were most common in oral squamous cell carcinoma. Multiparameter analysis of AgNOR in oral exfoliative smears is a simple, sensitive and cost-effective method for differentiating premalignant from malignant lesions and can be used in conjunction with routine cytomorphological evaluation.     

Ku-deficient yeast strains exhibit alternative states of silencing competence

In Saccharomyces cerevisiae, efficient silencer function requires telomere proximity, i.e. compartments of the nucleoplasm enriched in silencing factors. Accordingly, silencers located far from telomeres function inefficiently. We show here that cells lacking yKu balance between two mitotically stable states of silencing competence. In one, a partial delocalization of telomeres and silencing factors throughout the nucleoplasm correlates with enhanced silencing at a non-telomeric locus, while in the other, telomeres retain their focal pattern of distribution and there is no repression at the non-telomeric locus, as observed in wild-type cells. The two states also differ in their level of residual telomeric silencing. These findings indicate the existence of a yKu-independent pathway of telomere clustering and Sir localization. Interestingly, this pathway appears to be under epigenetic control.     

Capture and commercialization of blue land crabs ("guaiamum") Cardisoma guanhumi (Lattreille, 1825) along the coast of Bahia State, Brazil: an ethnoecological approach

Background

Blue Land Crab (Cardisoma guanhumi) is one of the most important crustacean species captured and commercialized in Brazil. Although this species is not considered to be threatened with extinction, populations of C. guanhumi are known to be rapidly diminishing due to heavy harvesting pressures and degradation of their natural habitats, highlighting the necessity of developing and implanting management and protection strategies for their populations. There have been no ethnozoological publications that have focused specifically on C. guanhumi, in spite of importance of this type of information for developing efficient management plans of resource utilization. So, the present work describes the ethnoecological aspects of the capture and commercialization of C. guanhumi by a fishing community in northeastern Brazil.

Methods

Field work was carried out in the municipality of Mucuri, Bahia in Brazil, between the months of January and March/2011 through the use of open semi-structured interviews with all of the crustacean harvesters in city who acknowledged their work in capturing this species, totaling 12 interviewees. The informants were identified through the use of the "snowball" sampling technique. In addition to the interviews themselves, the "guided tour" technique and direct observations was employed.

Results

According all the interviewees, the C. guanhumi is popularly called "guaiamum" and is collected in "apicum" zones. They recognize sexual dimorphism in the species based on three morphological characteristics and the harvesters also pointed two stages in the reproductive cycle during the year and another phase mentioned by the interviewees was ecdysis. All of the interviewed affirmed that the size and the quantities C. guanhumi stocks in Mucuri have been diminishing. All of the interviewees agreed that the species and other mangrove resources constituted their principal source of income. The harvesters dedicated three to five days a week to collect Blue Land Crabs and the principal technique utilized for capturing is a trap called a "ratoeira" (rat-trap).

Conclusions

The results of the present work demonstrated that the community retains a vast and important volume of knowledge about C. guanhumi that could subsidize both scientific studies and the elaboration of viable management and conservation strategies for this species.     

The Repetitive Domain of ScARP3d Triggers Entry of Spiroplasma citri into Cultured Cells of the Vector Circulifer haematoceps

Spiroplasma citri is a plant pathogenic mollicute transmitted by the leafhopper vector Circulifer haematoceps. Successful transmission requires the spiroplasmas to cross the intestinal epithelium and salivary gland barriers through endocytosis mediated by receptor-ligand interactions. To characterize these interactions we studied the adhesion and invasion capabilities of a S. citri mutant using the Ciha-1 leafhopper cell line. S. citri GII3 wild-type contains 7 plasmids, 5 of which (pSci1 to 5) encode 8 related adhesins (ScARPs). As compared to the wild-type strain GII3, the S. citri mutant G/6 lacking pSci1 to 5 was affected in its ability to adhere and enter into the Ciha-1 cells. Proteolysis analyses, Triton X-114 partitioning and agglutination assays showed that the N-terminal part of ScARP3d, consisting of repeated sequences, was exposed to the spiroplasma surface whereas the C-terminal part was anchored into the membrane. Latex beads cytadherence assays showed the ScARP3d repeat domain (Rep3d) to be involved, and internalization of the Rep3d-coated beads to be actin-dependent. These data suggested that ScARP3d, via its Rep3d domain, was implicated in adhesion of S. citri GII3 to insect cells. Inhibition tests using anti-Rep3d antibodies and competitive assays with recombinant Rep3d both resulted in a decrease of insect cells invasion by the spiroplasmas. Unexpectedly, treatment of Ciha-1 cells with the actin polymerisation inhibitor cytochalasin D increased adhesion and consequently entry of S. citri GII3. For the ScARPs-less mutant G/6, only adhesion was enhanced though to a lesser extent following cytochalasin D treatment. All together these results strongly suggest a role of ScARPs, and particularly ScARP3d, in adhesion and invasion of the leafhopper cells by S. citri.     

Analysis of Single Locus Trajectories for Extracting In Vivo Chromatin Tethering Interactions

Is it possible to extract tethering forces applied on chromatin from the statistics of a single locus trajectories imaged in vivo? Chromatin fragments interact with many partners such as the nuclear membrane, other chromosomes or nuclear bodies, but the resulting forces cannot be directly measured in vivo. However, they impact chromatin dynamics and should be reflected in particular in the motion of a single locus. We present here a method based on polymer models and statistics of single trajectories to extract the force characteristics and in particular when they are generated by the gradient of a quadratic potential well. Using numerical simulations of a Rouse polymer and live cell imaging of the MAT-locus located on the yeast Saccharomyces cerevisiae chromosome III, we recover the amplitude and the distance between the observed and the interacting monomer. To conclude, the confined trajectories we observed in vivo reflect local interaction on chromatin.     

DELAYED URIC ACID ACCUMULATION IN PLASMA PROVIDES ADDITIONAL ANTI-OXIDANT PROTECTION AGAINST IRON-TRIGGERED OXIDATIVE STRESS AFTER A WINGATE TEST

Reactive oxygen species are produced during anaerobic exercise mostly by Fe ions released into plasma and endothelial/muscle xanthine oxidase activation that generates uric acid (UA) as the endpoint metabolite. Paradoxically, UA is considered a major antioxidant by virtue of being able to chelate pro-oxidative iron ions. This work aimed to evaluate the relationship between UA and plasma markers of oxidative stress following the exhaustive Wingate test. Plasma samples of 17 male undergraduate students were collected before, 5 and 60 min after maximal anaerobic effort for the measurement of total iron, haem iron, UA, ferric-reducing antioxidant activity in plasma (FRAP), and malondialdehyde (MDA, biomarker of lipoperoxidation). Iron and FRAP showed similar kinetics in plasma, demonstrating an adequate pro-/antioxidant balance immediately after exercise and during the recovery period (5–60 min). Slight variations of haem iron concentrations did not support a relevant contribution of rhabdomyolysis or haemolysis for iron overload following exercise. UA concentration did not vary immediately after exercise but rather increased 29% during the recovery period. Unaltered MDA levels were concomitantly measured. We propose that delayed UA accumulation in plasma is an auxiliary antioxidant response to post-exercise (iron-mediated) oxidative stress, and the high correlation between total UA and FRAP in plasma (R-Square = 0.636; p = 0.00582) supports this hypothesis.