Porphyromonas gingivalis LPS stimulation downregulates DNMT1, DNMT3a, and JMJD3 gene expression levels in human HaCaT keratinocytes

Objective: The role of epigenetic regulation in inflammatory diseases such as periodontitis is poorly known. The aim of this study was to assess whether Porphyromonas gingivalis lipopolysaccharide (LPS) can modulate gene expression levels of the some enzymes that promote epigenetic events in cultures of the human keratinocytes and gingival fibroblasts. In addition, the same enzymes were evaluated in gingival samples from healthy and periodontitis-affected individuals. Materials and methods: Primary gingival fibroblast and keratinocyte (HaCaT) cultures were treated with medium containing P. gingivalis LPS or P. gingivalis LPS vehicle for 24 h. After this period, cell viability was assessed by MTT test and total RNA extracted to evaluate gene expression levels of the following enzymes by qRT-PCR: DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), histone demethylases Jumonji domain containing 3 (JMJD3) and ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX). To evaluate gene expression in healthy and periodontitis-affected individuals, total RNA was extracted from biopsies of gingival tissue from healthy and periodontitis sites, and gene expression of DNMT1, DNAMT3a, JMJD3, and UTX was evaluated by qRT-PCR. Results: No significant differences were found in the gene expression analysis between healthy and periodontitis-affected gingival samples. The results showed that LPS downregulated DNMT1 (p < 0. 05), DNMT3a (p < 0. 05), and JMJD3 (p < 0. 01) gene expression in HaCaT cells, but no modulation was observed in gingival fibroblasts. Conclusion: P. gingivalis LPS exposure to human HaCaT keratinocytes downregulates gene expression of the enzymes that promote epigenetic events. Clinical relevance: The advance knowledge about epigenetic modifications caused by periodontopathogens may to possibly led to the development of new periodontal therapies. © 2012 Springer-Verlag.

Cytogenetic mapping of H1 histone and ribosomal RNA genes in hybrids between catfish species Pseudoplatystoma corruscans and Pseudoplatystoma reticulatum

A physical chromosome mapping of the H1 histone and 5S and 18S ribosomal RNA (rRNA) genes was performed in interspecific hybrids of Pseudoplatystoma corruscans and P. reticulatum. The results showed that 5S rRNA clusters were located in the terminal region of 2 chromosomes. H1 histone and 18S ribosomal genes were co-localized in the terminal portion of 2 chromosomes (distinct from the chromosomes bearing 5S clusters). These results represent the first report of association between H1 histone and 18S genes in fish genomes. The chromosome clustering of ribosomal and histone genes was already reported for different organisms and suggests a possible selective pressure for the maintenance of this association. © 2012 S. Karger AG, Basel.

Supplementation with the histone deacetylase inhibitor trichostatin A during in vitro culture of bovine embryos

Summary Trichostatin A (TSA) is a histone deacetylase inhibitor that induces histone hyperacetylation and increases gene expression levels. The aim of the present study was to establish a suitable condition for the use of TSA in in vitro cultures of bovine embryos, and to determine whether TSA would increase blastocyst rates by improvement of chromatin remodelling during embryonic genome activation and by increasing the expression of crucial genes during early development. To test this hypothesis, 8-cell embryos were exposed to four concentrations of TSA for different periods of time to establish adequate protocols. In a second experiment, three experimental groups were selected for the evaluation of embryo quality based on the following parameters: apoptosis, total cell number and blastocyst hatching. TSA promoted embryonic arrest and degeneration at concentrations of 15, 25 and 50 nM. All treated groups presented lower blastocyst rates. Exposure of embryos to 5 nM for 144 h and to 15 nM for 48 h decreased blastocyst hatching. However, the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay (TUNEL) assay revealed similar apoptosis rates and total cell numbers in all groups studied. Although, in the present study, TSA treatment did not improve the parameters studied, the results provided background information on TSA supplementation during in vitro culture of bovine embryos and showed that embryo quality was apparently not affected, despite a decrease in blastocyst rate after exposure to TSA. © Cambridge University Press 2011.

Chromosomal Mapping of Repetitive DNAs in the Grasshopper Abracris flavolineata Reveal Possible Ancestry of the B Chromosome and H3 Histone Spreading

Supernumerary chromosomes (B chromosomes) occur in approximately 15% of eukaryote species. Although these chromosomes have been extensively studied, knowledge concerning their specific molecular composition is lacking in most cases. The accumulation of repetitive DNAs is one remarkable characteristic of B chromosomes, and the occurrence of distinct types of multigene families, satellite DNAs and some transposable elements have been reported. Here, we describe the organization of repetitive DNAs in the A complement and B chromosome system in the grasshopper species Abracris flavolineata using classical cytogenetic techniques and FISH analysis using probes for five multigene families, telomeric repeats and repetitive C0t-1 DNA fractions. The 18S rRNA and H3 histone multigene families are highly variable and well distributed in A. flavolineata chromosomes, which contrasts with the conservation of U snRNA genes and less variable distribution of 5S rDNA sequences. The H3 histone gene was an extensively distributed with clusters occurring in all chromosomes. Repetitive DNAs were concentrated in C-positive regions, including the pericentromeric region and small chromosomal arms, with some occurrence in C-negative regions, but abundance was low in the B chromosome. Finally, the first demonstration of the U2 snRNA gene in B chromosomes in A. flavolineata may shed light on its possible origin. These results provide new information regarding chromosomal variability for repetitive DNAs in grasshoppers and the specific molecular composition of B chromosomes. © 2013 Bueno et al.

Digestible lysine levels for laying hens and their effects on egg quality

Egg quality of semi-heavy laying hens fed on low protein diets (14.0% CP) and on different lysine levels is evaluated, while maintaining the same ratio of digestible amino acids / digestible lysine. Four hundred and twenty commercial strain Isa Brown laying hens, 28 weeks old, were divided into 42 experimental plots. A completely randomized design with six treatments and seven replicates was employed in four production cycles of 28 days each. Treatments comprised Control - 16.92% CP; 0.750% digestible lysine. Treatments 1 to 5, with CP levels 14% and digestible lysine levels 0.600, 0.675, 0.750, 0.825 and 0.900% respectively. Levels of Treatments 1 and 2 (0.546 and 0.640% digestible Met + Cys / 0.600 and 0.675% digestible lysine) provided smaller egg size. On the other hand, eggs had higher shell percentage when compared to control diet. When compared to other digestible amino acids, digestible lysine requirement may be estimated at 0.750% in a diet with 14% CP, which corresponds to the average daily intake of 876 mg dig. lysine hen-1 day-1 and 798 mg dig. Met + Cys hen-1 day-1, without jeopardizing performance and eggs' internal and external quality.

Scattered organization of the histone multigene family and transposable elements in Synbranchus

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Reevaluation of the digestible lysine requirement for broilers based on genetic potential

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Enhancing effect of lysine combined with other compounds on cephamycin C production in Streptomyces clavuligerus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The use of stable isotopes to investigate the effects of supplemental lysine and methionine on protein turnover and amino acid utilization in pacu, Piaractus mesopotamicus, juveniles

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Lysine in the diet of Rhamdia voulezi male broodstocks confined in net cages

The focus of this study was to evaluate the effect of lysine on the productive (weight and length gain, apparent feed conversion, specific growth rate and condition factor) and reproductive performance of Rhamdia voulezi males confined in net cages during the first reproductive cycle. The following parameters were assessed: seminal parameters (motility rate and duration, survival, sperm concentration, morphology, pH and osmolarity), hormonal parameters (cortisol and testosterone), testicular tissue (histomorphology), organosomatic indices (gonadosomatic, hepatosomatic and viscerosomatic indices) and composition of essential amino acids, crude protein and moisture of whole carcass. Four hundred fish were used, distributed in a random experimental design with four treatments and four replications in 16 net cages with 25 fish each. The treatments consisted of four different diets prepared so as to contain the following levels of lysine: 1.20, 1.40, 1.60 and 1.80%, with 30% crude protein and 3500 kcal kg(-1) digestible energy for 185 days (Jul./12-Jan./13). Eighteen males were selected per treatment, and they all released semen after slight abdominal pressure. The males were weighed, measured, submitted to hypophysation (2.5 mg kg(-1) carp pituitary extract), and then had their semen and blood collected. The fish were sacrificed by cervical dislocation, dissected, and the testes, liver, fat and guts were removed and weighed. The effects (p < 0.05) for the means of final weight, weight gain, apparent feed conversion and condition factor were observed for the analysis of productive performance. With regard to the reproductive parameters, only the seminal volume was affected (p < 0.05). Thus, the levels of testosterone showed quadratic effect (p < 0.05). The anatomy and the histomorphology of the testes were similar between the treatments during the spermiation period. With regard to the organosomatic indices, there was no influence (p > 0.05) between the treatments. The amino acids in the carcass were not affected (p > 0.05). The increment of lysine in the diet provided linear increase for weight gain and seminal volume and linear decrease for feed conversion in R. voulezi broodstocks confined in net cages. (C) 2014 Elsevier B.V. All rights reserved.

Modelling the maximum potential of nitrogen deposition and requirements of lysine for broilers

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Modelling of the nitrogen deposition and dietary lysine requirements of Redbro broilers

The aim of this study was to determine the coefficients of the Goettingen model for Redbro birds and estimate the digestible lysine requirements. To determine the model parameters, three nitrogen balance trials were performed in Periods I (14-28 days), II (42-56 days) and III (70-84 days), using 42 birds per trial. The birds were individually housed and subjected to six diets with increasing levels of nitrogen, with lysine as the limiting amino acid (deficient by 20% in relation to other amino acids). Dietary nitrogen concentrations were 8, 16, 24, 32, 40 and 48 g/kg. A control diet was added to confirm lysine as the first limiting amino acid. Nitrogen balance trials were divided into 5 days of adaptation and two periods of excreta collection, each one of 5 days. The response of the birds to a control diet confirmed that lysine was the first limiting amino acid. The adjustment of the exponential functions between nitrogen retention or excretion and nitrogen intake allowed estimation of parameters of the Goettingen model. The maximum potential for nitrogen retention was 3276, 2585 and 2603 mg/BWkg0.67.day, nitrogen maintenance requirement was 225, 135 and 122 mg/BWkg0.67.day and efficiency of nitrogen utilisation was 313 x 10(-6), 406 x 10(-6) and 415 x 10(-6) in the phases of 14-28, 42-56 and 70-84 days. The digestible lysine intake for Periods I, II and III, based on 60% of the maximum potential for nitrogen retention, was 711, 989 and 1272 mg/day (1.225%, 1.137% and 1.09% of lysine in the diet for a daily feed intake of 58, 87 and 117 g/day), respectively.

The Influence of Dietary Lysine on Yellow Perch Female Reproductive Performance and the Quality of Eggs

The objectives of this study were to (1) evaluate if dietary lysine (Lys) has an effect on the free amino acid (FAA) pool of Yellow Perch Perca flavescens eggs, and (2) determine how dietary Lys influences the reproductive performance and eggs fertilization rate (embryo survival) of female Yellow Perch. Two-year-old Yellow Perch of initial size of approximately 75 g were randomly distributed into six 400-L tanks at 32 +/- 1 fish per tank. This experiment included two wheat gluten-based diets in triplicate Lys-deficient ([-] Lys) and Lys-supplemented ([+]Lys; 2.23% in dry feed) diets. Females from the reference group were fed a commercial diet. Females from reference, (+) Lys, and (-) Lys groups were stripped and their eggs divided into 0.4-1.2-g portions and mixed with sperm (21.4 +/- 4.3 mu L) from either reference, (+) Lys, or (-) Lys males. The mean weight of Yellow Perch females and mean total weight of ovulated eggs were the greatest in the reference group compared with both (+) Lys and (-) Lys groups. There were no differences in the ratio of weight of eggs to female body weight as well as egg size among groups. There was no difference among treatments in the concentration of free amino acids except glutamic and aspartic acids in Yellow Perch eggs. There was significant effect of female dietary treatments on the egg fertilization rate averaged across all males. The higher fertilization rate was observed in the reference and (+) Lys groups compared with the (-) Lys group. The effect of female dietary treatment on the egg survival was also dependent on the dietary treatment of males.

Model to estimate lysine requirements of broilers

The purpose of this study is to describe the development of a model to predict the digestible lysine requirements of broilers using the factorial approach, and to evaluate the model using as reference the model presented in Brazilian Tables for Poultry and Swine. The model partitions the requirement for maintenance and growth for feather-free body protein and feather protein, in which the inputs are body and feather protein weight and the daily rates of protein deposition in the feather-free body and feathers. The parameters that express the lysine requirement for maintenance were obtained in metabolism trials with roosters, and those for the efficiency of lysine utilization in experiments with broilers from 1 to 42 d. Based on these results the model proposed was: Lys (mg/d) = [(151xBP(m)(-0.27)xBP(t)) + (0.01xP(t)x18)] + [(75xBPD/0.77) + (18xFPD/0.77)], where Lys = digestible lysine requirement (mg/d), BPm=body protein weight at maturity (kg), BPt=body protein weight at time t (kg), FPt=feather protein weight at time t (kg), BPD=body protein deposition (g/d), FPD = feather protein deposition (g/d). The model yields sensible predictions of the digestible lysine requirements of broilers of different strains and ages growing at their potential, and suggests a lower lysine requirement after 27 d than does the Brazilian model. The proposed model is the first step in the development of a simulation model that predicts the food intake of a broiler and hence the dietary amino acid content that would optimise performance.

Response of pullets to digestible lysine intake

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)