Effects of basic fibroblast growth factor on density and morphology of fibroblasts grown on root surfaces with or without conditioning with tetracycline or EDTA.

A study was conducted to evaluate in vitro the effect of root surface conditioning with basic fibroblast growth factor (b-FGF) on morphology and proliferation of fibroblasts. Three experimental groups were used: non-treated, and treated with 50 microg or 125 microg b-FGF/ml. The dentin samples in each group were divided into subgroups according to the chemical treatment received before application of b-FGF: none, or conditioned with tetracycline-HCl or EDTA. After contact with b-FGF for 5 min, the samples were incubated for 24 h with 1 ml of culture medium containing 1 x 10(5) cells/ml plus 1 ml of culture medium alone. The samples were then subjected to routine preparation for SEM, and random fields were photographed. Three calibrated and blind examiners performed the assessment of morphology and density according to two index systems. Classification and regression trees indicated that the root surfaces treated with 125 microg b-FGF and previously conditioned with tetracycline-HCl or EDTA presented a morphology more suggestive of cellular adhesion and viability (P = 0.004). The density of fibroblasts on samples previously conditioned with EDTA, regardless of treatment with b-FGF, was significantly higher than in the other groups (P < 0.001). The present findings suggest that topical application of b-FGF has a positive influence on both the density and morphology of fibroblasts.

Potencialidades da utilização de taninos na conservação de forragens e nutrição de ruminantes-revisão

This review present a discussion abouth the tannins. The tannins are compounds that have the ability to bind other macromolecules, reducing their availability to animal metabolism. This characteristic can be benefic or deleterious, depending on the tannin concentration degree in the plant and its structure. Positive effects are mainly related with a better use of the dietary protein and an increased efficiency of microbial protein synthesis in the rumen. The use of tannins to reduce ruminal methane emission has been subject of current researches, firmly indicating a decrease of ruminal methanogenesis. However, many of these studies, carried out to evaluate the effects of tannins on forage fermentation and animal metabolism, are based on legume plants, so their results can not be surpassed to studies concerning tanniniferous grasses. By improving the characterization of the profile and reactivity of tannins in tanniniferous forages, such as some sorghum hybrids, as well as conducting experiments to demonstrate the effects of tannins on animal metabolism and performance, the real potential of forages with tannin will be enlightened.

Pattern of silver nitrate-staining during meiosis and spermiogenesis in testicular lobes of Antiteuchus tripterus (Heteroptera: Pentatomidae)

The pattern of silver nitrate (Ag)-staining differed among testicular lobes of Antiteuchus tripterus. In general, these differences are in regard to the number, size, shape, coloring intensity, and location of the stained bodies or masses, observed during meiosis and spermiogenesis. These characteristics were similar in lobes 1-3. Lobes 4-6, however, differed from each other and from lobes 1-3 as well. Because the Ag-staining method is specific for nucleolar organizing regions and nucleolar material, the observations in meiosis of lobes 1-3 suggested the presence of a single pair of nucleolar organizing region-bearing chromosomes in A. tripterus, as previously found in other Pentatomidae species. In general, the amount of Ag-stained material seen in meiosis of the testicular lobes 1-3 of A. tripterus is smaller than in the other lobes. The differences among lobes observed during spermiogenesis included a striking variation in morphology of the Ag-stained material found in the head and tail of the spermatids. Given that the key role of the nucleolar material is to participate in protein synthesis, interlobular variations seem to be related to the different functions attributed to each lobe (reproduction to lobes 1-3 and basically nutrition to lobes 4-6). To our knowledge, this is the first time that the nucleolar material was studied in each testicular lobe during spermatogenesis. The present observations encourage further studies since, in addition to being of basic biological interest, several Pentatomidae species are agricultural pests and added knowledge of their biology, mainly in reproduction, may be important for the development of control strategies. ©FUNPEC-RP.

Therapeutic option for children with esophageal caustic stenosis

Caustic ingestion is a leading cause of esophageal stenosis in children. Herein we report four cases using mitomycin C (MMC), a drug that inhibits cell division, protein synthesis and fibroblast proliferation and has been used as an adjuvant therapy for caustic esophageal stenosis that is recalcitrant to conventional dilation techniques. A retrospective chart review was performed on four pediatric patients with severe, recurrent esophageal stricture after caustic ingestion. The patients had required six to 20 esophageal dilations over a 4-16-month period before MMC application. MMC was applied after an endoscopic dilation on saturated pledgets at a dose of 0.1 mg/mL for 2 min in the area where the strictures had been lyzed. From the four children treated with MMC, two have been asymptomatic for 16 and 20 months and two still require esophageal dilation, however, at longer intervals. All patients have shown satisfactory weight gain with food intake exclusively per oral. Although further studies are required, there is strong evidence that MMC is a safe and effective adjuvant therapy in the treatment of esophageal caustic stenosis. © 2008 The Authors.

Induction and secretion of elastinolytic and proteolytic activity in cultures of Paracoccidioides brasiliensis

P. brasiliensis parasitizes various human tissues and proteinases exported by this fungus may allow it to metabolize and invade host tissues. The influence of the culture medium on the production of proteinases by P. brasiliensis isolates was studied and the export of these enzymes was followed as a function of culture time. The fungus was grown in neopeptone, BSA, elastin or collagen medium. The culture medium was assayed for azocollytic, elastinolytic and caseinolytic activity. Proteolytic activity was also analysed by electrophoresis of the culture medium on gelatin and casein substrate gels. P. brasiliensis expressed relatively high levels of azocoll, elastin and casein degrading activity in all types of medium, except in neopeptone medium. Generally, expression of azocollytic activity peaked during the third week of culture and caseinolytic activity during the fourth week of culture. Azocollytic activity was highest at pH 4.0 and caseinolytic activity at pH 8.0. Elastinolytic activity was also highest at pH 8.0. This activity, as well as the others, may provide the fungus with a source of carbon and nitrogen and may also be responsible for the invasion of host tissues, such as pulmonary elastic fiber, by P. brasiliensis.

Degradação ruminal e síntese de proteína microbiana em bovines alimentados com silagem de sorgo contendo tanino suplementado com concentrado ou uréia

This work aimed to determine ruminal parameters of dry mater (DM), neutral detergent fiber (NDF) and protein synthesis in the rumen of animals fed diets based on sorghum with different tannin levels. In situ degradation was evaluated by rumen incubation of level 1 (L1) and level 2 (L2) sorghum silages during 0, 6, 24, 48 and 96 hours. A duplicated 4 × 4 Latin Square was used. Potential degradability (PD) of DM was similar among silages, whereas the effective degradability (ED) decreased when the diets were supplemented with concentrate. Microbial nitrogen flux and microbial synthesis efficiency were not affected by roughage source. The synthesis efficiency, expressed in organic matter and crude protein digested in rumen, was higher in L1 tannin diets supplemented with concentrate. There was not relationship between the presence of tannins and the parameters of ruminal degradation.

Mast cells modulate the inflammatory process in endotoxin-induced uveitis

Purpose: To investigate the role of mast cells and annexin-A1 (Anxa1) in endotoxin-induced uveitis (EIU). Methods: EIU was induced by injection of lipopolysaccharide (LPS) into the paws of rats, which were then sacrificed after 24 and 48 h. To assess EIU in the absence of mast cells, groups of animals were pretreated with compound 48/80 (c48/80) and sacrificed after 24 h after no treatment or EIU induction. The eyes were used for histological studies and the aqueous humor (AqH) pool was used for the analysis of transmigrated cells and Anxa1 levels. In inflammatory cells, Anxa1 expression was monitored by immunohistochemistry. Results: After 24 h, rats with EIU exhibited degranulated mast cells, associated with elevated numbers of infiltrating leukocytes and the high expression of Anxa1 in the AqH and the neutrophils. After 48 h of EIU, the mast cells were intact, indicating granule re-synthesis, and there was a reduction of neutrophil transmigration and an increase in the number of mononuclear phagocytic cells in ocular tissues. Anxa1 expression was decreased in neutrophils but increased in mononuclear phagocytic cells. In the animals pretreated with c48/80 and subjected to EIU, mast cells responded to this secretagogue by degranulating and few transmigrated neutrophils were observed. Conclustions: We report that mast cells are a potential source of pharmacological mediators that are strongly linked to the pathophysiology of EIU, and the endogenous protein Anxa1 is a mediator in the homeostasis of the inflammatory process with anti-migratory effects on leukocytes, which supports further studies of this protein as an innovative therapy for uveitis. © 2011 Molecular Vision.

Biostimulatory effect of low-level laser therapy on keratinocytes in vitro

Epithelial cells play an important role in reparative events. Therefore, therapies that can stimulate the proliferation and metabolism of these cells could accelerate the healing process. To evaluate the effects of low-level laser therapy (LLLT), human keratinocytes were irradiated with an InGaAsP diode laser prototype (LASERTable; 780 ± 3 nm; 40 mW) using 0.5, 1.5, 3, 5, and 7 J/cm2 energy doses. Irradiations were done every 24 h totaling three applications. Evaluation of cell metabolism (MTT assay) showed that LLLT with all energy doses promoted an increase of cell metabolism, being more effective for 0.5, 1.5, and 3 J/cm2. The highest cell counts (Trypan blue assay) were observed with 0.5, 3, and 5 J/cm2. No statistically significant difference for total protein (TP) production was observed and cell morphology analysis by scanning electron microscopy revealed that LLLT did not promote morphological alterations on the keratinocytes. Real-time polymerase chain reaction (qPCR) revealed that LLLT also promoted an increase of type I collagen (Col-I) and vascular endothelial growth factor (VEGF) gene expression, especially for 1.5 J/cm2, but no change on fibroblast growth factor-2 (FGF-2) expression was observed. LLLT at energy doses ranging from 0.5 to 3 J/cm2 promoted the most significant biostimulatory effects on cultured keratinocytes. © 2012 Springer-Verlag London Ltd.

Regulation of visfatin by microbial and biomechanical signals in PDL cells

Objectives: This in vitro study was established to examine whether visfatin thought to be a link between periodontitis and obesity is produced by periodontal ligament (PDL) cells and, if so, whether its synthesis is modulated by microbial and/or biomechanical signals. Materials and methods: PDL cells seeded on BioFlex® plates were exposed to the oral pathogen Fusobacterium nucleatum ATCC 25586 and/or subjected to biomechanical strain for up to 3 days. Gene expression of visfatin and toll-like receptors (TLR) 2 and 4 was analyzed by RT-PCR, visfatin protein synthesis by ELISA and immunocytochemistry, and NFκB nuclear translocation by immunofluorescence. Results: F. nucleatum upregulated the visfatin expression in a dose- and time-dependent fashion. Preincubation with neutralizing antibodies against TLR2 and TLR4 caused a significant inhibition of the F. nucleatum-upregulated visfatin expression at 1 day. F. nucleatum stimulated the NFκB nuclear translocation. Biomechanical loading reduced the stimulatory effects of F. nucleatum on visfatin expression at 1 and 3 days and also abrogated the F. nucleatum-induced NFκB nuclear translocation at 60 min. Biomechanical loading inhibited significantly the expression of TLR2 and TLR4 at 3 days. The regulatory effects of F. nucleatum and/or biomechanical loading on visfatin expression were also observed at protein level. Conclusions: PDL cells produce visfatin, and this production is enhanced by F. nucleatum. Biomechanical loading seems to be protective against the effects of F. nucleatum on visfatin expression. Clinical relevance: Visfatin produced by periodontal tissues could play a major role in the pathogenesis of periodontitis and the interactions with obesity and other systemic diseases. © 2013 Springer-Verlag Berlin Heidelberg.

Cyp17a1 and cyp19a1 in the zebrafish testis are differentially affected by oestradiol

Oestrogens can affect expression of genes encoding steroidogenic enzymes in fish gonads. However, little information is available on their effects at the protein level. In this context, we first analysed the expression of key steroidogenic enzyme genes and proteins in zebrafish testis, paying attention also to other cell types than Leydig cells. Gene expression was analysed by quantitative PCR on fluorescence-activated cell-sorting fractions coupled or not to differential plating, while protein synthesis was studied by immunohistochemistry using specific antibodies against zebrafish Cyp17a1, Cyp19a1a and Cyp19a1b. Furthermore, we have evaluated the effect of oestrogen treatment (17β-oestradiol (E2), 10 nM) on the localization of these enzymes after 7 and 14 days of in vivo exposure in order to study how oestrogen-mediated modulation of their expression is linked to oestrogen effects on spermatogenesis. The major outcomes of this study are that Leydig cells express Cyp17a1 and Cyp19a1a, while testicular germ cells express Cyp17a1 and both, Cyp19a1a and Cyp19a1b. As regards Cyp17a1, both protein and mRNA seem to be quantitatively dominating in Leydig cells. Moreover, E2 exposure specifically affects only Leydig cell Cyp17a1 synthesis, preceding the disruption of spermatogenesis. The oestrogen-induced suppression of the androgen production capacity in Leydig cells is a major event in altering spermatogenesis, while germ cell steroidogenesis may have to be fuelled by precursors from Leydig cells. Further studies are needed to elucidate the functionality of steroidogenic enzymes in germ cells and their potential role in testicular physiology. © 2013 Society for Endocrinology.

The Deoxyhypusine Synthase Mutant dys1-1 Reveals the Association of eIF5A and Asc1 with Cell Wall Integrity

The putative eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein among archaea and eukaryotes that has recently been implicated in the elongation step of translation. eIF5A undergoes an essential and conserved posttranslational modification at a specific lysine to generate the residue hypusine. The enzymes deoxyhypusine synthase (Dys1) and deoxyhypusine hydroxylase (Lia1) catalyze this two-step modification process. Although several Saccharomyces cerevisiae eIF5A mutants have importantly contributed to the study of eIF5A function, no conditional mutant of Dys1 has been described so far. In this study, we generated and characterized the dys1-1 mutant, which showed a strong depletion of mutated Dys1 protein, resulting in more than 2-fold decrease in hypusine levels relative to the wild type. The dys1-1 mutant demonstrated a defect in total protein synthesis, a defect in polysome profile indicative of a translation elongation defect and a reduced association of eIF5A with polysomes. The growth phenotype of dys1-1 mutant is severe, growing only in the presence of 1 M sorbitol, an osmotic stabilizer. Although this phenotype is characteristic of Pkc1 cell wall integrity mutants, the sorbitol requirement from dys1-1 is not associated with cell lysis. We observed that the dys1-1 genetically interacts with the sole yeast protein kinase C (Pkc1) and Asc1, a component of the 40S ribosomal subunit. The dys1-1 mutant was synthetically lethal in combination with asc1Δ and overexpression of TIF51A (eIF5A) or DYS1 is toxic for an asc1Δ strain. Moreover, eIF5A is more associated with translating ribosomes in the absence of Asc1 in the cell. Finally, analysis of the sensitivity to cell wall-perturbing compounds revealed a more similar behavior of the dys1-1 and asc1Δ mutants in comparison with the pkc1Δ mutant. These data suggest a correlated role for eIF5A and Asc1 in coordinating the translational control of a subset of mRNAs associated with cell integrity. © 2013 Galvão et al.

Platelet lysate 3D scaffold supports mesenchymal stem cell chondrogenesis: An improved approach in cartilage tissue engineering

Articular lesions are still a major challenge in orthopedics because of cartilage's poor healing properties. A major improvement in therapeutics was the development of autologous chondrocytes implantation (ACI), a biotechnology-derived technique that delivers healthy autologous chondrocytes after in vitro expansion. To obtain cartilage-like tissue, 3D scaffolds are essential to maintain chondrocyte differentiated status. Currently, bioactive 3D scaffolds are promising as they can deliver growth factors, cytokines, and hormones to the cells, giving them a boost to attach, proliferate, induce protein synthesis, and differentiate. Using mesenchymal stem cells (MSCs) differentiated into chondrocytes, one can avoid cartilage harvesting. Thus, we investigated the potential use of a platelet-lysate-based 3D bioactive scaffold to support chondrogenic differentiation and maintenance of MSCs. The MSCs from adult rabbit bone marrow (n=5) were cultivated and characterized using three antibodies by flow cytometry. MSCs (1×105) were than encapsulated inside 60μl of a rabbit platelet-lysate clot scaffold and maintained in Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 supplemented with chondrogenic inductors. After 21 days, the MSCs-seeded scaffolds were processed for histological analysis and stained with toluidine blue. This scaffold was able to maintain round-shaped cells, typical chondrocyte metachromatic extracellular matrix deposition, and isogenous group formation. Cells accumulated inside lacunae and cytoplasm lipid droplets were other observed typical chondrocyte features. In conclusion, the usage of a platelet-lysate bioactive scaffold, associated with a suitable chondrogenic culture medium, supports MSCs chondrogenesis. As such, it offers an alternative tool for cartilage engineering research and ACI. © 2013 Informa UK Ltd.

Digestibility, fermentation and rumen microbiota of crossbred heifers fed diets with different soybean oil availabilities in the rumen

The goal of this study was to evaluate the effects of different soybean oil availabilities on the intake and partial and total digestibility of dry matter (DM) and nutrients, rumen fermentation parameters, efficiency of microbial synthesis, and the rumen microbiota of crossbred beef heifers. Nine crossbred heifers fitted with rumen and duodenal cannulae were evaluated in a triple 3 × 3 Latin square design with three treatments and three periods in three simultaneous repetitions. Heifers approximately 18 months old, with mean initial and final body weights of 316.3±28.8 and 362.6±34.4 kg, respectively, were fed a diet containing 600. g/kg of corn silage and 400. g/kg concentrate with a 58.0. g/kg fat content in the total diet. The sources of lipids included soybean grain, rumen-protected fat, and soybean oil. The statistical analyses were conducted using PROC MIXED from SAS, and the means were compared using Tukey's test (P<0.05). Dietary lipid sources did not affect nutrient intake (P>0.05). Except the apparent digestibility of organic matter (P=0.024), the apparent digestibility of the other nutrients did not differ among the treatment groups. Regarding body nitrogen retention, the soybean grain treatment was more effective than the rumen-protected fat treatment (P=0.045); however, the soybean oil treatment did not differ from the other two treatments. In relation to the efficiency of microbial protein synthesis (g. N/kg of organic matter apparently digested in the rumen corrected for microbial organic matter), the soybean oil and soybean grain treatments were more efficient than the rumen-protected fat treatment (P=0.001). Animals fed rumen-protected fat had larger numbers of protozoa (P<0.001) and fungi (P<0.001) than those supplemented with soybean grain and soybean oil. The dietary lipid sources did not affect pH, the molar concentration of propionate and total volatile fatty acids (P>0.05), whereas the concentrations of ammonia nitrogen and acetate were higher in animals fed with rumen-protected fat than in those submitted to the other treatments. The use of different soybean oil availabilities did not affect nutrient intake; however, treatments with soybean oil and soybean grain were more efficient regarding nutrient intake than rumen-protected fat because they reduced the numbers of fungi and protozoa and consequently improved the efficiency of microbial protein synthesis. © 2013 Elsevier B.V.

Effects of zoledronic acid on odontoblast-like cells

The aim of the study was to evaluate the effects of a highly potent bisphosphonate, zoledronic acid (ZOL), on cultured odontoblast-like cells MDPC-23. The cells (1.5 × 104 cells/cm2) were seeded for 48 h in wells of 24-well dished. Then, the plain culture medium (DMEM) was replaced by fresh medium without fetal bovine serum. After 24 h, ZOL (1 or 5 μM) was added to the medium and maintained in contact with the cells for 24 h. After this period, the succinic dehydrogenase (SDH) enzyme production (cell viability-MTT assay), total protein (TP) production, alkaline phosphatase (ALP) activity, and gene expression (qPCR) of collagen type I (Col-I) and ALP were evaluated. Cell morphology was assessed by SEM. Five μM ZOL caused a significant decrease in SDH production. Both ZOL concentrations caused a dose-dependent significant decrease in TP production and ALP activity. ZOL also produced discret morphological alterations in the MDPC-23 cells. Regarding gene expression, 1 μM ZOL caused a significant increase in Col-I expression. Although 5 μM ZOL did not affect Col-I expression, it caused a significant alteration in ALP expression (ANOVA and Tukey's test, p < 0.05). ZOL presented a dose-dependent cytotoxic effect on the odontoblast-like cells, suggesting that under clinical conditions the release of this drug from dentin could cause damage to the pulpo-dentin complex. © 2012 Elsevier Ltd.

Polymorphisms in the ghrelin gene and their associations with milk yield and quality in water buffaloes

Ghrelin is a gastrointestinal hormone that acts in releasing growth hormone and influences the body general metabolism. It has been proposed as a candidate gene for traits such as growth, carcass quality, and milk production of livestock because it influences feed intake. In this context, the aim of this study was to verify the existence of polymorphisms in the ghrelin gene and their associations with milk, fat and protein yield, and percentage in water buffaloes (Bubalus bubalis). A group of 240 animals was studied. Five primer pairs were used and 11 single nucleotide polymorphisms (SNP) were found in the ghrelin gene by sequencing. The animals were genotyped for 8 SNP by PCR-RFLP. The SNP g.960G>A and g.778C>T were associated with fat yield and the SNP g.905T>C was associated with fat yield and percentage and protein percentage. These SNP are located in intronic regions of DNA and may be in noncoding RNA sites or affect transcriptional efciency. The ghrelin gene in buffaloes influences milk fat and protein synthesis. The polymorphisms observed can be used as molecular markers to assist selection. © 2013 American Dairy Science Association.