Soil bacterial phoD gene abundance and expression in response to applied phosphorus and long-term management

Bacterial transformation of phosphorus (P) compounds in soil is largely dependent on soil microbial community function, and is therefore sensitive to anthropogenic disturbances such as fertilization or cropping systems. However, the effect of soil management on the transcription of bacterial genes that encode phosphatases, such as phoD, is largely unknown. This greenhouse study examined the effect of long-term management and P amendment on potential alkaline phosphatase (ALP) activity and phoD gene (DNA) and transcript (RNA) abundance. Soil samples (0–15 cm) were collected from the Glenlea Long-term Rotation near Winnipeg, Manitoba, to compare organic, conventional and prairie management systems. In the greenhouse, pots of soil from each management system were amended with P as either soluble mineral fertilizer or cattle manure and then planted with Italian ryegrass (Lolium multiforum). Soils from each pot were sampled for analysis immediately and after 30 and 106 days. Significant differences among the soil/P treatments were detected for inorganic P, but not the organic P in NaHCO3-extracts. At day 0, ALP activity was similar among the soil/P treatments, but was higher after 30 days for all P amendments in soil from organically managed plots. In contrast, ALP activity in soils under conventional and prairie management responded to increasing rates of manure only, with significant effects from medium and high manure application rates at 30 and 106 days. Differences in ALP activity at 30 days corresponded to the abundance of bacterial phoD genes, which were also significantly higher in soils under organic management. However, this correlation was not significant for transcript abundance. Next-generation sequencing allowed the identification of 199 unique phoD operational taxonomic units (OTUs) from the metagenome (soil DNA) and 35 unique OTUs from the metatranscriptome (soil RNA), indicating that a subset of phoD genes was being transcribed in all soils.

In vitro osteogenesis on fluorcanasite glass-ceramic with three different chemical compositions

This study aimed at investigating in vitro osteogenesis on three fluorcanasite glass-ceramic compositions with different solubilities (K3, K5, and K8). Osteoblastic cells were obtained from human alveolar bone fragments and cultured under standard osteogenic condition until subconfluence. First passage cells were cultured on K3, K5, and K8 and on Bioglass (R) 45S5 (45S5-control). Cell adhesion was evaluated at 24 h. For proliferation and viability, cells were cultured for 1, 4, and 10 days. Total protein content and alkaline phosphatase (ALP) activity were measured at 7, 14, and 21 days. Cultures were stained with Alizarin red at 21 days, for detection of mineralized matrix. Data were compared by ANOVA followed by Duncan`s test. Cell adhesion, cell proliferation, viability, total protein content, and ALP activity were not affected by fluorcanasite glass-ceramic composition and solubility. Bone-like formation was similar on all fluorcanasite-glass ceramics and was reduced compared to 45S5. The changes in the chemical composition and consequently solubility of the fluorcanasite glass-ceramics tested here did not significantly alter the in vitro osteogenesis. Further modifications of the chemical composition of the fluorcanasite glass-ceramic would be required to improve bone response, making this biomaterial a good candidate to be employed as a bone substitute.

TNF-alpha Promotes an Odontoblastic Phenotype in Dental Pulp Cells

Dental pulp cells can differentiate toward an odontoblastic phenotype to produce reparative dentin beneath caries lesions. However, the mechanisms involved in pulp cell differentiation under pro-inflammatory stimuli have not been well-explored. Thus, we hypothesized that the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) could be a mediator involved in dental pulp cell differentiation toward an odontoblastic phenotype. We observed that TNF-alpha-challenged pulp cells exhibited increased mineralization and early and increased expression of dentin phosphoprotein (DPP), dentin sialoprotein (DSP), dentin matrix protein-1, and osteocalcin during a phase of reduced matrix metalloproteinase (MMP) expression. We investigated whether these events were related and found that p38, a mitogen-activated protein kinase, differentially regulated MMP-1 and DSP/DPP expression and mediated mineralization upon TNF-alpha treatment. These findings indicate that TNF-alpha stimulates differentiation of dental pulp cells toward an odontoblastic phenotype via p38, while negatively regulating MMP-1 expression.

Effects of the Association between a Calcium Hydroxide Paste and 0.4% Chlorhexidine on the Development of the Osteogenic Phenotype In Vitro

The present study aimed to evaluate whether the association between a calcium hydroxide paste (Calen paste) and 0.4% chlorhexidine (CHX) affects the development of the osteogenic phenotype in vitro. With rat calvarial osteogenic cell cultures, the following parameters were assayed: cell morphology and viability, alkaline phosphatase activity, total protein content, bone sialoprotein immunolocalization, and mineralized nodule formation. Comparisons were carried out by using the nonparametric Kruskal-Wallis test (level of significance, 5%). The results showed that the association between Calen paste and 0.4% CHX did not affect the development of the osteogenic phenotype. No significant changes were observed in terms of cell shape, cell viability, alkaline phosphatase activity, and the total amount of bone-like nodule formation among control, Calen, or Calen + CHX groups. The strategy to combine Ca(OH)(2) and CHX to promote a desirable synergistic antibacterial effect during endodontic treatment in vivo might not significantly affect osteoblastic cell biology. (J Endod 2008;34:1485-1489)

Effects of Cadmium on the Rat Salivary Glands, During Lactation

Cadmium (Cd) in air, drinking water and food has the potential to affect the health of people, mainly those who live in highly industrialized regions. Cd affects placental function, can cross the placental barrier and directly modify fetal development. Once the organism is particularly susceptible to the exposition to the Cd during the perinatal period, and that this metal can be excreted in the milk, the aim of the present work was to study the effects of the constant exposition to drinkable water containing low levels of Cd during the lactation, on the salivary glands of the rat. Female rats received ad libitum drinking water containing 300mg/l of CdCl2 throughout the whole lactation. Control animals received a similar volume of water without Cd. Lactant rats (21 day old) were killed by lethal dose of anesthetic. The salivary glands were separated, fixed in ""alfac"" solution for 24 h, and serially sectioned. The 6 mu m thick sections were stained with hematoxylin and eosin. Nuclear glandular parameters were estimated, as well as cytoplasm and cell volume, nucleus/cytoplasm ratio, number and surface density, diameters and cell thickness. Mean body weight was 34.86 g for the control group and 18.56 g for the Cd-treated group. Histologically, the glandular acini were significantly smaller, the gland ducts were similar in both groups studied. The connective tissue was more abundant. In conclusion, the salivary glands (submandibular, parotid and sublingual) showed retarded growth after Cd intoxication.

Immunohistochemical, tomographic and histological study on onlay bone graft remodeling. Part II: calvarial bone

Objectives Little information is available on the molecular events that occur during graft incorporation over time. The calvarial bone (Cb) grafts have been reported to produce greater responses compared with other donor regions in maxillofacial reconstructions, but the scientific evidences for this are still lacking. The objectives of this study are (1) to study the morphological pattern of Cb onlay bone grafts and compare them with the biological events through immunohistochemical responses and (2) to establish the effects of perforations in maintaining the volume and bone density of the receptor bed. Material and methods Sixty New Zealand White rabbits were submitted to Cb onlay bone grafts on the mandible. In 30 rabbits, the receptor bed was perforated (perforated group), while for the remaining animals the bed was kept intact (non-perforated group). Six animals from each group were sacrificed at 5, 7, 10, 20 and 60 days after surgery. Histological sections from the grafted area were prepared for immunohistochemical and histological analyses. Immuno-labeling was found for proteins Osteoprotegerin (OPG), receptor activator of nuclear factor-kappa beta ligand (RANKL), alkaline phosphatase (ALP), osteopontin (OPN), vascular endothelial growth factor (VEGF), tartrate-resistant acid phosphatase (TRAP), Type I collagen (COL I) and osteocalcin (OC). The tomography examination [computerized tomography (CT) scan] was conducted just after surgery and at the sacrifice. Results The histological findings revealed that the perforations contributed to higher bone deposition during the initial stages at the graft-receptor bed interface, accelerating the graft incorporation process. The results of the CT scan showed lower resorption for the perforated group (P < 0.05), and both groups showed high bone density rates at 60 days. This set of evidences is corroborated by the immunohistochemical outcomes indicating that proteins associated with revascularization and osteogenesis (VEGF, OPN, TRAP and ALP) were found in higher levels in the perforated group. Conclusions These findings indicate that the bone volume of calvarial grafts is better maintained when the receptor bed is perforated, probably resulting from more effective graft revascularization and greater bone deposition. The process of bone resorption peaked between 20 and 60 days post-operatively in both groups although significantly less in the perforated group. To cite this article:Pedrosa Jr WF, Okamoto R, Faria PEP, Arnez MFM, Xavier SP, Salata LA. Immunohistochemical, tomographic and histological study on onlay bone grafts remodeling. Part II: calvarial bone.Clin. Oral Impl. Res. 20, 2009; 1254-1264.doi: 10.1111/j.1600-0501.2009.01747.x.

Yerba Mate (Ilex paraguariensis) Aqueous Extract Decreases Intestinal SGLT1 Gene Expression but Does Not Affect Other Biochemical Parameters in Alloxan-Diabetic Wistar Rats

Yerba mate (Ilex paraguariensis) is rich in polyphenols, especially chlorogenic acids. Evidence suggests that dietary polyphenols could play a role in glucose absorption and metabolism. The aim of this study was to evaluate the antidiabetic properties of yerba mate extract in alloxan-induced diabetic Wistar rats. Animals (n = 41) were divided in four groups: nondiabetic control (NDC, n = 10), nondiabetic yerba mate (NDY, n = 10), diabetic control (DC, n = 11), and diabetic yerba mate (NDY, n = 10). The intervention consisted in the administration of yerba mate extract in a 1 g extract/kg body weight dose for 28 days; controls received saline solution only. There were no significant differences in serum glucose, insulin, and hepatic glucose-6-phosphatase activity between the groups that ingested yerba mate extract (NDY and DY) and the controls (NDC and DC). However, the intestinal SGLT1 gene expression was significantly lower in animals that received yerba mate both in upper (p = 0.007) and middle (p < 0.001) small intestine. These results indicate that bioactive compounds present in yerba mate might be capable of interfering in glucose absorption, by decreasing SGLT1 expression.

Endothelial and inducible nitric oxide synthases in oocytes of cattle

Nitric oxide (NO) is a chemical messenger generated by the activity of the nitric oxide synthases (NOS). The NOS/NO system appears to be involved in oocyte maturation, but there are few studies on gene expression and protein activity in oocytes of cattle. The present study aimed to investigate gene expression and protein activity of NOS in immature and in vitro matured oocytes of cattle. The influence of pre-maturation culture with butyrolactone I in NOS gene expression was also assessed. The following experiments were performed: (1) detection of the endothelial (eNOS) and inducible (iNOS) isoforms in the ovary by immunohistochemistry; (2) detection of eNOS and iNOS in the oocytes before and after in vitro maturation (W) by immunofluorescence; (3) eNOS and iNOS mRNA and protein in immature and in vitro matured oocytes, with or without pre-maturation, by real time PCR and Western blotting, respectively; and (4) NOS activity in immature and in vitro matured oocytes by NADPH-diaphorase. eNOS and iNOS were detected in oocytes within all follicle categories (primary, secondary and tertiary), and other compartments of the ovary and in the cytoplasm of immature and in vitro matured oocytes. Amount of mRNA for both isoforms decreased after IVM but was maintained after pre-maturation culture. The NOS protein was detected in immature (pre-mature or not) and was still detected in similar amount after pre-maturation and maturation for both isoforms. NOS activity was detected only in part of the immature oocytes. In conclusion, isoforms of NOS (eNOS and iNOS) are present in oocytes of cattle from early folliculogenesis up to maturation; in vitro maturation influences amount of mRNA and NOS activity. (C) 2009 Elsevier B.V. All rights reserved.

Light modulates the melanophore response to alpha-MSH in Xenopus laevis: An analysis of the signal transduction crosstalk mechanisms involved

Melanin granule (melanosome) dispersion within Xenopus laevis melanophores is evoked either by light or alpha-MSH. We have previously demonstrated that the initial biochemical steps of light and alpha-MSH signaling are distinct, since the increase in cAMP observed in response to alpha-MSH was not seen after light exposure. cAMP concentrations in response to alpha-MSH were significantly lower in cells pre-exposed to light as compared to the levels in dark-adapted melanophores. Here we demonstrate the presence of an adenylyl cyclase (AC) in the Xenopus melanophore, similar to the mammalian type IX which is inhibited by Ca(2+)-calmodulin-activated phosphatase. This finding supports the hypothesis that the cyclase could be negatively modulated by a light-promoted Ca(2+) increase. In fact, the activity of calcineurin PP2B phosphatase was increased by light, which could result in AC IX inhibition, thus decreasing the response to alpha-MSH. St-Ht31, a disrupting agent of protein kinase A (PKA)-anchoring kinase A protein (AKAP) complex totally blocked the melanosome dispersing response to alpha-MSH, but did not impair the photo-response in Xenopus melanophores. Sequence comparison of a melanophore AKAP partial clone with GenBank sequences showed that the anchoring protein was a gravin-like adaptor previously sequenced from Xenopus non-pigmentary tissues. Co-immunoprecipitation of Xenopus AKAP and the catalytic subunit of PKA demonstrated that PKA is associated with AKAP and it is released in the presence of alpha-MSH. We conclude that in X laevis melanophores, AKAP12 (gravin-like) contains a site for binding the inactive PKA thus compartmentalizing PKA signaling and also possesses binding sites for PKC. Light diminishes alpha-MSH-induced increase of cAMP by increasing calcineurin (PP2B) activity, which in turn inhibits adenylyl cyclase type IX, and/or by activating PKC, which phosphorylates the gravin-like molecule, thus destabilizing its binding to the cell membrane. (C) 2009 Elsevier Inc. All rights reserved.

Metabolic recovery of adipose tissue is associated with improvement in insulin resistance in a model of experimental diabetes

Obesity and insulin resistance are highly correlated with metabolic disturbances. Both the excess and lack of adipose tissue can lead to severe insulin resistance and diabetes. Adipose tissue plays an active role in energy homeostasis, hormone secretion, and other proteins that affect insulin sensitivity, appetite, energy balance, and lipid metabolism. Rats with streptozotocin-induced diabetes during the neonatal period develop the classic diabetic picture of hyperglycemia, hypoinsulinemia, and insulin resistance in adulthood. Low body weight and reduced epididymal (EP) fit mass were also seen in this model. The am) of this study was to investigate the glucose homeostasis and metabolic repercussions on the adipose tissue following chronic treatment with antidiabetic drugs in these animals. In the 4th week post birth, diabetic animals started an 8-week treatment with pioglitazone, metformin, or insulin.

Intensive insulin treatment induces insulin resistance in diabetic rats by impairing glucose metabolism-related mechanisms in muscle and liver

Insulin replacement is the only effective therapy to manage hyperglycemia in type 1 diabetes mellitus (T1DM). Nevertheless, intensive insulin therapy has inadvertently led to insulin resistance. This study investigates mechanisms involved in the insulin resistance induced by hyperinsulinization. Wistar rats were rendered diabetic by alloxan injection, and 2 weeks later received saline or different doses of neutral protamine Hagedorn insulin (1.5, 3, 6, and 9 U/day) over 7 days. Insulinopenic-untreated rats and 6U- and 9U-treated rats developed insulin resistance, whereas 3U-treated rats revealed the highest grade of insulin sensitivity, but did not achieve good glycemic control as 6U- and 9U-treated rats did. This insulin sensitivity profile was in agreement with glucose transporter 4 expression and translocation in skeletal muscle, and insulin signaling, phosphoenolpyruvate carboxykinase/glucose-6-phosphatase expression and glycogen storage in the liver. Under the expectation that insulin resistance develops in hyperinsulinized diabetic patients, we believe insulin sensitizer approaches should be considered in treating T1DM. Journal of Endocrinology (2011) 211, 55-64

MKP-1 mediates glucocorticoid-induced ERK1/2 dephosphorylation and reduction in pancreatic beta-cell proliferation in islets from early lactating mothers

Maternal pancreatic islets undergo a robust increase of mass and proliferation during pregnancy, which allows a compensation of gestational insulin resistance. Studies have described that this adaptation switches to a low proliferative status after the delivery. The mechanisms underlying this reversal are unknown, but the action of glucocorticoids (GCs) is believed to play an important role because GCs counteract the pregnancy-like effects of PRL on isolated pancreatic islets maintained in cell culture. Here, we demonstrate that ERK1/2 phosphorylation (phospho-ERK1/2) is increased in maternal rat islets isolated on the 19th day of pregnancy. Phospho-ERK1/2 status on the 3rd day after delivery (L3) rapidly turns to values lower than that found in virgin control rats (CTL). MKP-1, a protein phosphatase able to dephosphorylate ERK1/2, is increased in islets from L3 rats. Chromatin immunoprecipitation assay revealed that binding of glucocorticoid receptor (GR) to MKP-1 promoter is also increased in islets from L3 rats. In addition, dexamethasone (DEX) reduced phospho-ERK1/2 and increased MKP-1 expression in RINm5F and MIN-6 cells. Inhibition of transduction with cycloheximide and inhibition of phosphatases with orthovanadate efficiently blocked DEX-induced downregulation of phospho-ERK1/2. In addition, specific knockdown of MKP-1 with siRNA suppressed the downregulation of phosphoERK1/2 and the reduction of proliferation induced by DEX. Altogether, our results indicate that downregulation of phospho-ERK1/2 is associated with reduction in proliferation found in islets of early lactating mothers. This mechanism is probably mediated by GC-induced MKP-1 expression.

Smad5 regulates Akt2 expression and insulin-induced glucose uptake in L6 myotubes

Insulin-induced glucose uptake by skeletal muscle results from Akt2 activation and is severely impaired during insulin resistance Recently, we and others have demonstrated that BMP9 improves glucose homeostasis in diabetic and non-diabetic rodents. However, the mechanism by which BMP9 modulates insulin action remains unknown. Here we demonstrate that Smad5. a transcription factor activated by BMP9, and Akt2. are upregulated in differentiated L6 myotubes. Smad5, rather than Smad1/8, is downregulated ""in vivo"" and ""in vitro"" by dexamethasone Smad5 knockdown decreased Akt2 expression and serine phosphorylation and insulin-induced glucose uptake, and increased the expression of the lipid phosphatase Ship2. Additionally, binding of Smad5 to Akt2 gene is decreased in dexamethasone-treated rats and Increased in L6 myotubes compared to myoblasts The present study indicates that Smad5 regulates glucose uptake in skeletal muscle by controlling Akt2 expression and phosphorylation These finding reveals Smad5 as a potential target for the therapeutic of type 2 diabetes. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Hypermethioninemia provokes oxidative damage and histological changes in liver of rats

In the present study we evaluated the effect of chronic methionine administration on oxidative stress and biochemical parameters in liver and serum of rats, respectively. We also performed histological analysis in liver. Results showed that hypermethioninemia increased chemiluminescence, carbonyl content and glutathione peroxidase activity, decreased total antioxidant potential, as well as altered catalase activity. Hypermethioninemia increased synthesis and concentration of glycogen, besides histological studies showed morphological alterations and reduction in the glycogen/glycoprotein content in liver. Serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and glucose were increased in hypermethioninemic rats. These findings suggest that oxidative damage and histological changes caused by methionine may be related to the hepatic injury observed in hypermethioninemia. (C) 2009 Elsevier Masson SAS. All rights reserved.

O-GlcNAcylation contributes to the vascular effects of ET-1 via activation of the RhoA/Rho-kinase pathway

Aims Glycosylation with beta-N-acetylglucosamine (O-GlcNAcylation) is one of the most complex post-translational modifications. The cycling of O-GlcNAc is controlled by two enzymes: UDP-NAc transferase (OGT) and O-GlcNAcase (OGA). We recently reported that endothelin-1 (ET-1) augments vascular levels of O-GlcNAcylated proteins. Here we tested the hypothesis that O-GlcNAcylation contributes to the vascular effects of ET-1 via activation of the RhoA/Rho-kinase pathway. Methods and results Incubation of vascular smooth muscle cells (VSMCs) with ET-1 (0.1 mu M) produces a time-dependent increase in O-GlcNAc levels. ET-1-induced O-GlcNAcylation is not observed when VSMCs are previously transfected with OGT siRNA, treated with ST045849 (OGT inhibitor) or atrasentan (ET(A) antagonist). ET-1 as well as PugNAc (OGA inhibitor) augmented contractions to phenylephrine in endothelium-denuded rat aortas, an effect that was abolished by the Rho kinase inhibitor Y-27632. Incubation of VSMCs with ET-1 increased expression of the phosphorylated forms of myosin phosphatase target subunit 1 (MYPT-1), protein kinase C-potentiated protein phosphatase 1 inhibitor protein (protein kinase C-potentiated phosphatase inhibitor-17), and myosin light chain (MLC) and RhoA expression and activity, and this effect was abolished by both OGT siRNA transfection or OGT inhibition and atrasentan. ET-1 also augmented expression of PDZ-Rho GEF (guanine nucleotide exchange factor) and p115-Rho GEF in VSMCs and this was prevented by OGT siRNA, ST045849, and atrasentan. Conclusion We suggest that ET-1 augments O-GlcNAcylation and this modification contributes to increased vascular contractile responses via activation of the RhoA/Rho-kinase pathway.