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.

Intermittent Therapy with 1,25 Vitamin D and Calcitonin Prevents Cyclosporin-Induced Alveolar Bone Loss in Rats

Bone loss associated with cyclosporin A (CsA) therapy can result in serious morbidity to patients. Intermittent administration of 1,25 Vitamin D and calcitonin reduces osteopenia in a murine model of postmenopausal osteoporosis. The purpose of this study was to evaluate the effects of this therapeutic approach on CsA-induced alveolar bone loss in rats. Forty male Wistar rats were allocated to four experimental groups according to the treatment received during 8 weeks: (1) CsA (10 mg/kg/day, s.c.); (2) 1,25 Vitamin D (2 mu g/kg, p.o.; in weeks 1, 3, 5, and 7) plus calcitonin (2 mu g/kg, i.p.; in weeks 2, 4, 6, and 8); (3) CsA concurrently with intermittent 1,25 Vitamin D and calcitonin administration; and (4) the control treatment group (vehicle). At the end of the 8-week treatment period, serum concentrations of bone-specific alkaline phosphatase, tartrate-resistant acid phosphatase (TRAP-5b), osteocalcin, interleukin (IL)-1 beta, IL-6, and tumor necrosis factor alpha (TNF-alpha) were measured and an analysis of bone volume, bone surface, number of osteoblasts, and osteoclasts was performed. CsA administration resulted in significant alveolar bone resorption, as assessed by a lower bone volume and an increased number of osteoclasts, and increased serum bone-specific alkaline phosphatase, TRAP-5b, IL-1 beta, IL-6, and TNF-alpha concentrations. The intermittent administration of calcitriol and calcitonin prevented the CsA-induced osteopenic changes and the increased serum concentrations of TRAP-5b and inflammatory cytokines. Intermittent calcitriol/calcitonin therapy prevents CsA-induced alveolar bone loss in rats and normalizes the production of associated inflammatory mediators.

Simvastatin therapy in cyclosporine A-induced alveolar bone loss in rats

Background and Objective: Cyclosporine A treatment is important in the therapy of a number of medical conditions; however, alveolar bone loss is an important negative side-effect of this drug. As such, we evaluated whether concomitant administration of simvastatin would minimize cyclosporine A-associated alveolar bone loss in rats subjected, or not, to experimental periodontal disease. Material and Methods: Groups of 10 rats each were treated with cyclosporine A (10 mg/kg/day), simvastatin (20 mg/kg/day), cyclosporine A and simvastatin concurrently (cyclosporine A/simvastatin) or vehicle for 30 days. Four other groups of 10 rats each received a cotton ligature around the lower first molar and were treated similarly with cyclosporine A, simvastatin, cyclosporine A/simvastatin or vehicle. Calcium (Ca(2+)), phosphorus and alkaline phosphatase levels were evaluated in serum. Expression levels of interleukin-1 beta, prostaglandin E(2) and inducible nitric oxide synthase were evaluated in the gingivomucosal tissues. Bone volume and numbers of osteoblasts and osteoclasts were also analyzed. Results: Treatment with cyclosporine A in rats, with or without ligature, was associated with bone loss, represented by a lower bone volume and an increase in the number of osteoclasts. Treatment with cyclosporine A was associated with bone resorption, whereas simvastatin treatment improved cyclosporine A-associated alveolar bone loss in all parameters studied. In addition, simvastatin, in the presence of inflammation, can act as an anti-inflammatory agent. Conclusion: This study shows that simvastatin therapy leads to a reversal of the cyclosporine A-induced bone loss, which may be mediated by downregulation of interleukin-1 beta and prostaglandin E(2) production.

The pst operon of enteropathogenic Escherichia coli enhances bacterial adherence to epithelial cells

Enteropathogenic Escherichia coli (EPEC) adheres in vivo and in vitro to epithelial cells. Two main adhesins, the bundle-forming pilus and intimin, encoded by the Up operon and eae, respectively, are responsible for the localized and the intimate adherence phenotypes. Deletion of the pst operon of EPEC abolishes the transport of inorganic phosphate through the phosphate-specific transport system and causes the constitutive expression of the PHO regulon genes. In the absence of pst there is a decrease in the expression of the main EPEC adhesins and a reduction in bacterial adherence to epithelial cells in vitro. This effect is not related to PHO constitutivity, because a Delta pst phoB double mutant that is defective in the transcription of the PHO genes also displayed low levels of adherence and expression of adhesins. Likewise, a PHO-constitutive phoR mutation did not affect bacterial adherence. The expression of the per operon, which encodes the Up and ler regulators PerA and PerC, is also negatively affected by the pst deletion. Overall, the data presented here demonstrate that the pst operon of EPEC plays a positive role in the bacterial adherence mechanism by increasing the expression of perA and perC and consequently the transcription of bfp and eae.

Toxicokinetics and toxicological effects of single oral dose of fumonisin B1 containing Fusarium verticillioides culture material in weaned piglets

Toxicokinetics and the toxicological effects of culture material containing fumonisin B(1) (FB(1)) were studied in male weaned piglets by clinical, pathological, biochemical and sphingolipid analyses. The animals received a single oral dose of 5 mg FB(1)/kg of body weight. obtained from Fusarium verticillioides culture material. FB(1) was detected by H PLC in plasma collected at 1-h intervals up to 6 h and at 12-h intervals up to 96 h. FB(1) eliminated in feces and urine was quantified over a 96-h period and in liver samples collected 96 h post-intoxication. Blood samples were obtained at the beginning and end of the experiment to determine serum enzyme activity, total bilirubin, cholesterol, sphinganine (Sa), sphingosine (So) and the Sa/So ratio. FB(1) was detected in plasma between 30 min and 36 h after administration. The highest concentration of FB(1) was observed after 2 h, with a mean concentration of 282 mu g/ml. Only 0.93% of the total FB(1) was detected in urine between 75 min and 41 h after administration, the highest mean concentration (561 mu g/ml) was observed during the interval after 8 at 24 h. Approximately 76.5% of FB(1) was detected in feces eliminated between 8 and 84 h after administration, with the highest levels observed between 8 and 24 h. Considering the biochemical parameters, a significant increase only occurred in cholesterol, alkaline phosphatase and aspartate aminotransferase activities. In plasma and urine, the highest Sa and Sa/So ratios were obtained at 12 and 48 h, respectively. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Acute toxicity of a single gavage dose of fumonisin B(1) in rabbits

The aim of this study was to determine the clinical, pathological and mycotoxicological effects of oral administration of fumonisin B, (FBI) in rabbits. Eighteen rabbits were randomly assigned to two experimental groups: control group, 0 mg FB(1): fumonisin group. 31.5 mg FB(1)/kg body weight, corresponding to about 630 mg FB(1)/kg diet. Fumonisin administered as a single oral dose to rabbits resulted in acute toxicity, significantly interfering with body and liver weight. Serum biochemical analysis revealed a significant increase of total protein, alkaline phosphatase (AP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyltransferase (GGT), urea and creatinine in the group receiving FBI compared to control animals, a finding characterizing hepatic and renal injury in this group. Urinary protein concentrations were markedly elevated at 12,24,48 and 72 h after dosing, although visible pathological abnormalities were not observed, probably because of rapid repair of the damage. FBI was detected in feces, with a maximum concentration at 24h after administration, indicating that the enterohepatic circulation is important in rabbits. FBI concentrations found in urine were low, with peak elimination at 12 h after intoxication. The highest FBI concentrations were observed in feces compared to urine and liver, demonstrating that feces are the main routes of excretion. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Transcriptional Processing of the pst Operon of Escherichia coli

The pst operon of Escherichia coli is composed of five genes that encode a high-affinity phosphate transport system. pst belongs to the PHO regulon, which is a group of genes and operons that are induced in response to phosphate limitation. The pst operon also has a regulatory role in the repression of PHO genes` transcription under phosphate excess conditions. Transcription of pst is initiated at the promoter located upstream to the first gene, pstS. Immediately after its synthesis, the primary transcript of pst is cleaved into shorter mRNA molecules in a ribonuclease E-dependent manner. Other ribonucleases, such as RNase III and MazF, do not play a role in pst mRNA processing. RNase E is thus at least partially responsible for processing the pst primary transcript.

Stability of the pstS transcript of Escherichia coli

The pst operon of Escherichia coli is composed of five genes that encode a high-affinity phosphate transport system. As a member of the PHO regulon, pst transcription is activated under phosphate shortage conditions. Under phosphate-replete conditions, the pst operon also functions as a negative regulator of the PHO genes. Transcription of pst is initiated at the promoter located upstream to the first gene, pstS. Immediately after its synthesis, the primary transcript of pst is cleaved into shorter mRNA molecules. The transcription unit corresponding to pstS is significantly more abundant than the transcripts of the other pst genes due to stabilisation of pstS mRNA by a repetitive extragenic palindrome (REP) structure downstream to the pstS locus. The presence of the REP sequence also results in an increased level of PstS proteins. However, the surplus level of PstS proteins produced in the presence of REP does not contribute to the repressive role of Pst in PHO expression.

Deiodinase-mediated thyroid hormone inactivation minimizes thyroid hormone signaling in the early development of fetal skeleton

Thyroid hormone (TH) plays a key role on post-natal bone development and metabolism, while its relevance during fetal bone development is uncertain. To Study this, pregnant once were made hypothyroid and fetuses harvested at embryonic days (E) 12.5, 14.5, 16.5 and 18.5. Despite a marked reduction in fetal tissue concentration of both T4 and T3, bone development, as assessed at the distal epiphyseal growth plate of the femur and vertebra, was largely preserved Lip to E16.5. Only at E18.5, the hypothyroid fetuses exhibited a reduction in femoral type I and type X collagen and osteocalcin mRNA levels, in the length and area of the proliferative and hypertrophic zones, in the number of chondrocytes per proliferative column, and in the number of hypertrophic chondrocyres, in addition to a slight delay in endochondral and intramembranous ossification. This Suggests that LIP to E 16.5, thyroid hormone signaling in bone is kept to a minimum. In fact, measuring the expression level of the activating and inactivating iodothyronine deiodinases (D2 and D3) helped understand how this is achieved. D3 mRNA was readily detected as early as E14.5 and its expression decreased markedly (similar to 10-fold) at E18.5, and even more at 14 days after birth (P14). In contrast. D2 mRNA expression increased significantly by E18.5 and markedly (similar to 2.5-fold) by P14. The reciprocal expression levels of D2 and D3 genes during early bone development along with the absence of a hypothyroidism-induced bone phenotype at this time Suggest that coordinated reciprocal deiodinase expression keeps thyroid hormone signaling in bone to very low levels at this early stage of bone development. (c) 2008 Elsevier Inc. All rights reserved.

The importance of hormone receptor analysis in osteosarcoma cells growth submitted to treatment with estrogen in association with thyroid hormone

Bone tumor incidence in women peaks at age 50-60, coinciding with the menopause. That estrogen (E2) and triiodothyronine (T3) interact in bone metabolism has been well established. However, few data on the action of these hormones are available. Our purpose was to determine the role of E2 and T3 in the expression of bone activity markers, namely alkaline phosphatase (AP) and receptor activator of nuclear factor kappa B ligand (RANKL). Two osteosarcoma cell lines: MG-63 (which has both estrogen (ER) and thyroid hormone (TR) receptors) and SaOs-29 (ER receptors only) were treated with infraphysiological E2 associated with T3 at infraphysiological, physiological, and supraphysiological concentrations. Real-time RT-PCR was used for expression analysis. Our results show that, in MG-63 cells, infraphysiological E2 associated with supraphysiological T3 increases AP expression and decreases RANKL expression, while infraphysiological E2 associated with either physiological or supraphysiological T3 decreases both AP and RANKL expression. On the other hand, in SaOs-2 cells, the same hormone combinations had no significant effect on the markers` expression. Thus, the analysis of hormone receptors was shown to be crucial for the assessment of tumor potential growth in the face of hormonal changes. Special care should be provided to patients with T3 and E2 hormone receptors that may increase tumor growth. Copyright (C) 2007 John Wiley & Sons, Ltd.

Alternative promoters in the pst operon of Escherichia coli

The pst operon of Escherichia coli is composed of five genes pstS, pstC, pstA, pstB and phoU, that encode a high-affinity phosphate transport system and a negative regulator of the PHO regulon. Transcription of pst is induced under phosphate shortage and is initiated at the promoter located upstream of the first gene of the operon, pstS. Here, we show by four different technical approaches the existence of additional internal promoters upstream of pstC, pstB and phoU. These promoters are not induced by Pi-limitation and do not possess PHO-box sequences. Plasmids carrying the pst internal genes partially complement chromosomal mutations in their corresponding genes, indicating that they are translated into functional proteins.

The Thyroid Hormone Receptor (TR) beta-Selective Agonist GC-1 Inhibits Proliferation But Induces Differentiation and TR beta mRNA Expression in Mouse and Rat Osteoblast-Like Cells

Previous studies showed anabolic effects of GC-1, a triiodothyronine (T3) analogue that is selective for both binding and activation functions of thyroid hormone receptor (TR) beta 1 over TR alpha 1, on bone tissue in vivo. The aim of this study was to investigate the responsiveness of rat (ROS17/2.8) and mouse (MC3T3-E1) osteoblast-like cells to GC-1. As expected, T3 inhibited cellular proliferation and stimulated mRNA expression of osteocalcin or alkaline phosphatase in both cell lineages. Whereas equimolar doses of T3 and GC-1 equally affected these parameters in ROS17/2.8 cells, the effects of GC-1 were more modest compared to those of T3 in MC3T3-E1 cells. Interestingly, we showed that there is higher expression of TR alpha 1 than TR beta 1 mRNA in rat (similar to 20-90%) and mouse (similar to 90-98%) cell lineages and that this difference is even higher in mouse cells, which highlights the importance of TR alpha 1 to bone physiology and may partially explain the modest effects of GC-1 in comparison with T3 in MC3T3-E1 cells. Nevertheless, we showed that TR beta 1 mRNA expression increases (similar to 2.8- to 4.3-fold) as osteoblastic cells undergo maturation, suggesting a key role of TR beta 1 in mediating T3 effects in the bone forming cells, especially in mature osteoblasts. It is noteworthy that T3 and GC-1 induced TR beta 1 mRNA expression to a similar extent in both cell lineages (similar to 2- to 4-fold), indicating that both ligands may modulate the responsiveness of osteoblasts to T3. Taken together, these data show that TR beta selective T3 analogues have the potential to directly induce the differentiation and activity of osteoblasts.

Chitosan as a removing agent of beta-lactoglobulin from membrane models

Many chitosan biological activities depend on the interaction with biomembranes, but so far it has not been possible to obtain molecular-level evidence of chitosan action. In this article, we employ Langmuir phospholipid monolayers as cell membrane models and show that chitosan is able to remove beta-lactoglobulin (BLG) from negatively charged dimyristoyl phosphatidic acid (DMPA) and dipalmitoyl phosphatidyl glycerol (DPPG). This was shown with surface pressure isotherms and elasticity and PM-IRRAS measurements in the Langmuir monolayers, in addition to quartz crystal microbalance and fluorescence spectroscopy measurements for Langmuir-Blodgett (LB) films transferred onto solid substrates. Some specificity was noted in the removal action because chitosan was unable to remove BLG incorporated into neutral dipalmitoyl phosphatidyl choline (DPPC) and cholesterol monolayers and had no effect on horseradish peroxidase and urease interacting with DMPA. An obvious biological implication of these findings is to offer reasons that chitosan can remove BLG from lipophilic environments, as reported in the recent literature.

Using phospholipid Langmuir and Langmuir-Blodgett films as matrix for urease immobilization

The immobilization of enzymes in organized two-dimensional matrices is a key requirement for many biotechnological applications. In this paper, we used the Langmuir-Blodgett (LB) technique to obtain controlled architectures of urease immobilized in solid supports, whose physicochemical properties were investigated in detail. Urease molecules were adsorbed at the air-water interface and incorporated into Langmuir monolayers of the phospholipid dipalmitoyl phosphatidyl glycerol (DPPG). Incorporation of urease made DPPG monolayers more flexible and caused the reduction of the equilibrium and dynamic elasticity of the film. Urease and DPPG-urease mixed monolayers could be transferred onto solid substrates, forming LB films. A close packing arrangement of urease was obtained, especially in the mixed LB films, which was inferred with nanogravimetry and electrochemistry measurements. From the blocking effect of the LB films deposited onto indium tin oxide (ITO) substrates, the electrochemical properties of the LB films pointed to a charge transport controlled by the lipid architecture. (c) 2007 Elsevier Inc. All rights reserved.

In vitro effect of low-level laser on odontoblast-like cells

The aim of this study was to evaluate the metabolism of odontoblast-like MDPC-23 cells subjected to direct LLL irradiation. The cells were seeded (20,000 cells/well) in 24-well plates and incubated for 24 hours at 37 degrees C. After this period, the culture medium (DMEM) was replaced by fresh DMEM supplemented with 2 or 5% (stress induction by nutritional deficit) or 10% fetal bovine serum (FBS). The cells were exposed to laser doses of 2, 4, 10, 15 and 25 J/cm(2) from a near infrared InGaAsP diode laser prototype (LASERTable; 780 +/- 3 nm, 40 mW). One control group (sham irradiation) was established for each experimental condition (laser dose x FBS supplementation). Three and 72 hours after the last irradiation, cells were analyzed with respect to metabolism, morphology, total protein expression and alkaline phosphatase (ALP) activity. Higher metabolism and total protein expression were observed 72 hours after the last irradiation at the doses of 15 and 25 J/cm(2) (Mann-Whitney; p<0.05). Higher ALP activity was obtained with 5% FBS when the cells were irradiated with doses of 2 and 10 J/cm(2). For the dose of 25 J/cm(2), the highest ALP activity was observed with 10% FBS. It was concluded that the LLLT parameters used in this study stimulated the metabolic activity of the MDPC-23 cells, especially at the doses of 15 and 25 J/cm(2).