Vitellogenin content in fat body and ovary homogenates of workers and queens of Melipona quadrifasciata anthidioides during vitellogenesis

Vitellogenin (Vg) is an egg yolk protein that is produced primarily in the fat body of most female insects. In the advanced social structure of eusocial honeybees, the presence of the queen inhibits egg maturation in the workers ovaries. However in the stingless bee Melipona quadrifasciata, the workers always develop ovaries and lay a certain amount of eggs while provisioning the brood cells with larval food during what is known as the worker nurse phase. The present work is a comparative study of the presence of Vg in homogenates of the fat bodies and ovaries of the nurse workers, and the virgin and physogastric queens of M. quadrifasciata. The presence of Vg was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting using Apis mellifera anti-egg antibody. Vg was not detected in the fat bodies or ovaries of the workers, but it was found in the ovaries of virgin and physogastric queens and in the fat body of physogastric queens. The results are discussed, taking into account the reproductive state of the individuals and the other possible roles of Vg, such as a storage protein for metoabolism of other organs.

Increased Glyceride-Glycerol Synthesis in Liver and Brown Adipose Tissue of Rat: In-Vivo Contribution of Glycolysis and Glyceroneogenesis

We have previously shown that a high-protein, carbohydrate-free diet can decrease the production of glycerol-3-phosphate (G3P) from glucose and increase glyceroneogenesis in both brown (BAT) and epididymal (EAT) adipose tissue. Here, we utilized an in-vivo approach to examine the hypothesis that there is reciprocal regulation in the G3P synthesis from glucose (via glycolysis) and glyceroneogenesis in BAT, EAT and liver of fasted rats and cafeteria diet-fed rats. Glyceroneogenesis played a prominent role in the generation of G3P in the liver (similar to 70 %) as well as in BAT and EAT (similar to 80 %) in controls rats. The cafeteria diet induced an increase in the total glyceride-glycerol synthesis and G3P synthesis from glucose and a decrease in glyceroneogenesis in BAT; this diet did not affect either the total glyceride-glycerol synthesis or G3P generation from glyceroneogenesis or glycolysis in the liver or EAT. Fasting induced an increase in total glyceride-glycerol synthesis and glyceroneogenesis and a decrease in G3P synthesis from glucose in the liver but did not affect either the total glyceride-glycerol synthesis or G3P synthesis from glyceroneogenesis in BAT and EAT, despite a reduction in glycolysis in these tissues. These data demonstrate that reciprocal changes in the G3P generation from glucose and from glyceroneogenesis in the rat liver and BAT occur only when the synthesis of glycerides-glycerol is increased. Further, our data suggest that this increase may be essential for the systemic recycling of fatty acids by the liver from fasted rats and for the maintenance of the thermogenic capacity of BAT from cafeteria diet-fed rats.

Drug-targeting in combined cancer chemotherapy: tumor growth inhibition in mice by association of paclitaxel and etoposide with a cholesterol-rich nanoemulsion

Lipid nanoemulsions (LDE) may be used as carriers of paclitaxel (PTX) and etoposide (ETP) to decrease toxicity and increase the therapeutic action of those drugs. The current study investigates the combined chemotherapy with PTX and ETP associated with LDE. Four groups of 10-20 B16F10 melanoma-bearing mice were treated with LDE-PTX and LDE-ETP in combination (LDE-PTX + ETP), commercial PTX and ETP in combination (PTX + ETP), single LDE-PTX, and single LDE-ETP. PTX and ETX doses were 9 mu mol/kg administered in three intraperitoneal injections on three alternate days. In two control groups mice were treated with saline solution or LDE alone. Tumor growth, metastasis presence, cell-cycle distribution, blood cell counts and histological data were analyzed. Toxicity of all treatments was evaluated in mice without tumors. Tumor growth inhibition was similarly strong in all treatment groups. However, there was a greater reduction in the number of animals bearing metastases in the LDE-PTX + ETP group (30 %) in comparison to the PTX + ETP group (82 %, p < 0.05). Reduction of cellular density, blood vessels and increase of collagen fibers in tumor tissues were observed in the LDE-PTX + ETP group but not in the PTX + ETP group, and in both groups reduced melanoma-related anemia and thrombocytosis were observed. Flow cytometric analysis suggested that LDE-PTX + ETP exhibited greater selectivity to neoplastic cells than PTX-ETP, showing arrest (65 %) in the G(2)/M phase of the cell cycle (p < 0.001). Toxicity manifested by weight loss and myelosuppression was markedly milder in the LDE-PTX + ETP than in the PTX + ETP group. LDE-PTX + ETP combined drug-targeting therapy showed markedly superior anti-cancer properties and reduced toxicity compared to PTX + ETP.

Histomorphometrical and proliferative aspects of placenta and uterus of the collared peccary (Tayassu tajacu)

The histomorphometric and proliferative characteristics of the collared peccary (Tayassu tajacu) placenta and uterus were analyzed. The material was examined by standard histological techniques and histochemistry (PAS, Perls and Alcian Blue pH 0.5 and 2.5%) and the cellular proliferation by AgNORs and flow cytometry. All the analyzed morphometric variables differed between pregnant and non-pregnant uteri in the luteal phase using the Dunnet test. Height and gland diameter of uterine glands increased linearly during pregnancy, with an intense positive PAS and Perls reaction in all stages. The cells with more than seven AgNORs per nuclei and the cells in the G2M cell cycle phase in the maternal tissue also increased after 70 days of pregnancy. The uteroplacental ridges had a linear increase in size with two distinct areas, base and top, with uterine epithelium and trophoblastic cells changing their morphology following the placental ridge development. Flow cytometry analysis showed the percentage of cells in each cell cycle phase with a quadratic behavior for stages G2/M in the maternal tissue, suggesting an increase in proliferative capacity of maternal tissue after 65 days of pregnancy. The same quadratic effect was observed in the G0/G1 phase in both maternal and fetal tissues. Cells in apoptosis showed cubic behavior in both tissues. The morphometric and cellular dynamic aspects observed in this study have not been previously described and they extend our knowledge of functions relating to maternal-fetal dynamics in this species.

Time sequence of the intensification of the liver glucose production induced by high-fat diet in mice

It is well established that the development of insulin resistance shows a temporal sequence in different organs and tissues. Moreover, considering that the main aspect of insulin resistance in liver is a process of glucose overproduction from gluconeogenesis, we investigated if this metabolic change also shows temporal sequence. For this purpose, a well-established experimental model of insulin resistance induced by high-fat diet (HFD) was used. The mice received HFD (HFD group) or standard diet (COG group) for 1, 7, 14 or 56?days. The HFD group showed increased (P?<?0.05 versus COG) epididymal, retroperitoneal and inguinal fat weight from days 1 to 56. In agreement with these results, the HFD group also showed higher body weight (P?<?0.05 versus COG) from days 7 to 56. Moreover, the changes induced by HFD on liver gluconeogenesis were progressive because the increment (P?<?0.05 versus COG) in glucose production from l-lactate, glycerol, l-alanine and l-glutamine occurred 7, 14, 56 and 56 days after the introduction of the HFD schedule, respectively. Furthermore, glycaemia and cholesterolemia increased (P?<?0.05 versus COG) 14?days after starting the HFD schedule. Taken together, the results suggest that the intensification of liver gluconeogenesis induced by an HFD is not a synchronous all-or-nothing process but is specific for each gluconeogenic substrate and is integrated in a temporal manner with the progressive augmentation of fasting glycaemia. Copyright (c) 2012 John Wiley & Sons, Ltd.

Effects of the administration of pentoxifylline and prednisolone on the evolution of portal fibrogenesis secondary to biliary obstruction in growing animals: immunohistochemical analysis of the expression of TGF-BETA; and VEGF

OBJECTIVE: During the neonatal and infancy periods, some chronic liver diseases may lead to progressive hepatic fibrosis, which is a condition that can ultimately result in the loss of organ function and severe portal hypertension necessitating hepatic transplantation. In a previous report, pharmacological interventions were demonstrated to modulate hepatic fibrosis induced by bile duct ligation in young rats. The administration of pentoxifylline or prednisolone, or the combination of both, resulted in reduced fibrogenesis in portal spaces. The objectives of the present study were to evaluate the expression of transforming growth factor beta and vascular endothelial growth factor after bile duct ligation in young rats and to assess the effect of those same drugs on cytokine expression. METHODS: In this experimental study, 80 young rats (21 or 22 days old) were submitted either to laparotomy and common bile duct ligation or to sham surgery. The animals were allocated into four groups according to surgical procedure, and the following treatments were administered: (1) common bile duct ligation + distilled water, (2) sham surgery + distilled water, (3) common bile duct ligation + pentoxifylline, or (4) common bile duct ligation + prednisolone. After 30 days, a hepatic fragment was collected from each animal for immunohistochemical analysis using monoclonal antibodies against transforming growth factor beta and vascular endothelial growth factor. Digital morphometric and statistical analyses were performed. RESULTS: The administration of pentoxifylline reduced the transforming growth factor beta-marked area and the amount of transforming growth factor beta expressed in liver tissue. This effect was not observed after the administration of prednisolone. There was a significant reduction in vascular endothelial growth factor expression after the administration of either drug compared with the non-treatment group. CONCLUSIONS: The administration of pentoxifylline to cholestatic young rats resulted in the diminished expression of transforming growth factor beta and vascular endothelial growth factor in liver tissue. The administration of steroids resulted in the diminished expression of vascular endothelial growth factor only. These pathways may be involved in hepatic fibrogenesis in young rats submitted to bile duct ligation and exposed to pentoxifylline or prednisolone.

Biomonitoring of Microcystin and Aflatoxin Co-Occurrence in Aquaculture Using Immunohistochemistry and Genotoxicity Assays

This work investigated the effects of co-occurring aflatoxin B-1 (AFB(1)) and microcystin (MC) in aquaculture, using immunohistochemistry and genotoxicity methods. Tilapia (Oreochromis niloticus) were exposed to AFB(1) by intraperitoneal and MC (cell extract of Microcystis aeruginosa) by intraperitoneal and immersion routes. The interaction of MC-AFB(1) was evaluated co-exposing the intraperitoneal doses. Blood samples were collected after 8, 24, and 48h to analyze the micronucleus frequency and comet score. The interaction of MC-AFB(1) showed a synergic mutagenic response by higher micronucleus frequency of co-exposed group. A slight genotoxic synergism was also observed in the comet score. Immunohistochemistry detected MC in al lthe fish liver tissues exposed to MC by intraperitoneal route, and only the immersed group with the highest dose of MC showed a positive response. Although MC was non-detectable in the edible muscle, the combination of immunohistochemistry with genotoxicity assay was an attractive biomonitoring tool in aquaculture, where the animals were frequently exposed to co-occurring synergic hazards.

Antioxidant capacity and mineral content of pulp and peel from commercial cultivars of citrus from Brazil

Four Citrus species (C. sinensis, cvs. Pera and Lima; C. latifolia Tanaka cv. Tahiti; C limettioides Tanaka cv. Sweet lime and C. reticulate, cv. Ponkan) grown in Brazil were characterised in relation to contents of minerals, ascorbic acid, total polyphenols and antioxidant capacity of pulps and peels. In general, the peels demonstrated significantly higher contents of all compounds than the pulps (p < 0.05), with the exception of the Pera orange pulp that presented the highest acid ascorbic content (68 mg/100 ml), while the Tahiti lime peel presented the lowest (8 mg/100 g). Citrus showed high levels of potassium, calcium and magnesium, and the peels were considered sources of these minerals. The Ponkan mandarin peel presented the highest antioxidant capacity. The antioxidant capacity of citrus was correlated both to vitamin C and phenolics. Aside from citrus pulps, the peels are also good sources of bioactive compounds and minerals, and can be explored for their health promoting values in food products. (C) 2012 Elsevier Ltd. All rights reserved.

Thermal alteration and morphological changes of sound and demineralized primary dentin after Er:YAG laser ablation

The purpose of this study was to assess the influence of Er:YAG laser pulse repetition rate on the thermal alterations occurring during laser ablation of sound and demineralized primary dentin. The morphological changes at the lased areas were examined by scanning electronic microscopy (SEM). To this end, 60 fragments of 30 sound primary molars were selected and randomly assigned to two groups (n = 30); namely A sound dentin (control) and B demineralized dentin. Each group was divided into three subgroups (n = 10) according to the employed laser frequencies: I4 Hz; II6 Hz, and III10 Hz. Specimens in group B were submitted to a pH-cycling regimen for 21 consecutive days. The irradiation was performed with a 250 mJ pulse energy in the noncontact and focused mode, in the presence of a fine water mist at 1.5 mL/min, for 15 s. The measured temperature was recorded by type K thermocouples adapted to the dentin wall relative to the pulp chamber. Three samples of each group were analyzed by SEM. The data were submitted to the nonparametric Kruskal-Wallis test and to qualitative SEM analysis. The results revealed that the temperature increase did not promote any damage to the dental structure. Data analysis demonstrated that in group A, there was a statistically significant difference among all the subgroups and the temperature rise was directly proportional to the increase in frequency. In group B, there was no difference between subgroup I and II in terms of temperature. The superficial dentin observed by SEM displayed irregularities that augmented with rising frequency, both in sound and demineralized tissues. In conclusion, temperature rise and morphological alterations are directly related to frequency increment in both demineralized and sound dentin. Microsc. Res. Tech., 2011. (c) 2011 Wiley Periodicals, Inc.

Ciprofloxacin has antifibrotic effects in scleroderma fibroblasts via downregulation of Dnmt1 and upregulation of Fli1

Systemic sclerosis (SSc) is characterized by fibrosis of the skin and internal organs. The present study was undertaken to examine the effects of ciprofloxacin, a fluoroquinolone antibiotic implicated in matrix remodeling, on dermal and lung fibroblasts obtained from SSc patients. Dermal and lung fibroblasts from SSc patients and healthy subjects were treated with ciprofloxacin. Western blotting was used to analyze protein levels and RT-PCR was used to measure in RNA expression. The pharmacologic inhibitor UO126 was used to block Erk1/2 signaling. SSc dermal fibroblasts demonstrated a significant decrease in collagen type I mRNA and protein levels after antibiotic treatment, while healthy dermal fibroblasts were less sensitive to ciprofloxacin, downregulating collagen only at the protein levels. Connective tissue growth factor (CCN2) gene expression was significantly reduced and matrix metalloproteinase (MMPI) levels were enhanced after ciprofloxacin treatment to a similar extent in healthy and SSc fibroblasts. Ciprofloxacin induced Erk1/2 phosphorylation, and Erk1/2 blockade completely prevented MMP1 upregulation. However. Smad1 and Smad3 activation in response to TGF beta was not affected. The expression of friend leukemia integration factor 1 (Fli1). a transcriptional repressor of collagen, was increased after treatment with ciprofloxacin only in SSc fibroblasts, and this was accompanied by a decrease in the levels of DNA methyltransferase 1 (Dnmt1). Similar effects were observed in SSc-interstitial lung disease (ILD) lung fibroblasts. In summary, our study demonstrates that ciprofloxacin has antifibrotic actions in SSc dermal and lung fibroblasts via the downregulation of Dnmt1, the upregulation of Fli1 and induction of MMPI gene expression via an Erk1/2-dependent mechanism. Thus, our data suggest that ciprofloxacin may he an attractive therapy for SSc skin and lung fibrosis.

Upregulation of soluble and membrane-bound human leukocyte antigen G expression is primarily observed in the milder histopathological stages of chronic hepatitis C virus infection

Chronic hepatitis C virus (HCV) infection is a worldwide health problem that may evolve to cirrhosis and hepatocellular carcinoma. Incompletely understood immune system mechanisms have been associated with impaired viral clearance. The nonclassical class I human leukocyte antigen G (HLA-G) molecule may downregulate immune system cell functions exhibiting well-recognized tolerogenic properties. HCV genotype was analyzed in chronic HCV-infected patients. Because HLA-G expression may be induced by certain viruses, we evaluated the presence of HLA-G in the liver microenvironment obtained from 89 biopsies of patients harboring chronic HCV infection and stratified according to clinical and histopathological features. Overall, data indicated that HCV genotype 1 was predominant, especially subgenotype 1a, with a prevalence of 87%. HLA-G expression was observed in 45(51%) liver specimens, and it was more frequent in milder stages of chronic hepatitis (67.4%) than in moderate (27.8%; p = 0.009) and severe (36.0%; p = 0.021) stages of the disease. Altogether, these results suggest that the expression of HLA-G in the context of HCV is a complex process modulated by many factors, which may contribute to an immunologic environment favoring viral persistence. However, because the milder forms predominantly expressed HLA-G, a protective role of this molecule may not be excluded. (C) 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

The protective effect of cilostazol on isolated rabbit femoral arteries under conditions of ischemia and reperfusion: the role of the nitric oxide pathway

OBJECTIVES: The clinical significance of ischemia/reperfusion of the lower extremities demands further investigation to enable the development of more effective therapeutic alternatives. This study investigated the changes in the vascular reactivity of the rabbit femoral artery and nitric oxide metabolites under partial ischemia/reperfusion conditions following cilostazol administration. METHODS: Ischemia was induced using infrarenal aortic clamping. The animals were randomly divided into seven groups: Control 90 minutes, Ischemia/Reperfusion 90/60 minutes, Control 120 minutes, Ischemia/Reperfusion 120/90 minutes, Cilostazol, Cilostazol before Ischemia/Reperfusion 120/90 minutes, and Ischemia 120 minutes/Cilostazol/Reperfusion 90 minutes. Dose-response curves for sodium nitroprusside, acetylcholine, and the calcium ionophore A23187 were obtained in isolated femoral arteries. The levels of nitrites and nitrates in the plasma and skeletal muscle were determined using chemiluminescence. RESULTS: Acetylcholine- and A23187-induced relaxation was reduced in the Ischemia/Reperfusion 120/90 group, and treatment with cilostazol partially prevented this ischemia/reperfusion-induced endothelium impairment. Only cilostazol treatment increased plasma levels of nitrites and nitrates. An elevation in the levels of nitrites and nitrates was observed in muscle tissues in the Ischemia/Reperfusion 120/90, Cilostazol/Ischemia/Reperfusion, and Ischemia/Cilostazol/Reperfusion groups. CONCLUSION: Hind limb ischemia/reperfusion yielded an impaired endothelium-dependent relaxation of the femoral artery. Furthermore, cilostazol administration prior to ischemia exerted a protective effect on endothelium-dependent vascular reactivity under ischemia/reperfusion conditions.

Expression of VEGF and collagen using a latex biomembrane as bladder replacement in rabbits

Objective: To investigate the VEGF expression and collagen deposition using a latex biomembrane as bladder replacement in rabbits. Materials and Methods: After partial cystectomy, a patch of a non-vulcanized latex biomembrane (2 x 2 cm) was sewn to the bladder of rabbits with 5/0 monofilament polydioxanone sulfate sutures in a watertight manner. Groups of 5 animals were killed at 15, 45 and 90 days after surgery and the bladder was removed. Sections of 5 mu m were cut and stained with picrosirius-red in order to estimate the amount of extracellular matrix in the graft. To confirm the presence of VEGF in tissues, protein expression was determined by immunohistochemistry. Results: No death, urinary leakage or graft extrusion occurred in any group. All bladders showed a spherical shape. A progressive reduction in the amount of collagen occurred in the graft area and was negatively and linearly correlated with time (p < 0.001). VEGF expression was higher in grafted areas when compared to controls at 15 and 45 days after surgery and decreased with time (p < 0.001). Conclusion: The latex biomembrane as a matrix for partial bladder replacement in rabbits promotes temporary collagen deposition and stimulates the angiogenic process.

Expression Analysis of Wound Healing Genes in Human Periapical Granulomas of Progressive and Stable Nature

Introduction: Wound healing process involves the activation of extracellular matrix components, remodeling enzymes, cellular adhesion molecules, growth factors, cytokines and chemokines genes. However, the molecular patterns underlying the healing process periapical environment remain unclear. Here we hypothesized that endodontic infection might result in an imbalance in the expression of wound healing genes involved in the pathogenesis of periapical lesions. Furthermore, we suggest that differential expression of wound healing markers in active and latent granulomas could account for different clinical outcomes for such lesions. Methods: Study samples consisted of 93 periapical granulomas collected after endodontic surgeries and 24 healthy periodontal ligament tissues collected from premolars extracted for orthodontic purposes as control samples. Of these, 10 periapical granulomas and 5 healthy periapical tissues were used for expression analysis of 84 wound healing genes by using a pathway-specific real-time polymerase chain reaction array. The remaining 83 granulomas and all 24 control specimens were used to validate the obtained array data by real-time polymerase chain reaction. Observed variations in expression of wound healing genes were analyzed according to the classification of periapical granulomas as active/progressive versus inactive/stable (as determined by receptor activator for nuclear factor kappa B ligand/osteoprotegerin expression ratio). Results: We observed a marked increase of 5-fold or greater in SERPINE1, TIMP1, COL1A1, COL5A1, VTN, CTGF, FGF7, TGFB1, TNF, CXCL11, ITGA4, and ITGA5 genes in the periapical granulomas when compared with control samples. SERPINE1, TIMP1, COL1A1, TGFB1, and ITGA4 mRNA expression was significantly higher in inactive compared with active periapical granulomas (P < .001), whereas TNF and CXCL11 mRNA expression was higher in active lesions (P < .001). Conclusions: The identification of novel gene targets that curb the progression status of periapical lesions might contribute to a more accurate diagnosis and lead to treatment modalities more conducive to endodontic success. (J Endod 2012;38:185-190)

Early transplantation of human immature dental pulp stem cells from baby teeth to golden retriever muscular dystrophy (GRMD) dogs: Local or systemic?

Background:The golden retriever muscular dystrophy (GRMD) dogs represent the best available animal model for therapeutic trials aiming at the future treatment of human Duchenne muscular dystrophy (DMD). We have obtained a rare litter of six GRMD dogs (3 males and 3 females) born from an affected male and a carrier female which were submitted to a therapeutic trial with adult human stem cells to investigate their capacity to engraft into dogs muscles by local as compared to systemic injection without any immunosuppression. Methods Human Immature Dental Pulp Stem Cells (hIDPSC) were transplanted into 4 littermate dogs aged 28 to 40 days by either arterial or muscular injections. Two non-injected dogs were kept as controls. Clinical translation effects were analyzed since immune reactions by blood exams and physical scores capacity of each dog. Samples from biopsies were checked by immunohistochemistry (dystrophin markers) and FISH for human probes. Results and Discussion We analyzed the cells' ability in respect to migrate, engraftment, and myogenic potential, and the expression of human dystrophin in affected muscles. Additionally, the efficiency of single and consecutive early transplantation was compared. Chimeric muscle fibers were detected by immunofluorescence and fluorescent in situ hybridisation (FISH) using human antibodies and X and Y DNA probes. No signs of immune rejection were observed and these results suggested that hIDPSC cell transplantation may be done without immunosuppression. We showed that hIDPSC presented significant engraftment in GRMD dog muscles, although human dystrophin expression was modest and limited to several muscle fibers. Better clinical condition was also observed in the dog, which received monthly arterial injections and is still clinically stable at 25 months of age. Conclusion Our data suggested that systemic multiple deliveries seemed more effective than local injections. These findings open important avenues for further researches.