Nitric oxide participation in granulomatous response induced by Paracoccidioides brasiliensis infection in mice

The role of nitric oxide (NO) in granulomas of Paracoccidioides brasiliensis-infected inducible NO synthase-deficient C57BL/6 mice (iNOS KO) and their wild-type counterparts and its association with osteopontin (OPN) and matrix metalloproteinases (MMPs) was studied. At 15 days after infection (DAI), iNOS KO mice showed compact and necrotic granulomas with OPN+ macrophages and multinucleated giant cells, whereas wild-type mice developed loose granulomas with many fungi and OPN+ cells distributed throughout the tissue. In addition, high OPN levels and fungal load were observed in iNOS KO mice. Both experimental groups had MMP-9 activity. At 120 DAI, iNOS KO had smaller granulomas with OPN+ cells, lower OPN levels, lower fungal load and decreased MMP-9 activity compared with wild-type mice. These findings suggest that NO has an important role in granuloma modulation, by controlling OPN and MMP production, as well as by inducing loose granulomas formation and fungal dissemination, resulting, at later phases, in progression of paracoccidioidomycosis.

The role of endothelin pathway in modulation of airway reactivity to methacholine in C57Bl/6 and BALB/c mice

Endothelin peptides have been shown to increase cholinergic neurotransmission in the airway. Genetic differences in airway responsiveness to methacholine where reported in mice. The present study compared the airway reactivity to methacholine in C57Bl/6 and BALB/c mice, the involvement of endothelin on this reactivity and endothelin levels in lung homogenates. Whole airway reactivity was analyzed by means of an isolated lung preparation where lungs were perfused through the trachea with warm gassed Krebs solution at 5 ml/min, and changes in perfusion pressure triggered by methacholine at increasing bolus doses (0.1-100 mu g) were recorded. We found that the maximal airway response to methacholine was much greater in C57Bl/6 than in BALB/c (Emax 34 +/- 2 vs 12 +/- 1 cmH(2)O, respectively). Bosentan (mixed endothelin A/B receptor antagonist; 10 mg/kg, i.p., 30 min before sacrifice) reduced lung responsiveness to methacholine in C57Bl/6 (58% at EC50 level) but had no effect in BALB/c mouse strain. This effect seems to be mediated by the endothelin ETA receptor since it was significantly reduced by the selective endothelin ETA receptor antagonist, BQ 123. Immunoreactive endothelin levels were higher in C57Bl/6 than in BALB/c lungs (43 5 vs 19 +/- 5 pg/g of tissue). In conclusion, airway reactivity to methacholine and lung endothelins content varies markedly between C57Bl/6 and BALB/c strains. Endothelins upregulate lung responsiveness to methacholine only in C57Bl/6, an effect achieved through the endothelin ETA receptor. (C) 2008 Elsevier B.V. All rights reserved.

The variability of baroreflex sensitivity in juvenile, spontaneously hypertensive rats

In this study the baroreflex sensitivity of conscious, juvenile, spontaneously hypertensive rats (SHRs) was compared. The study population consisted of 19 eight-week-old male SHRs. The baroreflex sensitivity was quantified as the derivative of the variation in heart rate (HR) and the variation of mean arterial pressure (baroreflex sensitivity = Delta HR/Delta MAP). MAP was manipulated with sodium nitroprusside (SNP) and phenylephrine (PHE), administered via an inserted cannula in the right femoral vein. The SHRs were divided into four groups: (1) low bradycardic baroreflex (LB) where the baroreflex gain (BG) was between 0 and 1 bpm/mmHg with PHE; (2) high bradycardic baroreflex (HB), where the BG was < -1 bpm/mmHg with PHE; (3) low tachycardic baroreflex (LT) where the BC was between 0 and 3 bpm/mmHg with SNP; (4) high tachycardic baroreflex (HT) where the BG was > 3 bpm/mmHg with SNP. We noted that 36.8% of the rats presented with an increased bradycardic reflex, while 27.8% demonstrated an attenuated tachycardic reflex. No significant alterations were noted regarding the basal MAP and HR. There were significant differences in the baroreflex sensitivity between SHRs in the same laboratory. One should be careful when interpreting studies employing the SHR as a research model.

Assessment of inbreeding depression in a Guzerat dairy herd: Effects of individual increase in inbreeding coefficients on production and reproduction

Influences of inbreeding on daily milk yield (DMY), age at first calving (AFC), and calving intervals (CI) were determined on a highly inbred zebu dairy subpopulation of the Guzerat breed. Variance components were estimated using animal models in single-trait analyses. Two approaches were employed to estimate inbreeding depression: using individual increase in inbreeding coefficients or using inbreeding coefficients as possible covariates included in the statistical models. The pedigree file included 9,915 animals, of which 9,055 were inbred, with an average inbreeding coefficient of 15.2%. The maximum inbreeding coefficient observed was 49.45%, and the average inbreeding for the females still in the herd during the analysis was 26.42%. Heritability estimates were 0.27 for DMY and 0.38 for AFC. The genetic variance ratio estimated with the random regression model for CI ranged around 0.10. Increased inbreeding caused poorer performance in DMY, AFC, and CI. However, some of the cows with the highest milk yield were among the highly inbred animals in this subpopulation. Individual increase in inbreeding used as a covariate in the statistical models accounted for inbreeding depression while avoiding overestimation that may result when fitting inbreeding coefficients.

Population structure and inbreeding effects on growth traits of Santa Ines sheep in Brazil

The aim of this study was to describe the population structure, inbreeding and to quantify their effect for different weights, of Santa Ines sheep. For this reason, 6490 data of production and 17,097 animals in the pedigree data set were utilized to evaluate birth weight (BW), weight at 60 days (W60) and weight at 180 days (W180). The genetic structure analysis of the population was realized by the software ENDOG (v.4.6.), resulting in some level of inbreeding for 21.72% of the animals in the pedigree data, being 41.02% the maximum value, and average of 10.74% for the inbred individuals. The population average inbreeding was 2.33% and the average relatedness was 0.73%. The effective number of ancestors was 156 animals and the effective number of founders was 211 individuals. A significant depressive effect of the inbreeding can be verified for all traits. The monitored parameters related with the genetic variability on this population must be constant in order to prevent the decrease in the genetic progress. The utilization of a program for directed mating in the present flock is an appropriate alternative to keep the level of inbreeding under control. (C) 2010 Elsevier B.V. All rights reserved.

Immunomodulatory effects of Pteridium aquilinum on natural killer cell activity and select aspects of the cellular immune response of mice

Pteridium aquilinum (bracken fern) is one of the most common plants. Epidemiological studies have revealed a higher risk of certain types of cancers (i.e., esophageal, gastric) in people who consume bracken fern directly ( as crosiers or rhizomes) or indirectly through the consumption of milk from livestock that fed on the plant. In animals, evidence exists regarding the associations between chronic bracken fern intoxication, papilloma virus infection, and the development of carcinomas. While it is possible that some carcinogens in bracken fern could be responsible for these cancers in both humans and animals, it is equally plausible that the observed increases in cancers could be related to induction of an overall immunosuppression by the plant/its various constituents. Under the latter scenario, normal tumor surveillance responses against nascent (non-bracken-induced) cancers or responses against viral infections ( specifically those linked to induction of cancers) might be adversely impacted by continuous dietary exposure to this plant. Therefore, the overall objective of this study was to evaluate the immunomodulatory effects of bracken fern following daily ingestion of its extract by a murine host over a period of 14 ( or up to 30) days. In C57BL/6 mice administered ( by gavage) the extract, histological analyses revealed a significant reduction in splenic white pulp area. Among a variety of immune response parameters/functions assessed in these hosts and isolated cells, both delayed-type hypersensitivity (DTH) analysis and evaluation of IFN gamma. production by NK cells during T(H)1 priming were also reduced. Lastly, the innate response in these hosts-assessed by analysis of NK cell cytotoxic functionality-was also diminished. The results here clearly showed the immunosuppressive effects of P. aquilinum and that many of the functions that were modulated could contribute to the increased risk of cancer formation in exposed hosts.

Craniometaphyseal Dysplasia With Severe Craniofacial Involvement Shows Homozygosity at 6q21-22.1 Locus

Craniotubular dysplasias (CTD) are a heterogeneous group of genetic disorders of skeletal development, whose clinical and etiological classification is still much debated. One of the most common form is the autosomal dominant craniometaphyseal dysplasia (CMD) which is associated with mutation in the ANKH gene. In the literature a few families are reported with CMD phenotype that suggest an autosomal recessive (AR) pattern of inheritance. A candidate locus at 6q21-22 has been mapped in a large inbred Brazilian family, but the gene of the recessive form is still unknown. Our data on a female patient with CMD phenotype, born from healthy first degree cousins and displaying homozygosity for polymorphic markers at the 6q21-22 locus, further support the existence of an AR CMD, expanding its clinical spectrum to a more severe phenotype. (C) 2011 Wiley-Liss, Inc.

Unexpected genetic heterogeneity in a large consanguineous Brazilian pedigree presenting deafness

Nonsyndromic autosomal recessive deafness accounts for 80% of hereditary deafness. To date, 52 loci responsible for autosomal recessive deafness have been mapped and 24 genes identified. Here, we report a large inbred Brazilian pedigree with 26 subjects affected by prelingual deafness. Given the extensive consanguinity found in this pedigree, the most probable pattern of inheritance is autosomal recessive. However, our linkage and mutational analysis revealed, instead of an expected homozygous mutation in a single gene, two different mutant alleles and a possible third undetected mutant allele in the MYO15A gene (DFNB3 locus), as well as evidence for other causes for deafness in the same pedigree. Among the 26 affected subjects, 15 were homozygous for the novel c.10573delA mutation in the MYO15A gene, 5 were compound heterozygous for the mutation c.10573delA and the novel deletion c.9957_9960delTGAC and one inherited only a single c.10573delA mutant allele, while the other one could not be identified. Given the extensive consanguinity of the pedigree, there might be at least one more deafness locus segregating to explain the condition in some of the subjects whose deafness is not clearly associated with MYO15A mutations, although overlooked environmental causes could not be ruled out. Our findings illustrate a high level of etiological heterogeneity for deafness in the family and highlight some of the pitfalls of genetic analysis of large genes in extended pedigrees, when homozygosity for a single mutant allele is expected.

Expression of alpha-synuclein is increased in the hippocampus of rats with high levels of innate anxiety

A genomic region neighboring the alpha-synuclein gene, on rat chromosome 4, has been associated with anxiety- and alcohol-related behaviors in different rat strains. In this study, we have investigated potential molecular and physiological links between alpha-synuclein and the behavioral differences observed between Lewis (LEW) and Spontaneously Hypertensive (SHR) inbred rats, a genetic model of anxiety. As expected, LEW rats appeared more fearful than SHR rats in three anxiety models: open field, elevated plus maze and light/dark box. Moreover, LEW rats displayed a higher preference for alcohol and consumed higher quantities of alcohol than SHR rats. alpha-Synuclein mRNA and protein concentrations were higher in the hippocampus, but not the hypothalamus of LEW rats. This result inversely correlated with differences in dopamine turnover in the hippocampus of LEW and SHR rats, supporting the hypothesis that alpha-synuclein is important in the downregulation of dopamine neurotransmission. A novel single nucleotide polymorphism was identified in the 30-untranslated region (3`-UTR) of the alpha-synuclein cDNA between these two rat strains. Plasmid constructs based on the LEW 3`-UTR sequence displayed increased expression of a reporter gene in transiently transfected PC12 cells, in accordance with in-vivo findings, suggesting that this nucleotide exchange might participate in the differential expression of alpha-synuclein between LEW and SHR rats. These results are consistent with a novel role for alpha-synuclein in modulating rat anxiety- like behaviors, possibly through dopaminergic mechanisms. Since the behavioral and genetic differences between these two strains are the product of independent evolutionary histories, the possibility that polymorphisms in the alpha-synuclein gene may be associated with vulnerability to anxiety- related disorders in humans requires further investigation. Molecular Psychiatry (2009) 14, 894-905; doi: 10.1038/mp.2008.43; published online 22 April 2008

Nutrient Selection in the Absence of Taste Receptor Signaling

When allowed to choose between different macronutrients, most animals display a strong attraction toward carbohydrates compared with proteins. It remains uncertain, however, whether this food selection pattern depends primarily on the sensory properties intrinsic to each nutrient or, alternatively, metabolic signals can act independently of the hedonic value of sweetness to stimulate elevated sugar intake. Here we show that Trpm5(-/-) mice, which lack the cellular mechanisms required for sweet and several forms of L-amino acid taste transduction, develop a robust preference for D-glucose compared with isocaloric L-serine independently of the perception of sweetness. Moreover, a close relationship was found between glucose oxidation and taste-independent nutrient intake levels, with animals increasing intake as a function of glucose oxidation rates. Furthermore, microdialysis measurements revealed nutrient-specific dopaminergic responses in accumbens and dorsal striatum during intragastric infusions of glucose or serine. Specifically, intragastric infusions of glucose induced significantly higher levels of dopamine release compared with isocaloric serine in both ventral and dorsal striatum. Intragastric stimulation of dopamine release seemed to depend on glucose utilization, because administration of an anti-metabolic glucose analog resulted in lower dopamine levels in striatum, an effect that was reversed by intravenous glucose infusions. Together, our findings suggest that carbohydrate-specific preferences can develop independently of taste quality or caloric load, an effect associated with the ability of a given nutrient to regulate glucose metabolism and stimulate brain dopamine centers.

NEUREGULINS 1-ALPHA AND 1-BETA ON THE REGENERATION THE PERIPHERAL NERVES

Objective: To evaluate the effect of the neuregulins 1-alpha and 1-beta on the regeneration the sciatic nerves of male adult C57BL/6J mice, using the tubulization technique. Methods: Eighteen animals were used, divided into three groups. A polyethylene prosthesis was implanted in a 4.0 mm defect of the left sciatic nerve, as follows: group 1 containing only purified collagen (Vitrogen (R)); group 2, collagen with neuregulin 1-alpha; group 3, collagen with neuregulin 1-beta. The control group consisted of six segments of right sciatic nerves. After four weeks, the animals were sacrificed. A segment from the midpoint of the nerve regenerated inside the prostheses was extracted; histological sections were standardized, and slides were made up for histomorphometric analysis. Results: the results were statistically compared using the Tukey multiple comparisons test and The Student`s t test. The animals treated with neuregulins had greater numbers of myelinized axons, with a statistically significant difference in relation to the collagen-only group. There was no statistical difference between the neuregulin 1-alpha and 1-beta groups. Conclusion: The addition of neuregulins provided a significant increase in the number of myelinized fibers.

Role of SOCS-1 Gene on Melanoma Cell Growth and Tumor Development

Melanoma is the most aggressive form of skin cancer, and its incidence has increased dramatically over the years. The murine B16F10 melanoma in syngeneic C57Bl/6 mice has been used as a highly aggressive model to investigate tumor development. Presently, we demonstrate in the B16F10-Nex2 subclone that silencing of SOCS-1, a negative regulator of Jak/Stat pathway, leads to reversal of the tumorigenic phenotype and inhibition of melanoma cell metastasis. SOCS-1 silencing with short hairpin RNA affected tumor growth and cell cycle regulation with arrest at the S phase with large-sized nuclei, reduced cell motility, and decreased melanoma cell invasion through Matrigel. A clonogenic assay showed that SOCS-1 acted as a modulator of resistance to anoikis. In addition, down-regulation of SOCS-1 decreased the expression of epidermal growth factor receptor ( mainly the phosphorylated-R), Ins-R alpha, and fibroblast growth factor receptor. In vivo, silencing of SOCS-1 inhibited subcutaneous tumor growth and metastatic development in the lungs. Because SOCS-1 is expressed in most melanoma cell lines and bears a relation with tumor invasion, thickness, and stage of disease, the present results on the effects of SOCS-1 silencing in melanoma suggest that this regulating protein can be a target of cancer therapy.

Anabolic-androgenic steroid treatment induces behavioral disinhibition and downregulation of serotonin receptor messenger RNA in the prefrontal cortex and amygdala of male mice

Nandrolone is an anabolic-androgenic steroid (AAS) that is highly abused by individuals seeking enhanced physical strength or body appearance. Supraphysiological doses of this synthetic testosterone derivative have been associated with many physical and psychiatric adverse effects, particularly episodes of impulsiveness and overt aggressive behavior. As the neural mechanisms underlying AAS-induced behavioral disinhibition are unknown, we investigated the status of serotonergic system-related transcripts in several brain areas of mice receiving prolonged nandrolone administration. Male C57BL/6J mice received 15 mg/kg of nandrolone decanoate subcutaneously once daily for 28 days, and different sets of animals were used to investigate motor-related and emotion-related behaviors or 5-HT-related messenger RNA (mRNA) levels by real-time quantitative polymerase chain reaction. AAS-injected mice had increased body weight, were more active and displayed anxious-like behaviors in novel environments. They exhibited reduced immobility in the forced swim test, a higher probability of being aggressive and more readily attacked opponents. AAS treatment substantially reduced mRNA levels of most investigated postsynaptic 5-HT receptors in the amygdala and prefrontal cortex. Interestingly, the 5-HT(1B) mRNA level was further reduced in the hippocampus and hypothalamus. There was no alteration of 5-HT system transcript levels in the midbrain. In conclusion, high doses of AAS nandrolone in male mice recapitulate the behavioral disinhibition observed in abusers. Furthermore, these high doses downregulate 5-HT receptor mRNA levels in the amygdala and prefrontal cortex. Our combined findings suggest these areas as critical sites for AAS-induced effects and a possible role for the 5-HT(1B) receptor in the observed behavioral disinhibition.

Increased vascular O-GlcNAcylation augments reactivity to constrictor stimuli - Vasoactive Peptide Symposium

O-linked N-acetylglucosaminylation (O-GlcNAcylation) plays a role in many aspects of protein function. Whereas elevated O-GlcNAc levels contribute to diabetes-related end-organ damage, O-GlcNAcylation is also physiologically important. Because proteins that play a role in vascular tone regulation can be O-GlcNAcylated, we hypothesized that O-GlcNAcylation increases vascular reactivity to constrictor stimuli, Aortas front male Sprague-Dawley rats and C57BL/6 mice were incubated for 24 hours with vehicle or PugNAc (O-GlcNAcase inhibitor. 100 mu M). PugNAc incubation significantly increased O-GlcNAc proteins, as determined by Western blot. PugNAc also increased vascular contractions to phenylephrine and serotonin, an effect not observed in the presence of N(omega)-nitro-L-arginine methyl ester or in endothelium-denuded vessels. Acetylcholine-induced relaxation. but not that to sodium nitroprusside, was decreased by PugNAc treatment, an effect accompanied by decreased levels of phosphorylated endothelial nitric oxide synthase (eNOS)(Ser-1177) and Akt(Ser-473). Augmented O-GlcNAcylation increases vascular reactivity to constrictor stimuli, possibly due to its effects oil eNOS expression and activity, reinforcing the concept that O-GlcNAcylation modulates vascular reactivity and may play a role in pathological conditions associated with abnormal vascular function. J Am Soc Hypertens 2008:2(6): 410-417. (C) 2008 American Society of Hypertension. All rights reserved.

Donor bone marrow cells play a role in the prevention of accelerated graft rejection induced by semi-allogeneic spleen cells in transplantation

Spleen or spleen plus bone marrow cells from (BALB/c x C57Bl/6)F1 donors were transferred into BALB/c recipients 21 days before skin or cardiac transplantation. Prolonged graft survival was observed on recipients treated with the mixture of donor-derived cells as compared to those treated with spleen cells alone. We evaluated the expression of CD45RB and CD44 by splenic CD4(+) and CD8(+) T cells 7 and 21 days after donor cell transfer. The populations of CD8(+)CD45RB(low) and CD8(+)CD44(high) cells were significantly decreased in mice pre-treated with donor spleen and bone marrow cells as compared to animals treated with spleen cells only, although these cells expanded in both groups when compared to an earlier time-point. No differences were observed regarding CD4+ T cell population when recipients of donor-derived cells were compared. An enhanced production of IL-10 was observed seven days after transplantation in the supernatants of spleen cell cultures of mice treated with spleen and bone marrow cells. Taken together these data suggest that donor-derived bone marrow cells modulate the sensitization of the recipient by semi-allogeneic spleen cells in part by delaying the generation of activated/memory CD8(+) T cells leading to enhanced graft survival. (c) 2007 Elsevier B.V. All rights reserved.