net OR submit four copies of the application, in English, by regu

net OR submit four copies of the application, in English, by regular mail only to: The Trustees, The H.J. Eysenck Memorial Fund, PO Box 27824, London SE24 0WE Applications must be received by the 31st January 2014 and the successful candidate will be notified by the 1st May 2014. “
“Descending modulation from brainstem areas of spinal nociceptive transmission is a well-documented phenomenon. Most early studies describe a role for descending inhibitory control of spinal nociceptive activity mediated primarily by noradrenergic and serotonergic (5-HT) pathways,

but more recently, the role of descending facilitation from the brainstem, onto spinal nociceptive pathways, has stimulated intense research and, in particular, the role for 5-HT in

mediating this excitatory drive (Bannister et al., 2009 and Wei INCB018424 manufacturer et al., 2010). Serotonergic input to the dorsal horn of the spinal cord derives almost entirely from supraspinal sources, with a minor contribution from local spinal neurones. 5-HT pathways, running directly from the rostral ventromedial medulla (RVM; the site of origin of the serotonergic descending pathway) to the spinal cord, comprise one of the main neurotransmitter systems mediating descending modulation of spinal neuronal activity. Animal studies report variably on the function of descending controls from the RVM and of 5-HT in nociceptive transmission (Bannister Ribociclib et al., 2009 and Millan, 2002). Early studies investigating blockade of RVM activity and loss of 5-HT modulation have pointed to a loss of inhibitory control resulting in increased pain behaviours (Millan, 2002). However, in addition to descending inhibition, a wealth of evidence now exists for a descending excitatory drive from the RVM modulating spinal nociceptive transmission, which involves the activation of serotonergic pathways (Bannister et al., 2009, Dogrul et al., 2009 and Wei et al., 2010). The heterogeneous nature of the 5-HT receptor family underlies the bidirectional effect of the neurotransmitter. To date, seven different receptor subfamilies have been identified which vary with respect to their localisation, coupling and ligand

binding properties (Alexander et al., 2008). A number of reports have linked descending facilitation from the brainstem to activation of spinal 5-HT3 receptors (Dogrul et al., 2009, Rygh et al., 2006, Suzuki et al., 2002 and Svensson Cepharanthine et al., 2006). For instance, using in vivo electrophysiological methods, we have demonstrated a pro-nociceptive function for spinal 5-HT3 receptors on spinal neuronal activity since topical spinal application of the selective antagonist ondansetron significantly reduced spinal neuronal activity in normal and pathaphysiological conditions ( Rahman et al., 2004, Suzuki et al., 2002 and Suzuki et al., 2004). This pronociceptive role for spinal 5-HT3 receptors has also been borne out by behavioural and anatomical studies ( Dogrul et al., 2009, Oatway et al., 2004, Svensson et al., 2006 and Zeitz et al.

1% trifluoroacetic acid (TFA) in water (solvent A), and acetonitr

1% trifluoroacetic acid (TFA) in water (solvent A), and acetonitrile and solvent A (9:1) as solvent B. The separations were performed at a flow rate of 1 mL/min using a Shim-pack VP-ODS C-18 column (4.6 × 150 mm) and a 20–60% gradient of solvent B over 20 min. In all cases, elution was followed by ultraviolet absorption (214 nm). The scissile bonds in the peptides were determined by mass spectrometry analyses. The peptide fragments were detected by scanning from m/z 100 to m/z 1300 using an Esquire 3000 Plus Ion trap Mass Spectrometer with ESI and esquire CONTROL

software (Bruker Daltonics, MA, USA). Purified 18O-labeled or unlabeled oxidized W derivatives were dissolved in a mixture of 0.01% formic acid:acetonitrile (1:1) and infused into the mass (direct infusion pump) spectrometer at a flow rate of 240 μL/h. The skimmer voltage of the capillary was 40 kV, the dry gas was kept at 5.0 L/min, and the source Z-VAD-FMK datasheet temperature was maintained at 300 °C. After defining the natural peptides that were hydrolyzed by BjV, the ability of the other venoms to hydrolyze

angiotensin I (65 μM) was analyzed using 4 μL of each ATM/ATR inhibitor drugs one (B. alternatus [5.74 mg/mL], B. jararacussu [3.11 mg/mL], B. moojeni [0.86 mg/mL] and B. neuwiedi [0.11 mg/mL]). The scissile bonds found in angiotensin-I produced by these venoms were deduced by internal standardization of the HPLC system, using the results obtained with B. jararaca as reference. The ability of the antibothropic serum to neutralize the venoms proteolytic activities was estimated by incubating Rapamycin mouse it with Bothrops spp. venoms. Samples of Bothrops venoms were incubated, at room temperature, in the presence and absence of the antibothropic serum. The residual proteolytic activities of the venoms were measured as described above, using both FRETs substrates. The volume of the antibothropic serum and the pre-incubation time for serum neutralization of the proteolytic activities were established by using the B. jararaca venom. After establishing the best conditions to neutralize the metallo- and serine peptidases from the B. jararaca venom, the other Bothops spp venoms were tested (B. alternatus, B. jararacussu, B. moojeni and B. neuwiedi). The venoms were

used in volumes of 2.0 μL when the Abz-Metal was utilized as substrate and 0.2 μL for the kinetics with the Abz-Serine (see concentration on 2.5). For the maximum blocking effect of the proteolytic activity, the venoms were incubated with 10 μL of the antibothropic serum for 30 min at room temperature. After this period, 5 μM of each substrate was added and the residual activity was measured as described above. The experiments were made in triplicate. The same concentrations of Bothrops spp venoms described in the angiotensin-I degrading assays were utilized to determine the neutralizing potential of the commercial serum. Thus, after a pre-incubation time (venoms and antivenom), 65 μM of angiotensin I was added and after 1 h more samples were analyzed by HPLC reverse-phase.

, 2011) Thus, it is not surprising that a monotonic dose respons

, 2011). Thus, it is not surprising that a monotonic dose response was not found, particularly for endpoints related to the development of the embryos. Carls et al. (1999) stated, but did not demonstrate, that all measured aqueous PAH were freely dissolved and none were associated with oil droplets, which leads to the assumption that all the individual PAH in exposure water were bioconcentrated independent

of each other and other chemicals in the effluent. However, the analytical methods used by Carls et al. (1999) and in related studies, did not distinguish between freely dissolved and particulate oil (see Page et al., 2012). These assumptions are critical to the selection of TPAH as a dose metric and render the findings questionable because the effluents from the different oil-on-gravel loadings Volasertib contained different initial concentrations and compositions of the measured Selleckchem PCI 32765 alkanes and PAH that changed during both of the 16-day experiments. The presence of low solubility alkanes and high molecular weight alkyl PAH in the effluents from the oiled gravel column studies

(EVOSTC, 2009, Brannon et al., 2012 and Page et al., 2012; Supplementary data) is indicative of the presence of a non-dissolved or micro droplet oil phase in the column effluents that probably contained all or most of the higher molecular weight PAH (Faksness et al., 2004 and Redman et al., 2012). Therefore, the uptake and toxicity of PAH in the Carls et al. (1999) study likely cannot be attributed solely to a freely dissolved fraction of the oil PAH, and the likely presence of oil droplets represents an additional confounding factor that would affect the accumulated dose and that was not reported or discussed as part of the toxicology evaluation. Thus, total aqueous PAH, as measured, represented both freely dissolved and unknown amounts of PAH associated with oil droplets. TPAH concentrations in exposure water declined rapidly and PAH composition changed continuously over the course of the 16-day exposures in all doses of LWO and MWO (Carls et al., 1997, Carls et al., 1999 and EVOSTC, 2009; Supplementary data). The rapid decline of TPAH concentration in the LWO and MWO effluents during the 16-day

exposures (Fig. 1) was largely the result of losses medroxyprogesterone of lower molecular weight PAH, particularly naphthalene and alkyl-naphthalenes (Table 1). The relative concentrations of different individual PAH and PAH congener groups, as a percentage of TPAH concentration (%TPAH), changed in all effluent doses during the 16-day exposures, with percent alkyl-naphthalenes declining and percent alkyl-phenanthrenes, alkyl-dibenzothiophenes, and alkyl-chrysenes increasing in the low, mid, and high doses during the first 4 days of exposure (Table 1) and during the remainder of the two 16-day experiments. Thus, PAH exposure concentration declined and relative compositions were different for each dose during the course of the LWO and MWO experiments (EVOSTC, 2009).

gondii infection profoundly alters the manner in which rodents pe

gondii infection profoundly alters the manner in which rodents perceive and respond to stressful stimuli ( Webster,

2007), only two previous studies have investigated whether T. gondii is related to human anxiety ( Groer et al., 2011 and Miman et al., 2010). Groer et al. assessed whether T. gondii seropositivity NU7441 chemical structure and serointensity were associated with anxiety among a cohort of pregnant women enrolled in a study of postpartum thyroiditis, as assessed by the Profile of Mood Disorder States (POMS), a non-clinical diagnostic screening instrument ( Groer et al., 2011). Similar to our study, the authors found a positive correlation between T. gondii serointensity and the POMS tension-anxiety subscale score (r = 0.31, p < 0.04). However, use of the POMS limited Groer et al. to scoring participants on a 5-point anxiety scale, whereas our study utilized buy Cobimetinib a validated survey instrument that enabled us to assign subjects clinical diagnoses of GAD. In addition, generalizability of their findings were limited to pregnant women enrolled in a study of postpartum thyroiditis ( Groer et al., 2011), whereas we included a subset of individuals drawn from a population-based sample in our study. To our knowledge, only one prior study has examined associations between T. gondii and any anxiety disorder as diagnosed by DSM-IV criteria ( Miman et al., 2010). In a case-control study of 142

subjects, Miman et al. found that individuals with psychiatrist-diagnosed

obsessive–compulsive disorder (OCD) were more likely to be seropositive for T. gondii than were healthy controls (chi-square 12.12, p < 0.01). However, the authors did not report continuous or categorical antibody levels. Overall, our study is the first to demonstrate that, in addition to a positive association between T. gondii seropositivity and GAD, there may be a graded relationship between T. gondii IgG antibody levels and odds of GAD. While the underlying PTK6 mechanisms by which T. gondii specifically affects GAD but not PTSD or depression remain uncertain, potential anxiogenic pathways include histopathological, immunological, and neuromodulatory alterations ( Webster, 2007). Rodent studies have failed to uncover a highly selective tropism of T. gondii for a specific brain region; tissue cysts have been detected throughout the brain, with observed distribution patterns varying both between ( Berenreiterova et al., 2011, Haroon et al., 2012 and Vyas et al., 2007) and within ( Berenreiterova et al., 2011) studies. However, cyst density does not appear homogenous across brain regions ( Berenreiterova et al., 2011), while a recent study suggests that cysts may preferentially persist and increase in number in limbic regions known to mediate anxiety, including the amygdala and hypothalamus ( Haroon et al., 2012). In vivo studies of chronically infected rodents indicate that T.

For steady flows, the multizone model of flow between compartment

For steady flows, the multizone model of flow between compartments employs a semi-empirical closure model to relate the pressure drop with the average velocity through the holes. The approach

adopted here is consistent with other studies (see Chu et al., 2009, Mora Dapagliflozin mw et al., 2003 and Tan and Glicksman, 2005). The pressure difference between two neighbouring compartments [i1][j1][i1][j1] and [i2][j2][i2][j2] is equation(5) p[i1][j1]−p[i2][j2]=ξ[i1][j1],[i2][j2]ρ|f[i1][j1],[i2][j2]|f[i1][j1],[i2][j2]A[i1][j1],[i2][j2]2.Here ξ[i1][j1],[i2][j2]ξ[i1][j1],[i2][j2] is the local pressure loss coefficient between compartment [i1][j1][i1][j1] and [i2][j2][i2][j2], which is assumed to be constant. The pressure loss coefficient ξ is usually determined empirically. For instance, Talazoparib mouse for flow through a sharp-edged circle orifice (see Cao et al., 2011, Charles et al., 2005 and Chu et al., 2009) which is typical of the connection between compartments in ballast tanks, the pressure loss coefficient can be estimated by ( Chu et al., 2010) equation(6) ξ=2.58[1−exp(−60β)],ξ=2.58[1−exp(−60β)],where β is the ratio of the cross-sectional area of the orifice to the cross-sectional area of the partition wall. The fluid is transported

by the mean flow and mixed by turbulent dispersion. The mean flow is largest in the passage between compartments and is smallest within compartments. Integrating the flushed fraction over compartment [i][j][i][j], we Tacrolimus (FK506) have an approximate model describing the variation of the flushed fraction with time, i.e. equation(7) V[i][j]dC[i][j]dt=∑f[i][j],inC[i][j],in−∑f[i][j],outC[i][j],where C[i][j],inC[i][j],in

is the flushed fraction in the compartment(s) flowing into compartment [i][j]. The general multizone model that consists of (4), (5) and (7) for an m×n tank is described in more detail in Appendix A. The mathematical model generates a time series for the flushed fraction of water in each compartment. A set of diagnostic tools are required to quantify the timescale when each compartment is flushed and the rate at which they are flushed by the incoming water. The dimensionless characteristic time T1/2,[i][j]T1/2,[i][j] for flushing is identified when half of the original fluid in compartment [i  ][j  ] has been flushed out, mentioned as ‘half flushed time’ equation(8) T1/2,[i][j]=T|C[i][j]=1/2,T1/2,[i][j]=T|C[i][j]=1/2,and α1/2,[i][j]α1/2,[i][j] represents the characteristic flushing rate, at which compartment [i  ][j  ] is being flushed when half of its original fluid has been flushed out (that is, when T=T1/2,[i][j]T=T1/2,[i][j]) equation(9) α1/2,[i][j]=V[i][j]VdC[i][j]dT|T=T1/2,[i][j]. The flushing efficiency C¯, is defined as the fraction of the original fluid that has been flushed out of the whole tank, i.e. equation(10) C¯(T)=∑i∑jC[i][j]V[i][j]∑i∑jV[i][j].

msc org/track-a-fishery/fisheries-in-the-program/certified/pacifi; accessed 25th July 2013). If this sets

a precedent for certification of purse seine fisheries this may mark a move away from FAD fishing with renewed focus on pursuing free schools. The increase in the use of FADs over the past two decades has given rise to concern over their associated ecological impacts, yet management of FAD fishing is complicated by the compromise between achieving a reduction in these impacts and allowing the sustainable exploitation of healthy tuna stocks, namely skipjack tuna. This is complicated further by the current reliance of the purse seine fishery on this highly efficient fishing practice, which is likely to only increase further under a business-as-usual scenario as fishing operations

become more expensive Cabozantinib research buy and shrinking profit margins require an ever greater use of FADs. Metabolism inhibitor However, continued growth in FAD fishing might be expected to result in diminishing returns as the relative benefit of each FAD in the fishery is diluted. Explicit management of the use of FADs is undoubtedly a necessity to ensure future sustainability of the fishery. Whilst there are several options available to manage the use of FADs, each option is expected to produce a different response from the purse seine fleet. Time-area closures have already been implemented but with mixed success in reducing juvenile mortality due to the flexibility of the fleet in reallocating effort. Whilst larger (and longer) closures may achieve greater reductions in juvenile catch this would be at the expense of significant reductions in skipjack catch. This has major implications on the fishing and processing industries based in the Indian Ocean, with a realistic danger that many purse seiners would choose

to leave the Indian Ocean altogether. On the other hand, input controls such as limiting this website the number of actively monitored FADs or the number of sets made on floating objects directly address concerns about FAD fishing, if designed and implemented appropriately, but are likely to be challenging to negotiate within the IOTC and difficult to enforce. We are grateful to the Economic and Social Research Council and the Natural Environment Research Council for funding this research. This paper is a contribution from Imperial College’s Grand Challenges in Ecosystems and the Environment initiative. Generous thanks are also given to J. Pearce, L. Dagorn and A. Fonteneau for informative discussion on the current and future management of FAD fishing, and to J.J. Areso, several members of staff at the Seychelles Fishing Authority and a number of anonymous skippers who gave up their time to offer invaluable insight into the practical aspects of purse seine fishing. “
“Coastal communities throughout the developing world are recognised as being particularly vulnerable to environmental change [1], [2] and [3].

, 1998; Jangchud & Chinnan, 1999) However, soy protein films bec

, 1998; Jangchud & Chinnan, 1999). However, soy protein films became more resistant as the air temperature was increased up to 70 °C, when using higher RH ( Denavi et al., 2009). Here, the flour films plasticized with sorbitol exhibit larger TS values and lower E values than the films plasticized with glycerol, for all the drying conditions ( Tables 1 and 2). Tapia-Blácido et al. (2011) also verified that the flour film plasticized with sorbitol is more resistant to break and less flexible than the

film plasticized with glycerol. According to these authors, compared with sorbitol, glycerol is a more powerful plasticizer. This is because glycerol has smaller molecular mass (glycerol 92 mol g−1 and sorbitol 182 mol g−1), which makes it a more effective plasticizer for many edible films. Young’s modulus exhibits the same behavior as the TS as a function of T and RH (figure not shown). The larger YM values for films plasticized selleck compound with sorbitol are obtained at higher drying rates, so a different behavior is detected for the films plasticized with

glycerol. In the latter case, intermediate temperatures and a wide range of relative humidity give higher YM values. According to the analysis of variance (ANOVA), the linear, quadratic, and interaction parameters are statistically significant (p < 0.05). Therefore, these parameters were considered in the second-order model for the solubility (equations (12) and (13)). Because the F values were greater than Dasatinib cost the listed values, the models can be considered predictive. For glycerol: equation(12)

S=55.99−3.07X1−3.59X12−6.41X2−9.69X22−4.35X1X2(R2=0.87) For sorbitol: equation(13) S=47.35−7.59X2+2.16X12−7.33X22+5.10X1X2(R2=0.90) The solubility (S) response surface obtained for flour films plasticized with glycerol contains a maximum region ( Fig. 4a), which does not occur for the films plasticized with sorbitol ( Fig. 4b). The maximum solubility of the flour film plasticized with glycerol can be verified at T ranging from 30 to 40 °C and RH from 45 to 60%, so intermediate drying rates yield more soluble flour films. On the other hand, the solubility of flour films plasticized with sorbitol increases almost Ribose-5-phosphate isomerase in the full range of the RH when the films are dried at temperatures below 30 °C. However, at high T values (>40 °C), the solubility decreases when the RH values range from 33.8 to 40%, and from 70 to 76.2%. Thus, high drying rates as well as intermediate drying rates allow for the formation of films with low solubility. It can be assumed that these drying conditions promote hydrophobic interactions between lipid and proteins, as well as protein–protein and starch–starch interactions, with homogenous distribution of these interactions within the film matrix. All these interactions can culminate in lower solubility of the amaranth flour film.

Loops were optimized using MODLOOP ( Fiser and Sali, 2003b) based

Loops were optimized using MODLOOP ( Fiser and Sali, 2003b) based on the satisfaction of spatial restraints, without relying Selleck Small molecule library on a database of known protein structures. The DOPE potential was evaluated for all models, and the model with the lowest global score was selected for explicit solvent molecular dynamics simulation using the GROMACS package (

Lindahl et al., 2001) and the GROMOS-96 (43a1) force field to check its stability and consistency. The overall and local quality of the final model was assessed by VERIFY3D ( Eisenberg et al., 1997), PROSA ( Wiederstein and Sippl, 2007) and VADAR ( Willard et al., 2003). Three-dimensional structures were analyzed and compared using the program PyMoL ( The results obtained were expressed as the mean ± standard deviation (SD) and statistically analyzed by applying a one-way ANOVA, followed by the Tukey method. Differences with p < 0.05 were considered

statistically significant. A new proteinase isolated from the venom of Bothrops atrox, which is a snake native to the state of Pará in Brazil, was obtained by two chromatographic procedures. The first step consisted of gel filtration on a Sephadex G-75 column under alkaline conditions (pH 8.0). The chromatogram shown in Fig. 1A illustrates the five major fractions obtained (Ba I to Ba V). Fraction Ba III presented hemorrhagic activity. The SDS-PAGE analysis of the fraction content under reduced conditions ( Fig. 1A insert) shows that Ba III contained two proteins, with one main band presenting a molecular mass of approximately 27 kDa and the second band presenting a molecular mass of approximately 17 kDa. Ba III was submitted to a second purification procedure using anion exchange chromatography ( Fig. 1B). Unbound material was eluted in 50 mM ambic pH 7.4, whereas the bound proteins were

eluted with a linear gradient of increasing concentrations of ambic pH 7.4, up to 500 mM. The resulting fractions (ES I and ES II) were assayed for hemorrhagic activity, check details and fraction ES I was able to induce dorsal skin hemorrhage in mice. SDS-PAGE ( Fig. 1B insert) shows that ES I produced a single protein band of approximately 27 kDa under reducing conditions. To confirm the purity of the fraction, ES I was submitted to reverse phase chromatography on HPLC, which revealed a single homogenous peak ( Fig. 1C). In addition, isoelectric focusing produced a single protein band with a pI of 7.5 ( Fig. 1D). The MALDI-TOF mass spectrometry analysis, based on a single charged molecule, identified a protein with a molecular mass of 22.9 kDa (data not shown). Taken together, these results confirm the isolation of Batroxase, a new protein from Bothrops atrox snake venom. Batroxase was able to induce hemorrhaging after intradermal injection in the dorsal skin of mice, with a DMH of 10 μg (Fig. 2A).

PBMC were maintained in culture for 24 h in five different condit

PBMC were maintained in culture for 24 h in five different conditions:Phα1β (100 nM), ω-conotoxin MVIIA (100 nM), morphine (100 nM), lipopolysaccharides (LPS) (1 μg/ml;

positive control) and PBS (negative control). Flow cytometryc analyses were performed as previously described (Torres et al., 2005) with the following modifications: PBMC (2 × 105) were cultured (as described above) in 200 μl of culture media in 96-well plates for 24 h. After that, cells were then stained with antibodies labeled with fluorescein isothiocyanate (FITC) and phycoerytrin (PE) for 20 min (4 °C). Thereafter, PBMC were washed with 0.1% sodium azide in PBS, and fixed with 2% formaldehyde in PBS. The antibody used for extracellular staining was anti-CD14-FITC. After extracellular PR-171 mw staining, the cells were permeabilized with a solution of 0.5% saponin and stained for cytoplasmic selleck compound proteins during 30 min (room temperature) using PE anti-IL-1β, anti-IL-10 and anti-IL-6 antibodies. PBMC were washed with 0.5% saponin in PBS, and fixed with 2% formaldehyde in PBS. FITC and PE-labeled immunoglobulin isotype control

antibodies were included in all experiments. The stained cells were analyzed using a GUAVA EasyCyte plus (GE) and the CytoSoft 5.3 software. Leukocytes and monocytes were analyzed for their frequencies of surface markers and intracellular cytokines expression using the program GUAVA Express Pro (GE). Data of withdrawal response were presented as mean ± standard error of the mean (SEM) and were analyzed by two-way ANOVA followed by Bonferroni test. HR and BP were expressed as means ± standard deviation (SD) and were analyzed by one-way ANOVA followed by Student–Newman–Keuls test. GNS data were presented

as median and 25–75 interquartile range and analyzed with Newman–Keuls multiple 17-DMAG (Alvespimycin) HCl comparison test. Horizontal and vertical activity data of rats were expressed as mean ± SEM and were analyzed using Newman–Keuls multiple comparison test. Cytokines levels were expressed as median and 25–75 interquartile range and were analyzed using Kruskal–Wallis test followed by Dunn’s multiple comparison test. A value of P < 0.05 was assumed as statistically significant for all experiments. PBS was used as a control during the different treatments (toxins and morphine). The plantar incision produced a marked mechanical allodynia in the injured paw (Fig. 1; P < 0.05). Preemptive use of Phα1β (100 pmol/site) produced an antiallodynic effect from 2 to 6 h after the injection with a maximal effect of 60 ± 7% at 2 h ( Fig. 1a). An intrathecal administration of Phα1β (200 pmol/site) induced a long-lasting antinociception (24 h) and the maximal effect was 36 ± 5% at 3 h ( Fig. 1b).

1 ml/29 g body weight and samples of the parotid and submandibula

1 ml/29 g body weight and samples of the parotid and submandibular glands were collected. The animals were then sacrificed with an overdose of the anaesthetic according to the ethical guidelines of the Brazilian College of Animal Experimentation (COBEA). The salivary gland samples were analysed by fluorescence microscopy for the observation of INS-R and ER-alpha. Frozen samples of the salivary glands

were cut into 12-μm thick sections and incubated in blocking solution for 1 h at room temperature for the blockade of nonspecific protein–protein binding sites. Next, the material was incubated for 1 h with the primary rabbit polyclonal antibody against ER-alpha (Santa Cruz Biotechnology, San Diego, CA, USA). The slides were then washed in phosphate buffered saline and incubated with the PD-166866 cell line fluorescent conjugated TSA HDAC solubility dmso secondary antibody (goat anti-rabbit, Santa Cruz Biotechnology, San Diego, CA, USA). The sections were mounted in 1,4-diazabicyclo[2.2.2]-octane (Sigma, St. Louis, MO, USA). For the evaluation of insulin receptors, a similar protocol was applied using a primary antibody against INS-R (Santa Cruz Biotechnology, San Diego, CA, USA) diluted in blocking solution. After incubation

with the primary antibody, the sections were washed in phosphate buffered saline and the secondary fluorescein-conjugated antibody (goat anti-rabbit, Santa Cruz Biotechnology, San Diego, CA, USA) diluted in blocking solution was applied. The sections were then washed in phosphate buffered saline and mounted in 1,4-diazabicyclo[2.2.2]-octane (Sigma, St. Louis, MO, USA). The specimens were examined under a TNI-06T-PL fluorescence microscope at the Department of Morphology and Basic Pathology, Faculty of Medicine of Jundiaí. The images were acquired using 10× and 40× objectives. Sections in which the primary antibody was omitted served as negative controls. The intensity of staining was scored as intense, moderate and mild according

to the intensity and distribution of immunoexpression of the cellular receptors in the tissue sections.12 and 45 Benzatropine The mean glucose levels of untreated diabetic animals (group I) were ≥500 mg/dl. There was recovery of glucose levels in animals treated with insulin alone or combined with oestrogen, with mean levels of 190 mg/dl. These levels were similar to those of control animals (170 mg/dl). There was also a decrease of glucose levels in animals undergoing only oestrogen replacement therapy, with mean levels of 280 mg/dl. Mean blood oestrogen levels of groups III and IV (89.3 ± 18.2 pg/ml) were similar to that of the control group (group V) (93.8 ± 12.4 pg/ml). Mean oestrogen levels were 21.3 ± 7.2 pg/ml in animals of groups I and II. Group V showed intense expression of INS-R, mainly close to the acini and glandular ducts (Fig. 1A). Oestrogen receptor (ER-alpha) expression was mild and was localized in the nucleus of ductal cells (Fig. 1B and Table 1).