Effects of dopaminergic and cholinergic interactions on rat behavior.

The present work studied the effects of dopaminergic and muscarinic receptor agonists and antagonists on rat locomotor activity and catalepsy. Results showed that carbachol at the highest dose used (10 mg/kg, p.o.) decreased and pimozide at the dose used abolished locomotor activity. Atropine at a low dose (1 mg/kg, p.o.) increased and at a high dose decreased this parameter. Mazindol at a high dose also increased locomotor activity. A significant and dose-dependent increase in the time on the bar was observed in animals treated with carbachol or pimozide as compared to controls. The increase observed with pimozide was greater than 60 s. Effects of carbachol on locomotor activity were observed already after the first drug exposure, but the increased time on bar produced by this drug in the test of catalepsy was observed only after repeated exposure (7th day). The effect of the highest dose (10 mg/kg, p.o.) of atropine (decreased activity) as related to the lowest one was evident at the 7th day, but the increased locomotor activity seen at the low dose was detected already at the first day. There was a predominance of the effect of pimozide on the open field as well as on catalepsy after its association with each one of the three doses of carbachol. The association of atropine and mazindol did not seem to alter locomotor activity and catalepsy as related to each drug alone. Our results indicate that interactions between dopaminergic and cholinergic systems play an important role on behavior and motor functions.     

Structure–Function Relationship of New Crotamine Isoform from the <Emphasis Type="Italic">Crotalus durissus cascavella</Emphasis>

In this work we isolated a novel crotamine like protein from the Crotalus durissus cascavella venom by combination of molecular exclusion and analytical reverse phase HPLC. Its primary structure was:YKRCHKKGGHCFPKEKICLPPSSDLGKMDCRWKRK-CCKKGS GK. This protein showed a molecular mass of 4892.89 Da that was determined by Matrix Assisted Laser Desorption Ionization Time-of-flight (MALDI-TOF) mass spectrometry. The approximately pI value of this protein was determined in 9.9 by two-dimensional electrophoresis. This crotamine-like protein isolated here and that named as Cro 2 produced skeletal muscle spasm and spastic paralysis in mice similarly to other crotamines like proteins. Cro 2 did not modify the insulin secretion at low glucose concentration (2.8 and 5.6 mM), but at high glucose concentration (16.7 mM) we observed an insulin secretion increasing of 2.7–3.0-fold than to control. The Na⁺ channel antagonist tetrodoxin (6 mM) decreased glucose and Cro 2-induced insulin secretion. These results suggested that Na⁺ channel are involved in the insulin secretion. In this article, we also purified some peptide fragment from the treatment of reduced and carboxymethylated Cro 2 (RC-Cro 2) with cyanogen bromide and protease V8 from Staphylococcus aureus. The isolated pancreatic -cells were then treated with peptides only at high glucose concentration (16.7 mM), in this condition only two peptides induced insulin secretion. The amino acid sequence homology analysis of the whole crotamine as well as the biologically-active peptide allowed determining the consensus region of the biologically-active crotamine responsible for insulin secretion was KGGHCFPKE and DCRWKWKCCKKGSG.     

Evaluation of apoptosis markers in different cell lines infected with equine arteritis virus

Equine arteritis virus (EAV) induces apoptosis in infected cells. Cell death caused by EAV has been studied mainly using three cell lines, BHK-21, RK-13 and Vero cells. The mechanism of apoptosis varies among cell lines and results cannot be correlated owing to differences in EAV strains used. We evaluated different markers for apoptosis in BHK-21, RK-13 and Vero cell lines using the Bucyrus EAV reference strain. Acridine orange/ethidium bromide staining revealed morphological changes in infected cells, while flow cytometry indicated the extent of apoptosis. We also observed DNA fragmentation, but the DNA ladder was detected at different times post-infection depending on the cell line, i.e., 48, 72 and 96 h post-infection in RK-13, Vero and BHK-21 cells, respectively. Measurement of viral titers obtained with each cell line indicated that apoptosis causes interference with viral replication and therefore decreased viral titers. As an unequivocal marker of apoptosis, we measured the expression of caspase-3 and caspases-8 and -9 as extrinsic and intrinsic markers of apoptosis pathways, respectively. Caspase-8 in BHK-21 cells was the only protease that was not detected at any of the times assayed. We found that Bucyrus EAV strain exhibited a distinctive apoptosis pathway depending on the cell line.     

Blood shizonticidal activities of phenazines and naphthoquinoidal compounds against Plasmodium falciparum in vitro and in mice malaria studies

Due to the recent advances of atovaquone, a naphthoquinone, through clinical trials as treatment for malarial infection, 19 quinone derivatives with previously reported structures were also evaluated for blood schizonticide activity against the malaria parasite Plasmodium falciparum. These compounds include 2-hydroxy-3-methylamino naphthoquinones (2-9), lapachol (10), nor-lapachol (11), iso-lapachol (12), phthiocol (13) and phenazines (12-20). Their cytotoxicities were also evaluated against human hepatoma and normal monkey kidney cell lines. Compounds 2 and 5 showed the highest activity against P. falciparum chloroquine-resistant blood-stage parasites (clone W2), indicated by their low inhibitory concentration for 50% (IC₅₀) of parasite growth. The therapeutic potential of the active compounds was evaluated according to the selectivity index, which is a ratio of the cytotoxicity minimum lethal dose which eliminates 50% of cells and the in vitro IC₅₀. Naphthoquinones 2 and 5, with activities similar to the reference antimalarial chloroquine, were also active against malaria in mice and suppressed parasitaemia by more than 60% in contrast to compound 11 which was inactive. Based on their in vitro and in vivo activities, compounds 2 and 5 are considered promising molecules for antimalarial treatment and warrant further study.     

Rheumatoid factor and anti-citrullinated protein antibody positivity,but not level,are associated with increased mortality in patients with rheumatoid arthritis: results from two large independent cohorts

Introduction

This study aimed to investigate rheumatoid factor (RF) and anti-citrullinated protein antibody (ACPA) status and levels as predictors of mortality in two large cohorts of patients with early inflammatory arthritis (EIA).

Methods

Data from the Norfolk Arthritis Register (NOAR) and Leiden Early Arthritis Clinic (EAC) cohorts were used. At baseline, patients had demographic data and smoking status recorded; RF, ACPA and inflammatory markers were measured in the local laboratories. Patients were flagged with national death registers until death or censor date. Antibody status was stratified as negative, low or high positive by RF and ACPA levels individually. In addition, patients were grouped as seronegative, RF positive, ACPA positive or double antibody (RF and ACPA) positive. Cox regression models explored associations between antibody status and mortality adjusting for age, sex, smoking status, inflammatory markers and year of enrolment.

Results

A total of 4962 patients were included, 64% were female. Median age at onset was 56 (NOAR) and 54 (EAC) years. In NOAR and EAC respectively, 35% and 42% of patients were ACPA/RF positive. When antibody status was stratified as negative, low or high positive, there were no consistent findings between the two cohorts. Double antibody positivity was associated with excess mortality in both cohorts compared to seronegative patients: NOAR and EAC respective adjusted HR (95% confidence interval) 1.35 (1.09 to 1.68) and 1.58 (1.16 to 2.15).

Conclusions

Patients with EIA who are seropositive for both RF and ACPA have increased mortality compared to those who are single positive or seronegative. Antibody level in seropositive patients was not consistently associated with excess mortality.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0483-3) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">Escherichia coli</Emphasis> phylogenetic group determination and its application in the identification of the major animal source of fecal contamination

Background  

Escherichia coli strains are commonly found in the gut microflora of warm-blooded animals. These strains can be assigned to one of the four main phylogenetic groups, A, B1, B2 and D, which can be divided into seven subgroups (A₀, A₁, B1, B2₂, B2₃, D₁ and D₂), according to the combination of the three genetic markers chuA, yjaA and DNA fragment TspE4.C2. Distinct studies have demonstrated that these phylo-groups differ in the presence of virulence factors, ecological niches and life-history. Therefore, the aim of this work was to analyze the distribution of these E. coli phylo-groups in 94 human strains, 13 chicken strains, 50 cow strains, 16 goat strains, 39 pig strains and 29 sheep strains and to verify the potential of this analysis to investigate the source of fecal contamination.     

Alloxan diabetes blocks noradrenergic tachyphylaxis in the isolated rabbit kidney--effects of insulin.

Normal and alloxan treated diabetic rabbit kidneys were perfused with Krebs-Henseleit solution in a non-recirculating system and the effects of norepinephrine (NOR) 10(-6)M were tested by infusion of this drug for three subsequent periods of 20 min each, with an interval of 10 min for drug wash-out. In the control kidneys the infusion of NOR promoted an intense vasoconstriction, which was less intense during the second and the third periods. This was known as tachyphylaxis. In contrast to the controls, kidneys from diabetic animals did not show tachyphylaxis to NOR, but when insulin was added to the perfusate, tachyphylaxis appeared. Normal kidneys perfused with hyperosmolar solutions show, as in controls, the same phenomenon. The data presented here demonstrate a defect of adrenergic vascular receptors in alloxan treated kidneys, which can be acutely reversed by insulin. These facts are of importance for the understanding of the vascular disease in diabetes.     

alpha-Methyl-D-glucoside uptake in renal cortical slices of normal and alloxan diabetic rabbits.

The uptake of alpha-methyl-D-glucoside by slices of renal cortex was compared in normal and alloxan diabetic rabbits. Alloxanized rabbit tissue showed significantly higher levels of sugar accumulation than normal tissue. Diamide, which is known to oxidize intracellular glutathione (GSH), inhibited the uptake of alpha-methyl-D-glucoside by renal cortical slices. GSH stimulated sugar uptake and was also capable of reversing the inhibition of sugar accumulation caused by diamide. Mercaptoethanol and dithiothreitol, which are commonly used thiol compounds, were not as effective as GSH in stimulating sugar uptake. The level of GSH found in normal and alloxan diabetics rabbit kidneys shows a slightly decreased cortical GSH content in alloxanized animals. One can conclude that GSH participates in sugar uptake in kidney slices from both diabetic and normal rabbits.     

Insulin blockade of norepinephrine tachyphylaxis in the perfused kidney

Tachyphylaxis to norepinephrine (NOR) was determined in the rabbit kidney perfused with Krebs-Henseleit solution by using different calcium concentrations (2.5 mM; 5 mM; 12.5 mM) in the perfusate. The addition of insulin to the perfusion fluid causes a reversion of the tachyphylaxis which is seen at those Ca2+ concentrations. This effect is demonstrated mainly at 5 mM Ca2+. When kidneys were perfused with 12.5 mM calcium there was disappearance of NOR-mediated tachyphylaxis, both in the absence and in the presence of insulin. In this calcium concentration, insulin decreases vascular reactivity to NOR. These results suggest that insulin blockade of alpha adrenergic tachyphylaxis is a calcium-mediated effect which is thought to be due to an enhancement of calcium pumping inside the cells.