Characterization of pathology and parasite load in outbred and inbred mouse models of chronic Neospora caninum infection

Here, we analyzed histological findings and parasite burden in chronic Neospora caninum infection in BALB/c and ICR mice and studied the correlation between lesion severity and parasite load in brain. To obtain a better understanding of the infection, we examined the influence of various host pathogen factors. Groups of outbred (ICR) and inbred (BALB/c) mice were inoculated using several NC-1 parasite doses (4 x 10(5), 10(6), and 5 x 10(6) tachyzoites), inoculation routes (intraperitoneal and subcutaneous), and 3 immunosuppressive treatments (methylprednisolone, cyclophosphamide, and vinblastine). Lesion severity was analyzed in the liver, lung, heart, and brain tissues, and parasite load was measured by real-time polymerase chain reaction in brain tissue. The results indicated more severe cerebral lesions and higher brain parasite burdens in inbred than in outbred mice. Hepatic tissue was the primary lesion site in immunosuppressed ICR mice. We also observed that increased inoculum size was reflected in greater lesion severity and a higher cerebral parasite load. No difference was observed with respect to inoculation route. The study also showed an association between brain parasite burden and severity of cerebral lesions in BALB/c mice.     

Genetic screen for monitoring severe acute respiratory syndrome coronavirus 3C-like protease

A novel coronavirus (SCoV) is the etiological agent of severe acute respiratory syndrome. Site-specific proteolysis plays a critical role in regulating a number of cellular and viral processes. Since the main protease of SCoV, also termed 3C-like protease, is an attractive target for drug therapy, we have developed a safe, simple, and rapid genetic screen assay to monitor the activity of the SCoV 3C-like protease. This genetic system is based on the bacteriophage lambda regulatory circuit, in which the viral repressor cI is specifically cleaved to initiate the lysogenic-to-lytic switch. A specific target for the SCoV 3C-like protease, P1/P2 (SAVLQ/SGFRK), was inserted into the lambda phage cI repressor. The target specificity of the SCoV P1/P2 repressor was evaluated by coexpression of this repressor with a chemically synthesized SCoV 3C-like protease gene construct. Upon infection of Escherichia coli cells containing the two plasmids encoding the cI. SCoV P1/P2-cro and the beta-galactosidase-SCoV 3C-like protease constructs, lambda phage replicated up to 2,000-fold more efficiently than in cells that did not express the SCoV 3C-like protease. This simple and highly specific assay can be used to monitor the activity of the SCoV 3C-like protease, and it has the potential to be used for screening specific inhibitors.     

Hemizygosity at the NCF1 gene in patients with Williams-Beuren syndrome decreases their risk of hypertension

Williams-Beuren syndrome (WBS), caused by a heterozygous deletion at 7q11.23, represents a model for studying hypertension, the leading risk factor for mortality worldwide, in a genetically determined disorder. Haploinsufficiency at the elastin gene is known to lead to the vascular stenoses in WBS and is also thought to predispose to hypertension, present in approximately 50% of patients. Detailed clinical and molecular characterization of 96 patients with WBS was performed to explore clinical-molecular correlations. Deletion breakpoints were precisely defined and were found to result in variability at two genes, NCF1 and GTF2IRD2. Hypertension was significantly less prevalent in patients with WBS who had the deletion that included NCF1 (P=.02), a gene coding for the p47(phox) subunit of the NADPH oxidase. Decreased p47(phox) protein levels, decreased superoxide anion production, and lower protein nitrotyrosination were all observed in cell lines from patients hemizygous at NCF1. Our results indicate that the loss of a functional copy of NCF1 protects a proportion of patients with WBS against hypertension, likely through a lifelong reduced angiotensin II-mediated oxidative stress. Therefore, antioxidant therapy that reduces NADPH oxidase activity might have a potential benefit in identifiable patients with WBS in whom serious complications related to hypertension have been reported, as well as in forms of essential hypertension mediated by a similar pathogenic mechanism.     

Identification of a γ-hexachlorocyclohexane dehydrochlorinase (LinA) variant with improved expression and solubility properties

Under aerobic conditions, the enzyme γ-hexachlorocyclohexane dechlorinase (LinA) from Sphingomonas paucimobilis UT26 catalyses the elimination of chlorine atoms from the molecule of γ-hexachlorocyclohexane (γ-HCH) or lindane, a recalcitrant pesticide that is still widely used. In its native metabolic context, LinA starts the biodegradation process of lindane by transforming γ-HCH to 1,2,4 trichlorobenzene (TCB), a less persistent chemical. In an attempt to generate an improved version of this enzyme to be used in lindane bioremediation schemes, we have run an experimental evolution procedure on LinA, using Escherichia coli as the surrogate host. One round of random mutagenesis and subsequent screening for improved dechlorination in vivo sufficed to yield one mutant enzyme (LinAT10), bearing a single substitution C132R, that displayed a two-fold enhanced expression and three-fold enhanced solubility of the enzyme compared to the wild type protein. This resulted in a biological product with a six-fold increase in dechlorination ability when expressed in E. coli. The potential of this protein and its expression system for in situ bioremediation is discussed.     

Models of optimal foraging and resource partitioning: deep corollas for long tongues

We model the optimal foraging strategies for 2 nectarivore species,differing in the length of their proboscis, that exploit thenectar provided by 2 types of flowers, differing in the depthsof their corollas. When like flowers appear in clumps, nectarivoresmust decide whether to forage at a patch of deep or shallowflowers. If nectarivores forage optimally, at least one flowertype will be used by a single nectarivore species. Long-tonguedforagers will normally visit deep flowers and short-tonguedforagers shallow flowers, although extreme asymmetries in metaboliccosts may lead to the opposite arrangement. When deep and shallowflowers are randomly interspersed, nectarivores must decide,on encounter with a flower, whether to collect its nectar orcontinue searching. At low nectarivore densities, the optimalstrategy involves exploiting every encountered flower; however,as nectarivore densities increase and resources become scarce,long-tongued individuals should start concentrating on deepflowers and short-tongued individuals on shallow flowers. Therefore,regardless of the spatial distribution of flowers, corolla depthcan determine which nectarivore species exploit the nectar fromeach flower type in a given community. It follows that corollaelongation can evolve as a means to keep nectar thieves at bayif short-tongued visitors are less efficient pollinators thanlong-tongued visitors.     

Toxicity of diclofenac to Gyps vultures

Three endemic vulture species Gyps bengalensis, Gyps indicus and Gyps tenuirostris are critically endangered following dramatic declines in South Asia resulting from exposure to diclofenac, a veterinary drug present in the livestock carcasses that they scavenge. Diclofenac is widely used globally and could present a risk to Gyps species from other regions. In this study, we test the toxicity of diclofenac to a Eurasian (Gyps fulvus) and an African (Gyps africanus) species, neither of which is threatened. A dose of 0.8 mg kg(-1) of diclofenac was highly toxic to both species, indicating that they are at least as sensitive to diclofenac as G. bengalensis, for which we estimate an LD50 of 0.1-0.2 mg kg(-1). We suggest that diclofenac is likely to be toxic to all eight Gyps species, and that G. africanus, which is phylogenetically close to G. bengalensis, would be a suitable surrogate for the safety testing of alternative drugs to diclofenac.     

Enzymatic antioxidant response of a labrid fish (Coris julis) liver to environmental caulerpenyne

Exposure of marine animals to certain toxic compounds can enhance reactive oxygen species production with subsequent damage to macromolecules and alterations in oxidant defenses levels. Caulerpenyne is the major metabolite synthesized by Caulerpa species, used as chemical defense affecting several cellular and molecular targets. We assessed the changes produced by the presence of Caulerpa spp. in the activities of antioxidant enzymes as well as lipid peroxidation levels in liver of the teleost Coris julis. Fish were captured at two stations with Caulerpa species-Caulerpa taxifolia and Caulerpa prolifera-and at a region with the seagrass Posidonia oceanica as negative control. Caulerpenyne concentration was significantly higher in C. prolifera than in C. taxifolia (p<0.05). Glutathione S-transferase, glutathione peroxidase and glutathione reductase activities were significantly higher in both Caulerpa stations compared to the P. oceanica (p<0.05). No statistical difference (p>0.05) existed in catalase activity between groups. Glutathione reductase activity is significantly higher in C. prolifera station than in C. taxifolia (p<0.05). Despite the variations in the antioxidant enzyme activities, there was no significant difference in malondialdehyde concentration. In conclusion, the production of caulerpenyne by Caulerpa species could induce an antioxidant adaptation in the liver of C. julis in order to prevent oxidative damage.