Comparison between splenic and lymph node aspirations as sampling methods for the parasitological detection of Leishmania chagasi infection in dogs

The sensitivities of spleen and lymph node cultures for the diagnosis of canine visceral leishmaniasis were compared in 64 anti-Leishmania antibody positive dogs from an endemic area in Brazil. The sensitivity of spleen cultures for Leishmania detection was 97.9%; in lymph node cultures it was 25%. Positive spleen culture was more frequent (p = 0.048, Fisher's exact probability test) in symptomatic (28 out of 33 animals) than in asymptomatic animals (19 out of 31 animals). These results support the use of spleen instead of lymph node aspiration as the choice method for the parasitological diagnosis of the infection.     

Inhibition of voltage-gated calcium channels by sequestration of beta subunits

The auxiliary Ca(v)beta subunit is essential for functional expression of high-voltage activated Ca(2+) channels. Here, we describe a lure sequence designed to sequester the Ca(v)beta subunits in transfected bovine chromaffin cells. This sequence is composed of the extracellular and transmembrane domains of the alpha chain of the human CD8, the I-II loop of Ca(v)2.1 subunit, and EGFP. We showed that expressing the CD8-I-II-EGFP sequence in chromaffin cells led to a >50% decrease in overall Ca(2+) current density. Although this decrease involved all the Ca(2+) channel types (L, N, P/Q, R), the proportion of each type supporting the remaining current was altered. A similar effect was observed after transfection when measuring the functional role of Ca(2+) channels in catecholamine release by chromaffin cells: global decrease of release and change of balance between the different channel types supporting it. Possible explanations for this apparent discrepancy are further discussed.     

C-terminal deletion of AID uncouples class switch recombination from somatic hypermutation and gene conversion

Class-switch recombination (CSR), somatic hypermutation (SHM), and antibody gene conversion are distinct DNA modification reactions, but all are initiated by activation-induced cytidine deaminase (AID), an enzyme that deaminates cytidine residues in single-stranded DNA. Here we describe a mutant form of AID that catalyzes SHM and gene conversion but not CSR. When expressed in E. coli, AID(delta189-198) is more active in catalyzing cytidine deamination than wild-type AID. AID(delta189-198) also promotes high levels of gene conversion and SHM when expressed in eukaryotic cells, but fails to induce CSR. These results underscore an essential role for the C-terminal domain of AID in CSR that is independent of its cytidine deaminase activity and that is not required for either gene conversion or SHM.     

The effect of epistasis on the excess of the additive and nonadditive variances after population bottlenecks

The effect of population bottlenecks on the components of the genetic variance generated by two neutral independent epistatic loci has been studied theoretically (VA, additive; VD, dominant; VAA, additive x additive; VAD, additive x dominant; VDD; dominant x dominant components of variance). Nonoverdominance and overdominance models were considered, covering all possible types of marginal gene action at the single locus level. The variance components in an infinitely large panmictic population (ancestral components) were compared with their expected values at equilibrium, after t consecutive bottlenecks of equal size N (derived components). Formulae were obtained in terms of allele frequencies and effects at each locus and the corresponding epistatic value. An excess of VA after bottlenecks can be assigned to two sources: (1) the spatiotemporal changes in the marginal average effects of gene substitution alpha(i), which are equal to zero only for additive gene action within and between loci; and (2) the covariance between alpha2(i) and the heterozygosity at the loci involved, which is generated by dominance, with or without epistasis. Numerical examples were analyzed, indicating that an increase in VA after bottlenecks will only occur if its ancestral value is minimal or very small. For the nonoverdominance model with weak reinforcing epistasis, that increase has been detected only for extreme frequencies of the negative allele at one or both loci. With strong epistasis, however, this result can be extended to a broad range of intermediate frequencies. With no epistasis, the same qualitative results were found, indicating that dominance can be considered as the primary cause of an increase in VA following bottlenecks. In parallel, the derived total nonadditive variance exceeded its ancestral value (V(NA) = V(D) + V(AA) + V(AD) + V(DD)) for a range of combinations of allele frequencies covering those for an excess of VA and for very large frequencies of the negative allele at both loci. For the overdominance model, an increase in V(A) and V(NA) was respectively observed for equilibrium (intermediate) frequencies at one or both loci or for extreme frequencies at both loci. For all models, the magnitude of the change of V(A) and V(NA) was inversely related to N and t. At low levels of inbreeding, the between-line variance was not affected by the type of gene action. For the models considered, the results indicate that it is unlikely that the rate of evolution may be accelerated after population bottlenecks, in spite of occasional increments of the derived V(A) over its ancestral value.     

Extracellular products as mediators of the formation and detachment of Pseudomonas fluorescens biofilms

Pseudomonas fluorescens B52 produces substantial biofilms at the air/liquid/solid interface of glass coverslips clamped vertically and partly submerged in liquid medium at 21°C. Biofilm formation was maximal ca. 20–50 h after inoculation of the liquid medium and, as indicated by environmental scanning electron microscopy (ESEM), contained large numbers of bacterial cells that were embedded within an extensive exopolymeric matrix. Incubation beyond 50 h led to reductions in biofilm which ESEM related primarily to losses of exopolymer. Both biofilm formation and the subsequent decline in exopolymer deposition was more rapid, and occurred to greater extents, when supernatants from two-day old cultures of B52 were used as the initial growth media. The addition of N-acyl-hexanoyl homoserine lactone to fresh growth medium had a similar effect upon biofilm formation as using spent culture medium. Homoserine lactones could not be demonstrated in spent culture supernatants by an Agrobacterium tumefaciens bioassay. An exopolysaccharide lyase was detected in spent culture media taken from dense biofilm cultures whose action was specifically directed towards biofilm exopolysaccharide. Results suggest that (i) cell-cell signals such as homoserine lactones are associated with the formation of P. fluorescens biofilms, (ii) the enzymic degradation of exopolymers has a specific role in the detachment of cells under starvation conditions, and (iii) whilst short chain (C₆) exogenous homoserines can trigger such responses in P. fluorescens, its own signal substance is likely to possess a longer (>C₈) fatty acyl chain.     

Local Mixed-Effects Fitting for Detecting Reproductive Hormone Surge Times

Hormone secretion processes remain part of the biological mystery as they are highly regulated and individual specific. When hormone trajectories from multiple subjects are under investigation, both population-average mechanism and subject-specific deviations are of great interest. In particular, statistical methodologies that enable us not only to identify surge times and surge magnitudes but also to make inference on these biological features is in need. In this paper, we propose a local kernel smoothing method to perform the analysis of multiple hormone curves using the nonparametric mixed-effects model. We develop a local quadratic mixed-effects (LQME) fitting procedure that detects local maxima of the population-average profile curve and the individual profile curves. Related statistical inference is established to carry out a hypothesis test for the local surge and to construct a confidence interval for a detected surge feature. This method is illustrated by simulation studies and a reproductive hormone data analysis.     

Ovarian response in sheep superovulated after pretreatment with growth hormone and GnRH antagonists is weakened by failures in oocyte maturation

The administration of growth hormone (GH) or GH plus GnRH antagonists (GnRHa) in sheep allows the enhancement of the pool of gonadotrophin-responsive follicles present in the ovaries and may be useful to increase yields obtained in embryo programmes. The objective of the current study was to evaluate the ability of follicles recruited in response to treatment with GH and GnRHa to grow in response to exogenous follicle stimulating hormone (FSH) and the competence of their oocytes to resume meiosis. Seven females were treated with two doses of GnRHa (days 0 and 3) and three doses of 15 mg of GH (days 3, 4 and 5). Thereafter, this group and a second group (n = 7) were treated with three doses of 1.5 ml of FSH 12 h apart. A third group (control; n = 4) did not receive GH/GnRHa or FSH. The mean number of follicles aspirated on day 7 was higher in ewes treated with GH and GnRHa prior to the stimulation with exogenous FSH than in ewes treated with FSH without pretreatment and in untreated control sheep (20.4 +/- 2.6 vs 17.7 +/- 3.9 and 11.5 +/- 0.8, p < 0.05 and p < 0.01, respectively). The number of recovered cumulus-oocyte complexes after follicular aspiration was higher in the GH/GnRHa + FSH group (8.7 +/- 0.9 vs 6.8 +/- 1.3 in FSH group, n.s., and 4.5 +/- 0.8 in control, p < 0.05), but there were no differences found in the resumption of meiosis (63.1 +/- 9.5% for GH/GnRHa + FSH vs 79.5 +/- 6.3% for FSH and 60.0 +/- 8.8% for control). These results indicate that GH and GnRHa would be useful to increase the number of gonadotrophin-responsive follicles in the ovary, but adjustment of later FSH treatment allowing further development of follicles may be necessary prior to its use in superovulatory protocols.     

New records of pyrrolizidine alkaloid-feeding insects. Hemiptera and Coleoptera on Senecio brasiliensis

New records are reported for a Hemipteran (Largus rufipennis) and a Coleopteran (Chauliognathus fallax) feeding on Senecio brasiliensis (Asteraceae) and sequestering pyrrolizidine alkaloids (PAs). Both insects are warningly colored and rejected by predators.     

Purification and characterization of an adhesin from Pasteurella haemolytica

We purified an adhesin from Pasteurella. haemolytica by affinity chromatography using glutaraldehyde treated rabbit erythrocytes stroma. The adhesin is a protein of 68 kDa, as determined by SDS-PAGE, and the most abundant amino acids constituting this protein were Gly, Ser, Glx, and Ala, and low concentrations of Cys, Met, and Tyr residues were also found. The N-terminal sequence of the adhesin is ANEVNVYIYKQPYLI. No carbohydrate residues were detected. The adhesin agglutinated rabbit erythrocytes but when the latter were desialylated or pronase treated the agglutinating activity was abolished. The agglutinating activity of the adhesin was inhibited with N-acetyl-D-glucosamine (GlcNAc), and in a lesser degree with N-acetyl-neuraminic acid (NeuAc). GalNAc, N-glycolyl-neuraminic acid, N-deacetylated GlcNAc, or neutral sugars do not modify the activity of the adhesin. The equatorial -OH on C4 and the NH-acetylated group on C2 from GlcNAc, as well as the 4-OH and NH-acetylated group on C5 from NeuAc seem to be responsible for the interaction with the adhesin. The protein is divalent cation-dependent and thermolabile. As for the agglutinating activity, the adhesion of P.haemolytica to tracheal cell-cultures was inhibited by GlcNAc, NeuAc or the purified adhesin, strongly suggesting that the P.haemolytica adhesin plays an important role in infection.     

Activation of mu-opioid receptors transfers control of Galpha subunits to the regulator of G-protein signaling RGS9-2: role in receptor desensitization

In mouse periaqueductal gray matter (PAG) membranes, the mu-opioid receptor (MOR) coprecipitated the alpha-subunits of the Gi/o/z/q/11 proteins, the Gbeta1/2 subunits, and the regulator of G-protein signaling RGS9-2 and its partner protein Gbeta5. RGS7 and RGS11 present in this neural structure showed no association with MOR. In vivo intracerebroventricular injection of morphine did not alter MOR immunoreactivity, but 30 min and 3 h after administration, the coprecipitation of Galpha subunits with MORs was reduced by up to 50%. Furthermore, the association between Galpha subunits and RGS9-2 proteins was increased. Twenty-four hours after receiving intracerebroventricular morphine, the Galpha subunits left the RGS9-2 proteins and re-associated with the MORs. However, doses of the opioid able to induce tolerance promoted the stable transfer of Galpha subunits to the RGS9-2 control. This was accompanied by Ser phosphorylation of RGS9-2 proteins, which increased their co-precipitation with 14-3-3 proteins. In the PAG membranes of morphine-desensitized mice, the capacity of the opioid to stimulate G-protein-related guanosine 5'-O-(3-[35S]thiotriphosphate) binding as well as low Km GTPase activity was attenuated. The in vivo knockdown of RGS9-2 expression prevented morphine from altering the association between MORs and G-proteins, and tolerance did not develop. In PAG membranes from RGS9-2 knockdown mice, morphine showed full capacity to activate G-proteins. Thus, the tolerance that develops following an adequate dose of morphine is caused by the stabilization and retention of MOR-activated Galpha subunits by RGS9-2 proteins. This multistep process is initiated by the morphine-induced transfer of MOR-associated Galpha subunits to the RGS9-2 proteins, followed by Ser phosphorylation of the latter and their binding to 14-3-3 proteins. This regulatory mechanism probably precedes the loss of MORs from the cell membrane, which has been observed with other opioid agonists.