Plasmodium falciparum: generation of a cDNA library enriched in sporozoite-specific transcripts by directional tag subtractive hybridization

We have adapted the "directional tag subtractive hybridization" technique as a means of investigating stage-specific gene expression in Plasmodium falciparum. This technique utilizes unidirectional cDNA libraries cloned into separate lambda vectors and involves hydroxyapatite chromatographic separation of target antisense cDNA and driver sense strand cRNA followed by PCR amplification of cDNA sequences specific to the target stage. This technique enabled efficient subtraction of asexual blood stage sequences from a P. falciparum sporozoite cDNA library and led to identification of novel sporozoite sequences. This technique can be applied to study gene expression in parasite stages that are difficult to obtain routinely.     

Impairment of adenylate cyclase activity and G-proteins in human uterine leiomyoma

The mechanisms responsible for the growth of uterine leiomyoma (a frequent cause of infertility in women) are largely unknown. Some data supports that cAMP plays a role in the growth of uterine cells but there are no reports on the status of the cAMP producing system in this human benign neoplasia. In this study, biopsies from leiomyoma and the adjacent myometrium were taken from menstruating women subjected to total hysterectomy for leiomyoma. Adenylate cyclase activity was determined by a protein-binding method, and the expression of alpha(s), alphai1/2, alphai3 and alphai0) G-protein subunits was analysed by immunoblot. The leiomyoma samples exhibited a decreased expression of as and ai1/2 with respect to the adjacent myometrial tissue. No differences were observed in alphai3 and alphaio protein expression. The basal adenylate cyclase activity as well as the efficacy (as assessed by the maximal stimulation levels) of either forskolin or, to a lesser extent, Gpp[NH]p on stimulation the enzyme activity was significantly lower in leiomyoma than in myometrium, whereas the potency (as assessed by the ED50 values) of these two agents did not vary. Present data indicate that the human leiomyoma is associated with low levels of cAMP. It is conceivable that the loss of sensitivity of adenylate cyclase to endogenous regulatory molecules could be related to the pathogenesis of human leiomyomas given that cAMP inhibits the MAP-kinase cascade in uterine tissues.     

Characterization of SPACR, a sialoprotein associated with cones and rods present in the interphotoreceptor matrix of the human retina: immunological and lectin binding analysis

Rod and cone photoreceptors project from the outer retinal surface into a carbohydrate-rich interphotoreceptor matrix (IPM). Unique IPM glycoconjugates are distributed around rods and cones. Wheat germ agglutinin (WGA) strongly decorates the rod matrix domains and weakly decorates the cone matrix domains. This study characterizes the major WGA-binding glycoprotein in the human IPM, which we refer to as SPACR (sialoprotein associated with cones and rods). SPACR, which has a molecular weight of 147 kDa, was isolated and purified from the IPM by lectin affinity chromatography. A polyclonal antibody to SPACR was prepared that colocalizes in tissue preparations with WGA-binding domains in the IPM. Sequential digestion of SPACR with N- and O- glycosidases results in a systematic increase in electrophorectic mobility, indicating the presence of both N- and O-linked glycoconjugates. Complete deglycosylation results in a reduction in the relative molecular mass of SPACR by about 30%. Analysis of lectin binding allowed us to identify some of the structural characteristics of SPACR glycoconjugates. Treatment with neuraminidase exposes Galbeta1- 3GalNAc disaccharide as indicated by positive peanut agglutinin (PNA) staining, accompanied by the loss of WGA staining. Maackia amurensis agglutinins (MAA-1 and MAA-2), specific for sialic acid in alpha2-3 linkage to Gal, bind SPACR, while Sambucus nigra agglutinin (SNA), specific for alpha2-6 linked sialic acid, does not, indicating that the dominant glycoconjugate determinant on SPACR is the O-linked carbohydrate, NeuAcalpha2-3Galbeta1-3GalNAc. The abundance of sialic acid in SPACR suggests that this glycoprotein may contribute substantially to the polyanionic nature of the IPM. The carbohydrate chains present on SPACR could also provide sites for extensive crosslinking and participate in the formation of the ordered IPM lattice that surrounds the elongate photoreceptors projecting from the outer retinal surface.      

The structure, catalytic mechanism and regulation of adenylyl cyclase

The recent structure determinations of the mammalian effector enzyme adenylyl cyclase reveal the structure of its catalytic core, provide new insights into its catalytic mechanism and suggest how diverse signaling molecules regulate its activity.     

Prevalent HLA Class II Alleles in Mexico City Appear to Confer Resistance to the Development of Amebic Liver Abscess

Amebiasis is an endemic disease and a public health problem throughout Mexico, although the incidence rates of amebic liver abscess (ALA) vary among the geographic regions of the country. Notably, incidence rates are high in the northwestern states (especially Sonora with a rate of 12.57/100,000 inhabitants) compared with the central region (Mexico City with a rate of 0.69/100,000 inhabitants). These data may be related to host genetic factors that are partially responsible for resistance or susceptibility. Therefore, we studied the association of the HLA-DRB1 and HLA-DQB1 alleles with resistance or susceptibility to ALA in two Mexican populations, one each from Mexico City and Sonora. Ninety ALA patients were clinically diagnosed by serology and sonography. Genomic DNA was extracted from peripheral blood mononuclear cells. To establish the genetic identity of both populations, 15 short tandem repeats (STRs) were analyzed with multiplexed PCR, and the allelic frequencies of HLA were studied by PCR-SSO using LUMINEX technology. The allele frequencies obtained were compared to an ethnically matched healthy control group (146 individuals). We observed that both affected populations differed genetically from the control group. We also found interesting trends in the population from Mexico City. HLA-DQB1*02 allele frequencies were higher in ALA patients compared to the control group (0.127 vs 0.047; p= 0.01; pc= NS; OR= 2.9, 95% CI= 1.09-8.3). The less frequent alleles in ALA patients were HLA-DRB1*08 (0.118 vs 0.238 in controls; p= 0.01; pc= NS; OR= 0.42, 95% CI= 0.19-0.87) and HLA-DQB1*04 (0.109 vs 0.214; p= 0.02; pc= NS; OR= 0.40, 95% CI= 0.20-0.94). The haplotype HLA-DRB1*08/-DQB1*04 also demonstrated a protective trend against the development of this disease (0.081 vs. 0.178; p=0.02; pc=NS; OR= 0.40, 95% CI= 0.16-0.93). These trends suggest that the prevalent alleles in the population of Mexico City may be associated with protection against the development of ALA.     

Y Fuse? Sex Chromosome Fusions in Fishes and Reptiles

Chromosomal fusion plays a recurring role in the evolution of adaptations and reproductive isolation among species, yet little is known of the evolutionary drivers of chromosomal fusions. Because sex chromosomes (X and Y in male heterogametic systems, Z and W in female heterogametic systems) differ in their selective, mutational, and demographic environments, those differences provide a unique opportunity to dissect the evolutionary forces that drive chromosomal fusions. We estimate the rate at which fusions between sex chromosomes and autosomes become established across the phylogenies of both fishes and squamate reptiles. Both the incidence among extant species and the establishment rate of Y-autosome fusions is much higher than for X-autosome, Z-autosome, or W-autosome fusions. Using population genetic models, we show that this pattern cannot be reconciled with many standard explanations for the spread of fusions. In particular, direct selection acting on fusions or sexually antagonistic selection cannot, on their own, account for the predominance of Y-autosome fusions. The most plausible explanation for the observed data seems to be (a) that fusions are slightly deleterious, and (b) that the mutation rate is male-biased or the reproductive sex ratio is female-biased. We identify other combinations of evolutionary forces that might in principle account for the data although they appear less likely. Our results shed light on the processes that drive structural changes throughout the genome.     

Nucleosides Present on Phlebotomine Saliva Induce Immunossuppression and Promote the Infection Establishment

Background

Sand fly saliva plays a crucial role in establishing Leishmania infection. We identified adenosine (ADO) and adenosine monophosphate (AMP) as active pharmacologic compounds present in Phlebotomus papatasi saliva that inhibit dendritic cell (DC) functions through a PGE₂/IL 10-dependent mechanism.

Methodology/Principal Findings

Herein, we prepared a mixture of ADO and AMP in equimolar amounts similar to those present in the salivary-gland extract (SGE) form one pair of salivary glands of P. papatasi and co-injected it with Leishmania amazonensis or L. major into mouse ears. ADO+AMP mimicked exacerbative effects of P. papatasi saliva in leishmaniasis, increasing parasite burden and cutaneous lesions. Enzymatic catabolism of salivary nucleosides reversed the SGE-induced immunosuppressive effect associated with IL-10 enhancement. Immunosuppressive factors COX2 and IL-10 were upregulated and failed to enhance ear lesion and parasite burden in IL 10^-/- infected mice. Furthermore, nucleosides increased regulatory T cell (Treg) marker expression on CD4⁺CD25^- cells, suggesting induction of Tregs on effector T cells (T eff). Treg induction (iTreg) was associated with nucleoside-induced tolerogenic dendritic cells (tDCs) expressing higher levels of COX₂ and IL-10. In vitro generation of Tregs was more efficient in DCs treated with nucleosides. Suppressive effects of nucleosides during cutaneous leishmaniasis were mediated through an A2_AR-dependent mechanism. Using BALB/c mice deficient in A2A ADO receptor (A2_AR^–/–), we showed that co-inoculated mice controlled infection, displaying lower parasite numbers at infection sites and reduced iTreg generation.

Conclusion/Significance

We have demonstrated that ADO and AMP in P. papatasi saliva mediate exacerbative effects of Leishmania infection by acting preferentially on DCs promoting a tolerogenic profile in DCs and by generating iTregs in inflammatory foci through an A2_AR mechanism.     

Severity of Old World Cutaneous Leishmaniasis Is Influenced by Previous Exposure to Sandfly Bites in Saudi Arabia

BackgroundThe sandfly Phlebotomus papatasi is the vector of Leishmania major, the main causative agent of Old World cutaneous leishmaniasis (CL) in Saudi Arabia. Sandflies inject saliva while feeding and the salivary protein PpSP32 was previously shown to be a biomarker for bite exposure. Here we used recombinant PpSP32 to evaluate human exposure to Ph. papatasi bites, and study the association between antibody response to saliva and CL in endemic areas in Saudi Arabia.ConclusionsOur findings suggest a possible correlation between the type of immunity generated by the exposure to sandfly bites and disease outcome.