María de Lourdes Muñoz

Expression of Polymorphic msp1β Genes during Acute Anaplasma marginale Rickettsemia

ABSTRACT Immunization of cattle with native MSP1 induces protection againstAnaplasma marginale. The native immunogen is composed of a single MSP1a protein and multiple, undefined MSP1b polypeptides. In addition to the originally sequenced gene, designatedmsp1β(F1), we identified three completemsp1β genes in the Florida strain:msp1β(F2), msp1β(F3), andmsp1β(F4). Each of these polymorphic genes encodes a structurally unique MSP1b protein, and unique transcripts can be identified during acute A. marginale rickettsemia. The structural polymorphism is clustered in discrete variable regions, and each MSP1b protein results from a unique mosaic of five variable regions. Although each of the MSP1b proteins in the Florida strain contains epitopes recognized by serum antibody induced by protective immunization with the native MSP1 complex, the variable regions also include epitopes expressed by some but not all of the MSP1b proteins. These data support testing recombinant vaccines composed of the multiple antigenically and structurally unique MSP1b proteins combined with MSP1a in order to mimic the efficacy of native MSP1 immunization.

Entamoeba histolytica: proteinase secretion induced by collagen type I is dependent on cytoskeleton integrity

Abstractu2002Proteolytic activities of the protozoan parasite Entamoeba histolytica strain HM1:IMSS and the cytochalasin D-resistant mutant BG-3 were analyzed following stimulation with collagen type I and Ca2+, which induces electron-dense associated collagenase secretion. The mutant BG-3 had a protease activity of 73u2005kDa and secretion of total protease activity was not stimulated by collagen type I and Ca2+, which produced, in contrast, a 2-fold increase in protease secretion by the parental strain. This collagen-stimulated protease secretion was inhibited by cytochalasin D at a concentration of 1u2005μg/ml. Cytochalasin D did not have any effect on the protease activity released by the mutant BG-3. These findings suggest that cytoskeleton integrity is necessary for collagen-induced protease secretion.

Proteomic Identification of Dengue Virus Binding Proteins in Aedes aegypti Mosquitoes and Aedes albopictus Cells

The main vector of dengue in America is the mosquito Aedes aegypti, which is infected by dengue virus (DENV) through receptors of midgut epithelial cells. The envelope protein (E) of dengue virus binds to receptors present on the host cells through its domain III that has been primarily recognized to bind cell receptors. In order to identify potential receptors, proteins from mosquito midgut tissue and C6/36 cells were purified by affinity using columns with the recombinant E protein domain III (rE-DIII) or DENV particles bound covalently to Sepharose 4B to compare and evaluate their performance to bind proteins including putative receptors from female mosquitoes of Ae. aegypti. To determine their identity mass spectrometric analysis of purified proteins separated by polyacrylamide gel electrophoresis was performed. Our results indicate that both viral particles and rE-DIII bound proteins with the same apparent molecular weights of 57 and 67u2009kDa. In addition, viral particles bound high molecular weight proteins. Purified proteins identified were enolase, beta-adrenergic receptor kinase (beta-ARK), translation elongation factor EF-1 alpha/Tu, and cadherin.

Using msa-2b as a molecular marker for genotyping Mexican isolates of Babesia bovis

Variable merozoite surface antigens of Babesia bovis are exposed glycoproteins having a role in erythrocyte invasion. Members of this gene family include msa-1 and msa-2 (msa-2c, msa-2a(1), msa-2a(2) and msa-2b). To determine the sequence variation among B. bovis Mexican isolates using msa-2b as a genetic marker, PCR amplicons corresponding to msa-2b were cloned and plasmids carrying the corresponding inserts were purified and sequenced. Comparative analysis of nucleotide and deduced amino acid sequences revealed distinct degrees of variability and identity among the coding gene sequences obtained from 16 geographically different Mexican B. bovis isolates and a reference strain. Clustal-W multiple alignments of the MSA-2b deduced amino acid sequences performed with the 17 B. bovis Mexican isolates, revealed the identification of three genotypes with a distinct set each of amino acid residues present at the variable region: Genotype I represented by the MO7 strain (in vitro culture-derived from the Mexico isolate) as well as RAD, Chiapas-1, Tabasco and Veracruz-3 isolates; Genotype II, represented by the Jalisco, Mexico and Veracruz-2 isolates; and Genotype III comprising the sequences from most of the isolates studied, Tamaulipas-1, Chiapas-2, Guerrero-1, Nayarit, Quintana Roo, Nuevo Leon, Tamaulipas-2, Yucatan and Guerrero-2. Moreover, these three genotypes could be discriminated against each other by using a PCR-RFLP approach. The results suggest that occurrence of indels within the variable region of msa-2b sequences can be useful markers for identifying a particular genotype present in field populations of B. bovis isolated from infected cattle in Mexico.

Cinnabar-Preserved Bone Structures from Primary Osteogenesis and Fungal Signatures in Ancient Human Remains

The Red Queen remains (700 A.C.) found at Palenque, México, are examples of cinnabar (HgS) application to royal remains during pre-Hispanic times. The Red Queen remains are those of a ca. 30–35-yr-old female and present a striking similarity to the remains of another Mayan woman found at Copan, Honduras. Thus, covering the remains of royal women with HgS may have been a common practice in the Mayan civilization. High resolution microdiffraction and microscopic analysis of the Red Queen remains showed the presence of nanotubular organic minerals comparable in composition and molecular dimensions to collagen fibrils, and in spatial ordering to collagen fiber networks. Fungal structures are rare in the geological record because of poor preservation potential. Micrographs revealed the preservation of fungal signatures, with morphology comparable to parasitic fungal-coral matrix associations, consistent with the idea that fungal remains can be preserved in environments which contain high Hg concentrations. The well-preserved signatures of fungus-animal interactions and primary osteogenesis in the Red Queen remains are attributed to the long-term antibacterial activity of HgS and the association of sulfur components with nanotubular structures.

Mexican mestizo population sub-structure: effects on genetic and forensic statistical parameters

Since Mexican mestizos are an admixed population, it is necessary to determine the effects that the substructure of the population has on genetic and forensic parameters. With this aim, a study was performed with 15 STR loci (CODIS plus D2S1338 and D19S433) on 1,640 unrelated Mexican mestizos. We determine allele and genotypic frequencies observing departure from Hardy–Weinberg expectation (12 out of 15 loci, with an excess of homozygotes, Fisxa0>xa00), as well as pairs of loci in an apparent linkage disequilibrium (13 of 92 loci). We conducted a test for genetic population stratification, the results show that the Mexican mestizo population is substructured into three subgroups, which are in HW and linkage equilibrium. The combination of the 15 loci in the whole population has high forensic efficiency with the capacity to genetically discriminate one individual in one quintillion (1/1018). Our data potentially validates the use of these 15 STR loci to establish forensic identity and parentage testing for legal purposes, and offers a powerful tool for genetic variation analysis. However, given that the population is stratified, we highly recommend applying a correction with the inbreeding coefficient in calculations of paternity and forensic studies to avoid erroneous assumptions.

Immunolocalization of Clathrin During Electron-Dense Granule Secretion in Entamoeba histolytica

Entamoeba histolytica is characterized by the capacity of trophozoites to destroy human tissues. One of the major components in human tissues is collagen. Consequently, collagenase associated with electron-dense granules (EDG) from E. histolytica has been considered an important factor in the pathogenicity of this parasite (1). EDG secretion from E. histolytica is induced by incubation of trophozoites with collagen type I and Ca 2 1 . This induction is achieved through an increase of adhesion and tyrosine phosphorylation and activation of pp125 FAK and p42 MAPK , which results in cytoskeleton organization (2). It is known that some molecules, such as calmodulin, participate in the regulation of EDG secretion, and that the cytoskeleton is important in this biological process. However, the role of other molecules such as clathrin that participate in packaging of secretory granules in regulated secretion is unknown (3). In addition, this protein is the major coat protein of coated pits and vesicles that mediate the selective internalization and transport of receptors in mammalian cells (3). The gene of the heavy chain of this protein in trophozoites of E. histolytica has already been reported (EDZP0173). In this article, we present some evidence of the possible participation of clathrin in EDG secretion of E. histolytica.

Biochemical and immunological characterization of soluble antigens of Giardia lamblia.

Abstract The crude soluble antigens (CSA) of Giardia lamblia trophozoites and their analytically purified fractions were characterized biochemically and immunologically to determine the most immunogenic fraction and its localization on the parasite. Both Sephacryl S-300 column chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed the highly complex and heterogeneous nature of CSA. Gel filtration of CSA showed four fractions (FI–FIV) with molecular masses of 250, 150, 110, and 10u2009kDa for fractions I–IV, respectively. Protein profiles of CSA demonstrated 28 Coomassie-blue bands in the range of 200–14u2009kDa. Similar banding patterns with fewer polypeptides were observed in the FI fraction. However, fractions II and III showed polypeptide bands in the region of 97–14u2009kDa. The glycoprotein nature of CSA and its fractions were demonstrated in physicochemical analysis. In antigenic activity analysis the high-molecular-weight FI antigen was found to be 8 times more immunogenic than CSA as well as the other fractions. Major differences in the immunoreactivity of CSA and FI antigens were noted at 220, 30, and 22u2009kDa for the FI antigen and at 205, 84, 55, 43, and 20u2009kDa for CSA. Some of these FI polypeptides were found to be surface-associated as revealed by immunofluorescence and immunoblot assay. These results suggest the future use of the FI antigen in the serodiagnosis of and immunoprophylaxis against giardiasis.

Genetic Affiliation of Pre-Hispanic and Contemporary Mayas through Maternal Linage

abstract n Maya civilization developed in Mesoamerica and encompassed the Yucatan Peninsula, Guatemala, Belize, part of the Mexican states of Tabasco and Chiapas, and the western parts of Honduras and El Salvador. This civilization persisted approximately 3,000 years and was one of the most advanced of its time, possessing the only known full writing system at the time, as well as art, sophisticated architecture, and mathematical and astronomical systems. This civilization reached the apex of its power and influence during the Preclassic period, from 2000 BCE to 250 CE. Genetic variation in the pre-Hispanic Mayas from archaeological sites in the Mexican states of Yucatan, Chiapas, Quintana Roo, and Tabasco and their relationship with the contemporary communities in these regions have not been previously studied. Consequently, the principal aim of this study was to determine mitochondrial DNA (mtDNA) variation in the pre-Hispanic Maya population and to assess the relationship of these individuals with contemporary Mesoamerican Maya and populations from Asia, Beringia, and North, Central, and South America. Our results revealed interactions and gene flow between populations in the different archaeological sites assessed in this study. The mtDNA haplogroup frequency in the pre-Hispanic Maya population (60.53%, 34.21%, and 5.26% for haplogroups A, C, and D, respectively) was similar to that of most Mexican and Guatemalan Maya populations, with haplogroup A exhibiting the highest frequency. Haplogroup B most likely arrived independently and mixed with populations carrying haplogroups A and C based on its absence in the pre-Hispanic Mexican Maya populations and low frequencies in most Mexican and Guatemalan Maya populations, although this also may be due to drift. Maya and Ciboneys sharing haplotype H10 belonged to haplogroup C1 and haplotype H4 of haplogroup D, suggesting shared regional haplotypes. This may indicate a shared genetic ancestry, suggesting more regional interaction between populations in the circum-Caribbean region than previously demonstrated. Haplotype sharing between the pre-Hispanic Maya and the indigenous populations from Asia, the Aleutian Islands, and North, Central, and South America provides evidence for gene flow from the ancestral Amerindian population of the pre-Hispanic Maya to Central and South America.

A DNA Microarray-Based Assay to Detect Dual Infection with Two Dengue Virus Serotypes

Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV) serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples.