Graciela M. Panzetta-Dutari

Two Different Routes of Colonization of Aedes aegypti in Argentina from Neighboring Countries

ABSTRACT Aedes aegypti L. (Diptera, Culicidae) is the main vector of dengue and yellow fever. In Argentina, the species was apparently eradicated approximately in 1964; by 1986, it was reintroduced. To identify different gene pools in geographical populations of the species and to ascertain the possible routes of colonization, we analyzed the diversity of mitochondrial DNA haplotypes in 572 specimens from Argentina and neighboring countries. We found that the restriction fragment length polymorphism-polymerase chain reaction screening of a large DNA fragment including the A+T-rich region was the best strategy to reconstruct the colonization pattern of Ae. aegypti in Argentina. Twenty haplotypes were recognized; levels of genetic similarity varied among populations from different geographical locations. The haplotype network constructed on the basis of genetic distances showed three well differentiated groups. Two of them exhibited a well defined spatial distribution and populations in these groups presented an isolation-by-distance pattern. The persistence of relictual populations after the last eradication campaigns would explain the high levels of haplotype diversity and the presence of exclusive haplotypes in urban centers from northwestern Argentina. Eastern Argentine populations showed one prevalent haplotype, also predominant in Brazil and Paraguay. Our results highlight the need for efficient surveys and control campaigns, given the strong effect of land trade on genetic exchange among mosquito populations from Argentina and neighboring countries where dengue is endemic.

Chlorpyrifos modifies the expression of genes involved in human placental function.

The effects of organophosphate pesticides on human placenta remain poorly investigated although an increased risk of pregnancy alterations has been reported in women chronically exposed to these pesticides. Here, we have addressed whether chlorpyrifos (CPF) modifies the expression of genes relevant for placental function. Human placental JEG-3 cells were exposed to increasing CPF concentrations up to 100 μM for 24 and 48 h and cell viability, mRNA, protein and hormone levels were analyzed. Quantitative RT-PCR assays revealed that CPF increased the expression of ABCG2, GCM1 and, even more significantly, βhCG mRNAs in conditions where cell viability and morphology were not compromised. In addition, βhCG protein synthesis and secretion were time-dependently augmented. Present results may reflect a CPF nocive effect on placenta cells or a placental-defense mechanism to preserve its function. These novel CPF trophoblast target genes should be considered in future studies of pregnancy outcomes associated with in vivo exposures.

Restriction fragment-length polymorphism of the mtDNA A+T-Rich region as a genetic marker in Aedes Aegypti (Diptera: Culicidae)

Abstract The usefulness of the control region of mtDNA as a tool for the study of population structure in the mosquito Aedes aegypti (L.) was analyzed. Population samples were taken from several geographic areas of Argentina; one additional sample from the city of San Juan, Puerto Rico, was included for comparison. In individual mosquitoes, the A+T-rich and the COII-COIII regions were amplified by polymerase chain reaction using universal insect primers. Restriction fragment-length polymorphisms were investigated from amplification products. In the A+T region, three amplified fragments of different total length were obtained (of ≈2,500, 2,300, and 2,100 bp). The enzymes Ssp I, Dra I, Pac I, and Apo I produced polymorphic patterns. Including total fragment length as a variable, eight different haplotypes were resolved for Argentinian populations, some presenting a restricted geographic distribution. The coding COII-COIII region revealed very low polymorphism. Although 11 restriction enzymes were employed to analyze this region, only two different haplotypes were found, one of them shared by populations as distant as Buenos Aires and San Juan. We demonstrated that total length and restriction fragment-length polymorphisms within the noncoding A+T-rich region, but not the coding COII-COIII fragment, are informative to discriminate Aedes aegypti populations. Mean FST value (0.48, P < 0.001) indicates an important degree of differentiation among populations. Absence of an isolation by distance pattern was demonstrated.

Expression and Localization of StarD7 in Trophoblast Cells

The StAR-related lipid transfer (START) domain is defined as a motif of around 200 amino acids implicated in lipid/sterol binding. In a previous study, we identified the StarD7 transcript encoding one of the 15 family members with START domain present in the human genome. This transcript was found to be overexpressed in choriocarcinoma JEG-3 cells. In addition, we demonstrated that the recombinant StarD7 protein forms stable Gibbs and Langmuir monolayers at the air-buffer interface, showing marked surface activity and interaction with phospholipid monolayers, mainly with phosphatidylserine, cholesterol and phosphatidylglycerol. This study was undertaken to evaluate the expression and localization of StarD7 protein in trophoblastic samples. Here, we show for the first time the presence of StarD7 protein in human trophoblast cells. Western blot assays revealed a unique specific 34 kDa protein in JEG-3 cell line, choriocarcinoma tissue, complete hydatidiform mole, early and normal term placenta. Immunohistochemical data from early and normal term placentas and complete hydatidiform moles showed that this protein is abundant in the syncytiotrophoblasts, mainly at the apical side of the syncytium, with a weak and focal reaction in the cytotrophoblast cells. Furthermore, an increased StarD7 mRNA and protein expression, as well as a change in its sub-cellular localization was observed in in vitro differentiating cytotrophoblast isolated from normal term placenta. Taken together, these findings support and allow future studies to explore the possibility that StarD7 protein mediates transplacental lipid transport and/or is involved in syncytialization.

Krüppel-Like Factor 6 Expression Changes during Trophoblast Syncytialization and Transactivates ßhCG and PSG Placental Genes

Background Krüppel-like factor-6 (KLF6) is a widely expressed member of the Sp1/KLF family of transcriptional regulators involved in differentiation, cell cycle control and proliferation in several cell systems. Even though the highest expression level of KLF6 has been detected in human and mice placenta, its function in trophoblast physiology is still unknown. Methodology/Principal Findings Herein, we explored KLF6 expression and sub-cellular distribution in human trophoblast cells differentiating into the syncytial pathway, and its role in the regulation of genes associated with placental development and pregnancy maintenance. Confocal immunofluorescence microscopy demonstrated that KLF6 is expressed throughout human cytotrophoblast differentiation showing no evident modifications in its nuclear and cytoplasmic localization pattern. KLF6 transcript and protein peaked early during the syncytialization process as determined by qRT-PCR and western blot assays. Overexpression of KLF6 in trophoblast-derived JEG-3 cells showed a preferential nuclear signal correlating with enhanced expression of human β-chorionic gonadotropin (βhCG) and pregnancy-specific glycoprotein (PSG) genes. Moreover, KLF6 transactivated βhCG5, PSG5 and PSG3 gene promoters. Deletion of KLF6 Zn-finger DNA binding domain or mutation of the consensus KLF6 binding site abolished transactivation of the PSG5 promoter. Conclusions/Significance Results are consistent with KLF6 playing a role as transcriptional regulator of relevant genes for placental differentiation and physiology such as βhCG and PSG, in agreement with an early and transient increase of KLF6 expression during trophoblast syncytialization.

The Role of Pregnancy‐Specific Glycoprotein 1a (PSG1a) in Regulating the Innate and Adaptive Immune Response

Among several explanations for the acceptance of the fetus, the one that suggests that the maternal immune system is suppressed or modified has been the subject of many studies. Thus, it has been proposed that the cells of innate immune system might be able to distinguish the pregnant from the non‐pregnant state producing a signal, the so‐called signal P. We have previously proposed that pregnancy‐specific glycoprotein 1a (PSG1a), a representative member of the main glycoprotein family secreted by placental trophoblast, may modulate the activation of antigen‐presenting cells promoting the T‐cell shift of the maternal cell immunity toward a less harmful phenotype. In this review, we summarize current knowledge concerning the contribution of pregnancy‐specific glycoprotein 1a (PSG1a) to modulate the maternal innate and adaptive immune response in order to assure a successful pregnancy.

Absence of the 9-bp Deletion of Mitochondrial DNA in Pre-Hispanic Inhabitants of Argentina

We investigated the incidence of the Region V mitochondrial DNA 9-base-pair (bp) deletion from human remains recovered from several archaeological sites and contexts throughout Argentina. Of the 34 samples analyzed, 24 yielded DNA extractions that gave clear amplification results. All of the individuals carried two repeats of the 9 bp, one of which has been shown to be deleted in some individuals of Asian origin and defines mitochondrial lineage B. Although most of the modern Amerindian groups in the region exhibit the deletion in high frequencies, the absence of the 9-bp deletion among ancient populations of South America seems to be the rule rather than the exception, as was reported by several studies involving extinct populations. The evidence gathered until now suggests that the earliest settlers of this region of South America did not carry mitochondrial lineage B.

A new Comamonas testosteroni steroid-inducible gene: Cloning and sequence analysis

Comamonas testosteroni can grow on a variety of steroid compounds as the sole carbon and energy source. In a previous study, we cloned and sequenced the testosterone-inducible betahsd gene from C. testosteroni (Genti-Raimondi, S., Tolmasky, M., Patrito, L., Flury, A. and Actis, L., Molecular cloning and expression of the beta-hydroxysteroid dehydrogenase gene from Pseudomonas testosteroni. Gene, 1991, 105, 43-49.). Herein we report the cloning and characterization of another steroid-inducible gene (stdC), located 2400 bp upstream of betahsd. Nucleotide sequencing of a region encompassing the stdC gene revealed an open reading frame 546 bp long including the stop codon TGA with significant similarity to the orf4, orf1 and orf4 of unknown function described in the polyhydroxyalkanoic acid (PHA) cluster of Chromatium vinosum, Rhizobium meliloti and Thiocystis violacea, respectively. The aminoacid sequence deduced from the nucleotide sequence predicts a putative protein of 181 amino acids with a molecular weight of 20715 Da. Northern blot experiments indicate that the stdC gene was transcribed as a monocistronic mRNA with an apparent molecular size of 670 nt. The stdC transcript was abundant in C. testosteroni cells grown with different steroid carbon sources harvested in the exponential phase and was found to be under catabolite repression.

The lipid transfer protein StarD7: structure, function, and regulation

The steroidogenic acute regulatory (StAR) protein-related lipid transfer (START) domain proteins constitute a family of evolutionarily conserved and widely expressed proteins that have been implicated in lipid transport, metabolism, and signaling. The 15 well-characterized mammalian START domain-containing proteins are grouped into six subfamilies. The START domain containing 7 mRNA encodes StarD7, a member of the StarD2/phosphatidylcholine transfer protein (PCTP) subfamily, which was first identified as a gene overexpressed in a choriocarcinoma cell line. Recent studies show that the StarD7 protein facilitates the delivery of phosphatidylcholine to the mitochondria. This review summarizes the latest advances in StarD7 research, focusing on the structural and biochemical features, protein-lipid interactions, and mechanisms that regulate StarD7 expression. The implications of the role of StarD7 in cell proliferation, migration, and differentiation are also discussed.

Nucleotide sequence of a pregnancy-specific β1 glycoprotein gene family member

The pregnancy-specific β1 glycoprotein (PSG) genes encode a group of heterogeneous proteins produced in large amounts by the human syncytiotrophoblast. Their expression seems to be regulated at the transcriptional level during normal pregnancy. In the present work, we isolated from a human placental library a 17 kb genomic fragment corresponding to a member of the PSG multigene family. DNA sequence analysis of 1190 nucleotides upstream of the translational start and of the first intron, revealed the presence of several putative regulatory sequences. In a transient chloramphenicol acetyltransferase expression assay, 5′ flanking sequences within 123 nucleotides upstream to the first major transcription initiation site, functioned as a strong promoter in COS-7 cells. Meanwhile, sequences 5′ further upstream had the ability to abolish this promoter activity. The sequence analyzed did not contain any obvious TATA-like boxes or G+C-rich regions, suggesting the existence of unique promoter elements implicated in transcription initiation and regulation of this PSG gene family member.