Mitochondrial phylogeography and molecular evolution of the rhodopsin visual pigment in troglobitic populations of Astyanax mexicanus (De Filippi, 1853).

Cave-adapted animals provide a unique opportunity to study the evolutionary mechanisms underlying phenotypic, metabolic, behavioral, and genetic evolution in response to cave environments. The Mexican tetra ( Astyanax mexicanus) is considered a unique model system as it shows both surface and cave-dwelling morphs. To date, at least 33 different cave populations have been identified, with phylogenetic studies suggesting an origin from at least two independent surface lineages, thereby providing a unique opportunity to study parallel evolution. In the present study, we carried out the most exhaustive phylogeographic study of A. mexicanus to date, including cave and surface localities, using two mitochondrial markers (cytochrome b (cyt b) and cytochrome c oxidase subunit I ( COI)) and nuclear rhodopsin visual pigment ( rho). Additionally, we inferred the molecular evolution of rho within the two contrasting environments (cave and surface) and across three geographic regions (Sierra de El Abra, Sierra de Guatemala, and Micos). In total, 267 individuals were sequenced for the two mitochondrial fragments and 268 individuals were sequenced for the rho visual pigment from 22 cave and 46 surface populations. Phylogeographic results based on the mitochondrial data supported the two-lineage hypothesis, except for the Pachón and Chica caves, whose introgression has been largely documented. The Sierra de El Abra region depicted the largest genetic diversity, followed by the Sierra de Guatemala region. Regarding the phylogeographic patterns of rho, we recovered exclusive haplogroups for the Sierra de El Abra (Haplogroup I) and Sierra de Guatemala regions (Haplogroup IV). Moreover, a 544 bp deletion in the rho gene was observed in the Escondido cave population from Sierra de Guatemala, reducing the protein from seven to three intramembrane domains. This change may produce a loss-of-function (LOF) but requires further investigation. Regarding nonsynonymous ( dN) and synonymous ( dS) substitution rates (omega values ω), our results revealed the prevailing influence of purifying selection upon the rho pigment for both cave and surface populations (ω<1), but relaxation at the El Abra region. Notably, in contrast to the other two regions, we observed an increase in the number of dN mutations for Sierra de El Abra. However, given that a LOF was exclusively identified in the Sierra de Guatemala region, we cannot dismiss the possibility of a pleiotropic effect on the Rho protein.

Optimization of Orally Bioavailable Antileishmanial 2,4,5-Trisubstituted Benzamides.

Leishmaniasis, a neglected tropical disease caused by Leishmania species parasites, annually affects over 1 million individuals worldwide. Treatment options for leishmaniasis are limited due to high cost, severe adverse effects, poor efficacy, difficulty of use, and emerging drug resistance to all approved therapies. We discovered 2,4,5-trisubstituted benzamides (4) that possess potent antileishmanial activity but poor aqueous solubility. Herein, we disclose our optimization of the physicochemical and metabolic properties of 2,4,5-trisubstituted benzamide that retains potency. Extensive structure-activity and structure-property relationship studies allowed selection of early leads with suitable potency, microsomal stability, and improved solubility for progression. Early lead 79 exhibited an 80% oral bioavailability and potently blocked proliferation of Leishmania in murine models. These benzamide early leads are suitable for development as orally available antileishmanial drugs.

Antioxidant activity of kafirin hydrolysates on UVB irradiated human keratinocyte cells and in silico identification.

BACKGROUND: Ultraviolet B (UVB) causes photoaging of the skin, the appearance of wrinkles, spots, and alteration of the skin barrier. The main cells in the most superficial layer of the skin are the keratinocytes; these cells play an important role in protecting this organ. OBJECTIVE: The present study aimed to investigate the antioxidant activity of the hydrolysates from kafirin to inhibit UVB-induced responses in human keratinocytes cells (HaCaT). METHODS: Kafirin hydrolysates were produced by enzymatic hydrolysis with alcalase. The activity of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), in the HaCaT cell line in the presence of UVB and the effects of the hydrolysates against the UVB-induced response were evaluated. Furthermore, the peptides that were generated by hydrolysis were identified in silico using the BIOPEP database. RESULTS: Two protein sequences were identified (α-kafirin and the precursor protein of α-kafirin), in the kafirin extract. A degree of hydrolysis of 18.8% was obtained by hydrolyzing the kafirin extract with alcalase. The kafirin hydrolysates avoided the decrease in endogenous antioxidant enzymes such as SOD, CAT, and GPx reducing the oxidative stress generated by UVB. Using the BIOPEP-UWM database, we found 102 peptide sequences, and it has shown that the peptides have a large amount of hydrophobic amino acids such as proline, alanine, and glutamine, and amino acids with high antioxidant capacity. CONCLUSION: These results suggest that the kafirin hydrolysates can be used as antioxidant agents to ameliorate UVB-induced skin keratinocytes cells' response in vitro, providing an alternative against UVB-induced photoaging.

Diagnosis of tuberous sclerosis complex in adulthood: A case report.

Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder. TSC consists of a wide variety of clinical manifestations, with neurological and dermatological symptoms being the most frequent. This study aims to describe the clinical characteristics and management of a pregnant patient incidentally diagnosed with TSC.

Species Delimitation of Scavenger Flies in the Valley of Mexico.

Identification of species involved in cadaveric decomposition, such as scavenger Diptera, is a fundamental step for the use of entomological evidence in court. Identification based on morphology is widely used in forensic cases; however, taxonomic knowledge of scavenger fauna is poor for many groups and for many countries, particularly Neotropical ones. A number of studies have documented the utility of a DNA barcoding strategy to assist in the identification of poorly known and diverse groups, particularly in cases involving immature states or fragmented organisms. To provide baseline knowledge of the diversity of scavenger Diptera in the Valley of Mexico, we generated a DNA barcode collection comprised of sequences of the cytochrome c oxidase subunit 1 (COI) gene for all families sampled at a nature reserve located in this region. We collected and identified specimens on the basis of morphology and a species delimitation analysis. Our analyses of 339 individuals delineated 42 species distributed across nine families of Diptera. The richest families were Calliphoridae (9 species), Sarcophagidae (7 species), and Phoridae (6 species). We found many of the species previously recorded for the Valley of Mexico, plus 18 new records for the region. Our study highlights the utility of DNA barcoding as a first-step strategy to assess species richness of poorly studied scavenger fly taxa.

A cytoskeletal protein complex is essential for division of intracellular amastigotes of Leishmania mexicana.

Previous studies in Leishmania mexicana have identified the cytoskeletal protein KHARON as being important for both flagellar trafficking of the glucose transporter GT1 and for successful cytokinesis and survival of infectious amastigote forms inside mammalian macrophages. KHARON is located in three distinct regions of the cytoskeleton: the base of the flagellum, the subpellicular microtubules, and the mitotic spindle. To deconvolve the different functions for KHARON, we have identified two partner proteins, KHAP1 and KHAP2, which associate with KHARON. KHAP1 is located only in the subpellicular microtubules, whereas KHAP2 is located at the subpellicular microtubules and the base of the flagellum. Both KHAP1 and KHAP2 null mutants are unable to execute cytokinesis but are able to traffic GT1 to the flagellum. These results confirm that KHARON assembles into distinct functional complexes and that the subpellicular complex is essential for cytokinesis and viability of disease-causing amastigotes but not for flagellar membrane trafficking.

Touching the Surface: Diverse Roles for the Flagellar Membrane in Kinetoplastid Parasites.

While flagella have been studied extensively as motility organelles, with a focus on internal structures such as the axoneme, more recent research has illuminated the roles of the flagellar surface in a variety of biological processes. Parasitic protists of the order Kinetoplastida, which include trypanosomes and Leishmania species, provide a paradigm for probing the role of flagella in host-microbe interactions and illustrate that this interface between the flagellar surface and the host is of paramount importance. An increasing body of knowledge indicates that the flagellar membrane serves a multitude of functions at this interface: attachment of parasites to tissues within insect vectors, close interactions with intracellular organelles of vertebrate cells, transactions between flagella from different parasites, junctions between the flagella and the parasite cell body, emergence of nanotubes and exosomes from the parasite directed to either host or microbial targets, immune evasion, and sensing of the extracellular milieu. Recent whole-organelle or genome-wide studies have begun to identify protein components of the flagellar surface that must mediate these diverse host-parasite interactions. The increasing corpus of knowledge on kinetoplastid flagella will likely prove illuminating for other flagellated or ciliated pathogens as well.

Glucose Transporters and Virulence in Leishmania mexicana.

Glucose transporters are important for viability and infectivity of the disease-causing amastigote stages of Leishmania mexicana The Δgt1-3 null mutant, in which the 3 clustered glucose transporter genes, GT1, GT2, and GT3, have been deleted, is strongly impaired in growth inside macrophages in vitro We have now demonstrated that this null mutant is also impaired in virulence in the BALB/c murine model of infection and forms lesions considerably more slowly than wild-type parasites. Previously, we established that amplification of the PIFTC3 gene, which encodes an intraflagellar transport protein, both facilitated and accompanied the isolation of the original Δgt1-3 null mutant generated in extracellular insect-stage promastigotes. We have now isolated Δgt1-3 null mutants without coamplification of PIFTC3 These amplicon-negative null mutants are further impaired in growth as promastigotes, compared to the previously described null mutants containing the PIFTC3 amplification. In contrast, the GT3 glucose transporter plays an especially important role in promoting amastigote viability. A line that expresses only the single glucose transporter GT3 grows as well inside macrophages and induces lesions in animals as robustly as do wild-type amastigotes, but lines expressing only the GT1 or GT2 transporters replicate poorly in macrophages. Strikingly, GT3 is restricted largely to the endoplasmic reticulum in intracellular amastigotes. This observation raises the possibility that GT3 may play an important role as an intracellular glucose transporter in the infectious stage of the parasite life cycle.IMPORTANCE Glucose transport plays important roles for in vitro growth of insect-stage promastigotes and especially for viability of intramacrophage mammalian host-stage amastigotes of Leishmania mexicana However, the roles of the three distinct glucose transporters, GT1, GT2, and GT3, in parasite viability inside macrophages and virulence in mice have not been fully explored. Parasite lines expressing GT1 or GT2 alone were strongly impaired in growth inside macrophages, but lines expressing GT3 alone infected macrophages and caused lesions in mice as robustly as wild-type parasites. Notably, GT3 localizes to the endoplasmic reticulum of intracellular amastigotes, suggesting a potential role for salvage of glucose from that organelle for viability of infectious amastigotes. This study establishes the unique role of GT3 for parasite survival inside host macrophages and for robust virulence in infected animals.

Dominant-negative STAT5B mutations cause growth hormone insensitivity with short stature and mild immune dysregulation.

Growth hormone (GH) insensitivity syndrome (GHIS) is a rare clinical condition in which production of insulin-like growth factor 1 is blunted and, consequently, postnatal growth impaired. Autosomal-recessive mutations in signal transducer and activator of transcription (STAT5B), the key signal transducer for GH, cause severe GHIS with additional characteristics of immune and, often fatal, pulmonary complications. Here we report dominant-negative, inactivating STAT5B germline mutations in patients with growth failure, eczema, and elevated IgE but without severe immune and pulmonary problems. These STAT5B missense mutants are robustly tyrosine phosphorylated upon stimulation, but are unable to nuclear localize, or fail to bind canonical STAT5B DNA response elements. Importantly, each variant retains the ability to dimerize with wild-type STAT5B, disrupting the normal transcriptional functions of wild-type STAT5B. We conclude that these STAT5B variants exert dominant-negative effects through distinct pathomechanisms, manifesting in milder clinical GHIS with general sparing of the immune system.

KHARON Is an Essential Cytoskeletal Protein Involved in the Trafficking of Flagellar Membrane Proteins and Cell Division in African Trypanosomes.

African trypanosomes and related kinetoplastid parasites selectively traffic specific membrane proteins to the flagellar membrane, but the mechanisms for this trafficking are poorly understood. We show here that KHARON, a protein originally identified in Leishmania parasites, interacts with a putative trypanosome calcium channel and is required for its targeting to the flagellar membrane. KHARON is located at the base of the flagellar axoneme, where it likely mediates targeting of flagellar membrane proteins, but is also on the subpellicular microtubules and the mitotic spindle. Hence, KHARON is probably a multifunctional protein that associates with several components of the trypanosome cytoskeleton. RNA interference-mediated knockdown of KHARON mRNA results in failure of the calcium channel to enter the flagellar membrane, detachment of the flagellum from the cell body, and disruption of mitotic spindles. Furthermore, knockdown of KHARON mRNA induces a lethal failure of cytokinesis in both bloodstream (mammalian host) and procyclic (insect vector) life cycle stages, and KHARON is thus critical for parasite viability.

Flagellar membrane proteins in kinetoplastid parasites.

All kinetoplastid parasites, including protozoa such as Leishmania species, Trypanosoma brucei, and Trypanosoma cruzi that cause devastating diseases in humans and animals, are flagellated throughout their life cycles. Although flagella were originally thought of primarily as motility organelles, flagellar functions in other critical processes, especially in sensing and signal transduction, have become more fully appreciated in the recent past. The flagellar membrane is a highly specialized subdomain of the surface membrane, and flagellar membrane proteins are likely to be critical components for all the biologically important roles of flagella. In this review, we summarize recent discoveries relevant to flagellar membrane proteins in these parasites, including the identification of such proteins, investigation of their biological functions, and mechanisms of selective trafficking to the flagellar membrane. Prospects for future investigations and current unsolved problems are highlighted.

Kharon1 null mutants of Leishmania mexicana are avirulent in mice and exhibit a cytokinesis defect within macrophages.

In a variety of eukaryotes, flagella play important roles both in motility and as sensory organelles that monitor the extracellular environment. In the parasitic protozoan Leishmania mexicana, one glucose transporter isoform, LmxGT1, is targeted selectively to the flagellar membrane where it appears to play a role in glucose sensing. Trafficking of LmxGT1 to the flagellar membrane is dependent upon interaction with the KHARON1 protein that is located at the base of the flagellar axoneme. Remarkably, while Δkharon1 null mutants are viable as insect stage promastigotes, they are unable to survive as amastigotes inside host macrophages. Although Δkharon1 promastigotes enter macrophages and transform into amastigotes, these intracellular parasites are unable to execute cytokinesis and form multinucleate cells before dying. Notably, extracellular axenic amastigotes of Δkharon1 mutants replicate and divide normally, indicating a defect in the mutants that is only exhibited in the intra-macrophage environment. Although the flagella of Δkharon1 amastigotes adhere to the phagolysomal membrane of host macrophages, the morphology of the mutant flagella is often distorted. Additionally, these null mutants are completely avirulent following injection into BALB/c mice, underscoring the critical role of the KHARON1 protein for viability of intracellular amastigotes and disease in the animal model of leishmaniasis.

Molecular identification and characterization of an essential pyruvate transporter from Trypanosoma brucei.

Pyruvate export is an essential physiological process for the bloodstream form of Trypanosoma brucei as the parasite would otherwise accumulate this end product of glucose metabolism to toxic levels. In the studies reported here, genetic complementation in Saccharomyces cerevisiae has been employed to identify a gene (TbPT0) that encodes this vital pyruvate transporter from T. brucei. Expression of TbPT0 in S. cerevisiae reveals that TbPT0 is a high affinity pyruvate transporter. TbPT0 belongs to a clustered multigene family consisting of five members, whose expression is up-regulated in the bloodstream form. Interestingly, TbPT family permeases are related to polytopic proteins from plants but not to characterized monocarboxylate transporters from mammals. Remarkably, inhibition of the TbPT gene family expression in bloodstream parasites by RNAi is lethal, confirming the physiological relevance of these transporters. The discovery of TbPT0 reveals for the first time the identity of the essential pyruvate transporter and provides a potential drug target against the mammalian life cycle stage of T. brucei.

Expansion of human hematopoietic cells from umbilical cord blood using roller bottles in CO2 and CO2-free atmosphere.

In this work, we evaluated the expansion of human hematopoietic stem cells from umbilical cord blood in roller bottles. The Iscove's modified Dulbecco's medium, the Stem Pro 34-SFM medium, and the L-15 Leibovitz's medium for cultures in CO(2)-free atmosphere were assessed. At day 5 of culture, total colony forming unit expansions of 14.44 ± 3.74, 11.20 ± 6.37, and 17.25 ± 3.65-folds were attained, respectively. The expansion reached using L-15 medium in roller bottles was around 10 times higher than that achieved in the static control cultures. To our knowledge, this is the first report of cultures in CO(2)-free atmosphere to expand cord blood human hematopoietic stem cells and it opens a new branch of possibilities for culturing and clinical applications.

Purine restriction induces pronounced translational upregulation of the NT1 adenosine/pyrimidine nucleoside transporter in Leishmania major.

Leishmania and other parasitic protozoa are unable to synthesize purines de novo and are reliant upon purine nucleoside and nucleobase transporters to import preformed purines from their hosts. To study the roles of the four purine permeases NT1-NT4 in Leishmania major, null mutants in each transporter gene were prepared and the effect of each gene deletion on purine uptake was monitored. Deletion of the NT3 purine nucleobase transporter gene or both NT3 and the NT2 nucleoside transporter gene resulted in pronounced upregulation of adenosine and uridine uptake mediated by the NT1 permease and also induced up to a 200-fold enhancement in the level of the NT1 protein but not mRNA. A similar level of upregulation of NT1 was achieved in wild-type promastigotes that were transferred to medium deficient in purines. Pulse labelling and treatment of cells with the translation inhibitor cycloheximide revealed that control of NT1 expression occurs primarily at the level of translation and not protein turnover. These observations imply the existence of a translational control mechanism that enhances the ability of Leishmania parasites to import essential purines when they are present at limiting concentrations.

Arsenic transport by zebrafish aquaglyceroporins.

BACKGROUND: Arsenic is one of the most ubiquitous toxins and endangers the health of tens of millions of humans worldwide. It is a mainly a water-borne contaminant. Inorganic trivalent arsenic (AsIII) is one of the major species that exists environmentally. The transport of AsIII has been studied in microbes, plants and mammals. Members of the aquaglyceroporin family have been shown to actively conduct AsIII and its organic metabolite, monomethylarsenite (MAsIII). However, the transport of AsIII and MAsIII in in any fish species has not been characterized. RESULTS: In this study, five members of the aquaglyceroporin family from zebrafish (Danio rerio) were cloned, and their ability to transport water, glycerol, and trivalent arsenicals (AsIII and MAsIII) and antimonite (SbIII) was investigated. Genes for at least seven aquaglyceroporins have been annotated in the zebrafish genome project. Here, five genes which are close homologues to human AQP3, AQP9 and AQP10 were cloned from a zebrafish cDNA preparation. These genes were named aqp3, aqp3l, aqp9a, aqp9b and aqp10 according to their similarities to the corresponding human AQPs. Expression of aqp9a, aqp9b, aqp3, aqp3l and aqp10 in multiple zebrafish organs were examined by RT-PCR. Our results demonstrated that these aquaglyceroporins exhibited different tissue expression. They are all detected in more than one tissue. The ability of these five aquaglyceroporins to transport water, glycerol and the metalloids arsenic and antimony was examined following expression in oocytes from Xenopus leavis. Each of these channels showed substantial glycerol transport at equivalent rates. These aquaglyceroporins also facilitate uptake of inorganic AsIII, MAsIII and SbIII. Arsenic accumulation in fish larvae and in different tissues from adult zebrafish was studied following short-term arsenic exposure. The results showed that liver is the major organ of arsenic accumulation; other tissues such as gill, eye, heart, intestine muscle and skin also exhibited significant ability to accumulate arsenic. The zebrafish larvae also accumulate considerable amounts of arsenic. CONCLUSION: This is the first molecular identification of fish arsenite transport systems and we propose that the extensive expression of the fish aquaglyceroporins and their ability to transport metalloids suggests that aquaglyceroporins are the major pathways for arsenic accumulation in a variety of zebrafish tissues. Uptake is one important step of arsenic metabolism. Our results will contribute to a new understanding of aquatic arsenic metabolism and will support the use of zebrafish as a new model system to study arsenic associated human diseases.

An acid-activated nucleobase transporter from Leishmania major.

Parasitic protozoa are unable to synthesize purines de novo and must import preformed purine nucleobases or nucleosides from their hosts. Leishmania major expresses two purine nucleobase transporters, LmaNT3 and LmaNT4. Previous studies revealed that at neutral pH, LmaNT3 is a broad specificity, high affinity nucleobase transporter, whereas LmaNT4 mediates the uptake of only adenine. Because LmaNT4 is required for optimal viability of the amastigote stage of the parasite that lives within acidified phagolysomal vesicles of mammalian macrophages, the function of this permease was examined under acidic pH conditions. At acidic pH, LmaNT4 acquires the ability to transport adenine, hypoxanthine, guanine, and xanthine with Km values in the micromolar range, indicating that this transporter is activated at low pH. Thus, LmaNT4 is an acid-activated purine nucleobase transporter that functions optimally under the physiological conditions the parasite is exposed to in the macrophage phagolysosome. In contrast, LmaNT3 functions optimally at neutral pH. Two-electrode voltage clamp experiments performed on LmaNT3 and LmaNT4 expressed in Xenopus oocytes revealed substrate-induced inward directed currents at acidic pH, and application of substrates induced acidification of the oocyte cytosol. These observations imply that LmaNT3 and LmaNT4 are nucleobase/proton symporters.

Two novel nucleobase/pentamidine transporters from Trypanosoma brucei.

African trypanosomes are unable to synthesize purines de novo and must salvage preformed purine nucleosides and nucleobases from their hosts. The Trypanosoma brucei genome project has identified 12 members of the equilibrative nucleoside transporter family, most of which have been characterized previously as nucleoside and/or nucleobase transporters. Here the 11th member of this family, TbNT11.1, has been functionally expressed in null mutants of Leishmania that are deficient in purine nucleoside or nucleobase uptake and identified as a high-affinity purine nucleobase transporter. Expression of TbNT11.1 in Xenopus oocytes revealed that it is also a transporter for the diamidine drug pentamidine that is the principal drug employed to treat early stage human African trypanosomiasis and may thus contribute to the uptake of this therapeutically important compound. In addition, characterization of the 12th member of the family, TbNT12.1, reveals that it is an adenine/pentamidine transporter.

Molecular genetic analysis of purine nucleobase transport in Leishmania major.

Leishmania major and all other parasitic protozoa are unable to synthesize purines de novo and are therefore reliant upon uptake of preformed purines from their hosts via nucleobase and nucleoside transporters. L. major expresses two nucleobase permeases, NT3 that is a high affinity transporter for purine nucleobases and NT4 that is a low affinity transporter for adenine. nt3((-/-)) null mutant promastigotes were unable to replicate in medium containing 10 microM hypoxanthine, guanine, or xanthine and replicated slowly in 10 microM adenine due to residual low affinity uptake of that purine. The NT3 transporter mediated the uptake of the anti-leishmanial drug allopurinol, and the nt3((-/-)) mutants were resistant to killing by this drug. Expression of the NT3 permease was profoundly downregulated at the protein but not the mRNA level in stationary phase compared with logarithmic phase promastigotes. The nt4((-/-)) null mutant was quantitatively impaired in survival within murine bone marrow-derived macrophages. Extensive efforts to generate an nt3((-/-))/nt4((-/-)) dual null mutant were not successful, suggesting that one of the two nucleobase permeases must be retained for robust growth of the parasite. The phenotypes of these null mutants underscore the importance of purine nucleobase transporters in the Leishmania life cycle and pharmacology.

Estradiol valerate and alcohol intake: dose-response assessments.

BACKGROUND: An injection of estradiol valerate (EV) provides estradiol for a prolonged period. Recent research indicates that a single 2.0 mg injection of EV modifies a female rat's appetite for alcoholic beverages. This research extends the initial research by assessing 8 doses of EV (from .001 to 2.0 mg/female rat), as well assessing the effects of 2.0 mg EV in females with ovariectomies. RESULTS: With the administration of EV, there was a dose-related loss of bodyweight reaching the maximum loss, when it occurred, at about 4 days after injections. Subsequently, rats returned to gaining weight regularly. Of the doses tested, only the 2.0 mg dose produced a consistent increase in intake of ethanol during the time previous research indicated that the rats would show enhanced intakes. There was, however, a dose-related trend for smaller doses to enhance intakes. Rats with ovariectomies showed a similar pattern of effects, to intact rats, with the 2 mg dose. After extensive histories of intake of alcohol, both placebo and EV-treated females had estradiol levels below the average measured in females without a history of alcohol-intake. CONCLUSION: The data support the conclusion that pharmacological doses of estradiol can produce enduring changes that are manifest as an enhanced appetite for alcoholic beverages. The effect can occur among females without ovaries.