Stage-specific proteinases of Leishmania mexicana mexicana promastigotes

Four distinct bands of cysteine proteinase activity were detected when stationary-phase populations of Leishmania mexicana mexicana were subjected to gelatin-SDS-PAGE. The highest mobility band contained at least three isoforms separable by mono Q anion exchange chromatography. These high mobility activities were distinct from all the major amastigote enzymes. Stationary-phase promastigote populations also contained two acid-activable precursor forms of the promastigote-specific band. It is suggested that these promastigote-specific activities occur in the infective metacyclic stage of the parasite and may have a role in parasite survival upon inoculation into a mammal.     

Differential phagocytosis of Leishmania mexicana promastigotes and amastigotes by monocyte‐derived dendritic cells

Leishmania species are dimorphic protozoan parasites that live and replicate in the gut of sand flies as promastigotes or in mammalian hosts as amastigotes. Different immune cells, including DCs, and receptors differ in their involvement in phagocytosis of promastigotes and amastigotes and in recognition of different Leishmania species. In the case of L. mexicana, differences in phagocytosis of promastigotes and amastigotes by DCs and participation of C‐type lectin receptors (CLRs) have not been established. In the present study, flow cytometry and confocal microscopy were used to investigate the phagocytosis by monocyte‐derived dendritic cells (moDCs) of L. mexicana promastigotes and amastigotes in the presence or absence of immune serum during various periods of time. Blocking antibodies against mannose receptors and DC‐SIGN were used to explore the participation of these receptors in the phagocytosis of L. mexicana by moDC. The major differences in interactions of L. mexicana promastigotes and amastigotes with moDC were found to occur within the first 3 hr, during which phagocytosis of promastigotes predominated as compared with opsonization of promastigotes and amastigotes. However, after 6 hr of incubation, opsonized promastigotes were preferentially phagocytosed as compared with unopsonized promastigotes and amastigotes and after 24 hr of incubation there were no differences in the phagocytosis of promastigotes and amastigotes. Finally, after 3 hr incubation, DC‐SIGN was involved in the phagocytosis of promastigotes, but not of amastigotes.     

Chromatographic separation of DNA dependent RNA polymerases and molecular properties of RNA polymerase II from a Leishmania Spp

Multiple forms of DNA-dependent RNA polymerases have been isolated and characterized from Leishmania strain UR6 promastigotes. RNA polymerases from this organism fail to resolve into multiple forms by conventional chromatography on DEAE-Sephadex A25, but could be separated by a modification of the method using CM-Sephadex C25. The CM-Sephadex bound enzyme is resistant toamanitin even up to a concentration of 250g/ml. The activity which flows through CM-Sephadex further resolves into two forms upon chromatography on DEAE-Sephadex A25. These forms are sensitive to -amanitin to different extent. Enzyme activity in peak I is 50% inhibited by 3g/ml and in peak II by 50g/ml of the drug respectively. The enzyme in peak I has been further purified by heparin agarose and fast performance liquid chromatography (FPLC) on MonoQ. The enzyme has Stoke's radius of 70å, a sedimentation coefficient of 17.6S and an f/fo of 1.35. Analysis of ammonium sulfate and met n peak I, relative activities with Mn+2 versus Mg+2 and template specificities gave results similar to those reported for other type II RNA polymerases in eukaryotes. The MonoQ purified enzyme resolves into 16 polypeptides on denaturing polyacrylamide gel and densitometric analysis suggests that 9 major bands are present in the stoichiometry expected of RNA polymerase subunits having molecular weights: 154000; 104000; 77000; 64000; 52000; 48000; 46000; 45000 and 39000 respectively.     

Analysis of the cysteine proteinases of Leishmania mexicana and Trypanosoma cruzi using specific antisera

Abstract Antisera raised against papain and cysteine proteinases (CPs) purified from Leishmania mexicana and Trypanosoma cruzi have been used to study the proteins in the two parasites. The antisera against the major CP of T. cruzi (cruzipain) not only cross-reacted with known CPs of L. mexicana but also detected stage-specific molecules that may represent previously unrecognised CPs. The binding of the same abtisera to extracts of different life cycle stages of T. cruzi suggested that the stages possess different isoforms of cruzipain. The lack of cross-reactivity of anti-papain antiserum against cruzipain suggests that the major immunogenic epitopes of these CPs are different, whereas the detection of the major CPs of L. mexicana with both heterologous antisera shows that the parasite's enzymes share epitopes with the other CPs.     

Polymorphic microsatellite repeats are not conserved between Leishmania donovani and Leishmania major

Thirteen sets of polymerase chain reaction (PCR) primers were designed to amplify microsatellite loci identified in the genome sequence of Leishmania major. Polymorphisms were detected in L. major at all loci. In Leishmania donovani only two of these loci were informative for classification purposes with this data set. The PCR products of all loci from one L. donovani strain were sequenced and it was found that the number of repeats in the microsatellite loci were either substantially reduced with respect to L. major or absent altogether. Consequently it is unlikely to be possible to use the genome sequence of L. major to identify polymorphic microsatellite loci in other Leishmania species.     

Effect of the Antiphagocytic Agent Cytochalasin B on Macrophage Invasion by Leishmania mexicana Promastigotes and Trypanosoma cruzi Epimastigotes*

Unstimulated mouse peritoneal exudate cells were cultured on coverslips in Medium 199 containing 10% (v/v) calf serum. Cytochalasin B dissolved in dimethyl sulphoxide (DMSO) and diluted in Medium 199 was added to cultures to give final concentrations of 1, 5 and 10 μg/ml. Equal numbers of Leishmania mexicana promastigotes, Trypanosoma cruzi epimastigotes and sheep red cells were added to 24 hr cultures incubated at 37 C. The macrophage monolayers were fixed and stained at various time intervals. L. mexicana promastigotes and sheep red blood cells were found to attach to macrophages in the presence of the drug but did not enter the cells. When the medium containing the Cytochalasin was replaced with normal medium phagocytosis of the adherent parasites and red cells followed rapidly. T. cruzi epimastigotes were found inside macrophages in both drug-treated and drug-free cultures although the number found to be intracellular in the latter was significantly greater. This study suggests that L. mexicana promastigotes enter macrophages by being phagocytosed, whereas T. cruzi epimastigotes can actively penetrate these cells.     

Assessing the importance of four sandfly species (Diptera: Psychodidae) as vectors of Leishmania mexicana in Campeche,Mexico

Localized cutaneous leishmaniasis represents a public health problem in many areas of Mexico, especially in the Yucatan Peninsula. An understanding of vector ecology and bionomics is of great importance in evaluations of the transmission dynamics of Leishmania parasites. A field study was conducted in the county of Calakmul, state of Campeche, during the period from November 2006 to March 2007. Phlebotomine sandfly vectors were sampled using Centers for Disease Control light traps, baited Disney traps and Shannon traps. A total of 3374 specimens were captured in the two villages of Once de Mayo (93.8%) and Arroyo Negro (6.1%). In Once de Mayo, the most abundant species were Psathyromyia shannoni, Lutzomyia cruciata, Bichromomyia olmeca olmeca and Psychodopygus panamensis (all: Diptera: Psychodidae). The Shannon trap was by far the most efficient method of collection. The infection rate, as determined by Leishmania mexicana‐specific polymerase chain reaction, was 0.3% in Once de Mayo and infected sandflies included Psy. panamensis, B. o. olmeca and Psa. shannoni. There were significant differences in human biting rates across sandfly species and month of sampling. Ecological niche modelling analyses showed an overall overlap of 39.1% for the four species in the whole state of Campeche. In addition, the finding of nine vector–reservoir pairs indicates a potential interaction. The roles of the various sandfly vectors in Calakmul are discussed.     

Seroprevalence of Trypanosoma cruzi and Leishmania mexicana in Free‐Ranging Howler Monkeys in Southeastern Mexico

Natural infection of wild mammals by protozoa parasites is quite common in nature. For Neotropical Primates different infections of parasites that are etiological agent of disease in human have been identified. In particular, infections by Trypanosoma cruzi and Leishmania sp., have been reported for some New World primate species, but there are no reports of infection with these parasites in any primate species in Mexico. A serological study was conducted on two howler monkey species (Alouatta pigra and A. palliata) from the Mexican states of Campeche and Tabasco. A total of 55 serum samples (20 samples from A. pigra, 20 samples from A. palliata, and 15 samples from semifree ranging A. palliata of Los Tuxtlas, Veracruz as negative controls) were analyzed for the detection of immunoglobulin G antibodies against T. cruzi and Leishmania mexicana through enzyme linked immunosorbent assay test, indirect immunofluorescence assay and Western blot. The overall prevalence of antibodies in howler monkeys was 17.5% for T. cruzi and 30% for L. mexicana. Our results also indicate that A. pigra is more susceptible to develop leishmaniasis than A. palliata. Finally, the finding of positive serology in these primates should be given serious consideration for public health, given the potential role of these primate species as wild reservoirs for these diseases and the increasing contact of monkeys with human populations due to habitat loss and fragmentation. Am. J. Primatol. 75:161‐169, 2013. © 2012 Wiley Periodicals, Inc.     

Multiple functions of DNA polymerases

The primary role of DNA polymerases is to accurately and efficiently replicate the genome in order to ensure the maintenance of the genetic information and its faithful transmission through generations. This is not a simple task considering the size of the genome and its constant exposure to endogenous and environmental DNA damaging agents. Thus, a number of DNA repair pathways operate in cells to protect the integrity of the genome. In addition to their role in replication, DNA polymerases play a central role in most of these pathways. Given the multitude and the complexity of DNA transactions that depend on DNA polymerase activity, it is not surprising that cells in all organisms contain multiple highly specialized DNA polymerases, the majority of which have only recently been discovered. Five DNA polymerases are now recognized in Escherichia coli, 8 in Saccharomyces cerevisiae, and at least 15 in humans. While polymerases in bacteria, yeast and mammalian cells have been extensively studied much less is known about their counterparts in plants. For example, the plant model organism Arabidopsis thaliana is thought to contain 12 DNA polymerases, whose functions are mostly unknown. Here we review the properties and functions of DNA polymerases focusing on yeast and mammalian cells but paying special attention to the plant enzymes and the special circumstances of replication and repair in plant cells.     

Effect of the anti-microtubule compound tubulozole on Leishmania protozoan parasites in vitro

Abstract For the cis and trans stereoisomers of the synthetic anti-microtubule compound tubulozole, at micromolar concentrations, tubulozole-C is cytotoxic to mammalian cells whereas tubulozole-T is not. The effect of tubulozoles on the parasitic protozoan Leishmania was tested. For the promastigote stage of L. mexicana amazonensis , both isomers inhibited parasite growth. For the amastigote stage of L. mexicana amazonensis and L. major , within murine J774 macrophage line as host cells in vitro, tubulozole-T reduced the infective index. Despite the observation of macrophage cytotoxicity of tubulozole-T, this compound may be a potentially useful and novel anti-leishmanial drug.     

DNA聚合酶在维持基因组稳定性中的多重功能及其相关疾病

遗传物质的稳定传递是生命繁衍的根本。基因组DNA的精确复制和分配是遗传物质传递的基础,也是细胞周期两大最核心的生物学事件。DNA聚合酶作为催化合成DNA双链的酶,是复制过程中最重要的因子之一。尽管对这类酶的研究已有将近60年的历史,但依然是生命科学基础研究的前沿之一。真核生物中已知的DNA聚合酶有十几种,它们不仅参与正常基因组DNA合成过程,也参与DNA损伤情况下多种修复过程。如此众多的具有不同特性的DNA聚合酶在细胞内是如何分工与合作的,在正常细胞传代与环境胁迫等情况下维护基因组稳定性中的关键作用及其分子机制又是什么。更有意思的是,最近的肿瘤细胞比较基因组数据表明,多种DNA聚合酶基因突变与某些肿瘤和遗传疾病相关,从而为这些疾病致病机理研究与诊治提供了新的思路和方法。对上述DNA聚合酶相关核心问题的最新研究进展进行了综述。     

Nitric oxide production by Peromyscus yucatanicus (Rodentia) infected with Leishmania (Leishmania) mexicana

Peromyscus yucatanicus (Rodentia: Cricetidae) is a primary reservoir of Leishmania (Leishmania) mexicana (Kinetoplastida: Trypanosomatidae). Nitric oxide (NO) generally plays a crucial role in the containment and elimination of Leishmania. The aim of this study was to determine the amount of NO produced by P. yucatanicus infected with L. (L.) mexicana. Subclinical and clinical infections were established in P. yucatanicus through inoculation with 1 x 10² and 2.5 x 10⁶ promastigotes, respectively. Peritoneal macrophages were cultured alone or co-cultured with lymphocytes with or without soluble Leishmania antigen. The level of NO production was determined using the Griess reaction. The amount of NO produced was significantly higher (p ≤ 0.0001) in co-cultured macrophages and lymphocytes than in macrophages cultured alone. No differences in NO production were found between P. yucatanicus with subclinical L. (L.) mexicana infections and animals with clinical infections. These results support the hypothesis that the immunological mechanisms of NO production in P. yucatanicus are similar to those described in mouse models of leishmaniasis and, despite NO production, P. yucatanicus is unable to clear the parasite infection.     

Amplification efficiency of thermostable DNA polymerases

The amplification efficiencies of several polymerase chain reaction (PCR) enzymes were compared using real-time quantitative PCR with SYBR Green I detection. Amplification data collected during the exponential phase of PCR are highly reproducible, and PCR enzyme performance comparisons based upon efficiency measurements are considerably more accurate than those based on endpoint analysis. DNA polymerase efficiencies were determined under identical conditions using five different amplicon templates that varied in length or percentage GC content. Pfu- and Taq-based formulations showed similar efficiencies when amplifying shorter targets (<900 bp) with 45 to 56% GC content. However, when amplicon length or GC content was increased, Pfu formulations with dUTPase exhibited significantly higher efficiencies than Taq, Pfu, and other archaeal DNA polymerases. We discuss the implications of these results.     

Synergy between DNA polymerases increases polymerase chain reaction inhibitor tolerance in forensic DNA analysis

The success rate of diagnostic polymerase chain reaction (PCR) analysis is lowered by inhibitory substances present in the samples. Recently, we showed that tolerance to PCR inhibitors in crime scene saliva stains can be improved by replacing the standard DNA polymerase AmpliTaq Gold with alternative DNA polymerase-buffer systems (Hedman et al., BioTechniques 47 (2009) 951-958). Here we show that blending inhibitor-resistant DNA polymerase-buffer systems further increases the success rate of PCR for various types of real crime scene samples showing inhibition. For 34 of 42 “inhibited” crime scene stains, the DNA profile quality was significantly improved using a DNA polymerase blend of ExTaq Hot Start and PicoMaxx High Fidelity compared with AmpliTaq Gold. The significance of the results was confirmed by analysis of variance. The blend performed as well as, or better than, the alternative DNA polymerases used separately for all tested sample types. When used separately, the performance of the DNA polymerases varied depending on the nature of the sample. The superiority of the blend is discussed in terms of complementary effects and synergy between the DNA polymerase-buffer systems.     

Distribution and roles of X-family DNA polymerases in eukaryotes

Four types of DNA polymerase (Pol beta, Pol lambda, Pol mu and TdT) have been identified in eukaryotes as members of the polymerase X-family. Only vertebrates have all four types of enzyme. Plants and fungi have one or two X-family polymerases, while protostomes, such as fruit flies and nematodes, do not appear to have any. It is possible that the well-known metabolic pathways in which these enzymes are involved are restricted to the vertebrate world. The distribution of the DNA polymerases involved in DNA repair across the various biological kingdoms differs from that of the DNA polymerases involved in chromosomal DNA replication. In this review, we focus on the interesting pattern of distribution of the X-family enzymes across biological kingdoms and speculate on their roles.     

Characterization of marine X-family DNA polymerases and comparative analysis of base excision repair proteins

While mammalian DNA polymerase β (Pol β), which is a member of the Pol X family, play important roles in base excision repair (BER) that efficiently removes DNA base lesions arising from both endogenous and exogenous agents, this protein has been found only a subset of animals. To understand natural evolution of this enzyme, we isolated and characterized Pol β from jellyfish Aurelia sp.1. (AsPol β). Despite of phylogenetic distance and environmental differences between jellyfish and mammals, in vitro assays showed biochemical characteristics of AsPol β were very similar to those of a mammalian counterpart. We also searched two other homologs of mammalian genes that were involved in short patch (sp) BER in the nucleotide sequence database, and found that both of these homologs were encoded in the genomes of a lineage from Cnidarians through mammals and Arthropods. This study suggests that a DNA repair mechanism resembling mammalian sp-BER may be largely limited to a subset of animals. On the basis of our findings and previous reports, we discuss possible evolutional model of Pol β and the other members of the Pol X family.     

Structural diversity of the Y-family DNA polymerases

The Y-family translesion DNA polymerases enable cells to tolerate many forms of DNA damage, yet these enzymes have the potential to create genetic mutations at high rates. Although this polymerase family was defined less than a decade ago, more than 90 structures have already been determined so far. These structures show that the individual family members bypass damage and replicate DNA with either error-free or mutagenic outcomes, depending on the polymerase, the lesion and the sequence context. Here, these structures are reviewed and implications for polymerase function are discussed.     

Regulatory volume decrease in Leishmania mexicana: effect of anti-microtubule drugs

The trypanosomatid cytoskeleton is responsible for the parasite''s shape and it is modulated throughout the different stages of the parasite''s life cycle. When parasites are exposed to media with reduced osmolarity, they initially swell, but subsequently undergo compensatory shrinking referred to as regulatory volume decrease (RVD). We studied the effects of anti-microtubule (Mt) drugs on the proliferation of Leishmania mexicana promastigotes and their capacity to undergo RVD. All of the drugs tested exerted antiproliferative effects of varying magnitudes [ansamitocin P3 (AP3)> trifluoperazine > taxol > rhizoxin > chlorpromazine]. No direct relationship was found between antiproliferative drug treatment and RVD. Similarly, Mt stability was not affected by drug treatment. Ansamitocin P3, which is effective at nanomolar concentrations, blocked amastigote-promastigote differentiation and was the only drug that impeded RVD, as measured by light dispersion. AP3 induced 2 kinetoplasts (Kt) 1 nucleus cells that had numerous flagella-associated Kts throughout the cell. These results suggest that the dramatic morphological changes induced by AP3 alter the spatial organisation and directionality of the Mts that are necessary for the parasite''s hypotonic stress-induced shape change, as well as its recovery.     

The hyperthermophilic euryarchaeota Pyrococcus abyssi likely requires the two DNA polymerases D and B for DNA replication

DNA polymerases carry out DNA synthesis during DNA replication, DNA recombination and DNA repair. During the past five years, the number of DNA polymerases in both eukarya and bacteria has increased to at least 19 and multiple biological roles have been assigned to many DNA polymerases. Archaea, the third domain of life, on the other hand, have only a subset of the eukaryotic-like DNA polymerases. The diversity among the archaeal DNA polymerases poses the intriguing question of their functional tasks. Here, we focus on the two identified DNA polymerases, the family B DNA polymerase B (PabpolB) and the family D DNA polymerase D (PabpolD) from the hyperthermophilic euryarchaeota Pyrococcus abyssi. Our data can be summarized as follows: (i) both Pabpols are DNA polymerizing enzymes exclusively; (ii) their DNA binding properties as tested in gel shift competition assays indicated that PabpolD has a preference for a primed template; (iii) PabPolD is a primer-directed DNA polymerase independently of the primer composition whereas PabpolB behaves as an exclusively DNA primer-directed DNA polymerase; (iv) PabPCNA is required for PabpolD to perform efficient DNA synthesis but not PabpolB; (v) PabpolD, but not PabpolB, contains strand displacement activity; (vii) in the presence of PabPCNA, however, both Pabpols D and B show strand displacement activity; and (viii) we show that the direct interaction between PabpolD and PabPCNA is DNA-dependent. Our data imply that PabPolD might play an important role in DNA replication likely together with PabpolB, suggesting that archaea require two DNA polymerases at the replication fork.