Trypanosoma rangeli protein tyrosine phosphatase is associated with the parasite's flagellum

Protein tyrosine phosphatases (PTPs) play an essential role in the regulation of cell differentiation in pathogenic trypanosomatids. In this study, we describe a PTP expressed by the non-pathogenic protozoan Trypanosoma rangeli (TrPTP2). The gene for this PTP is orthologous to the T. brucei TbPTP1 and Trypanosoma cruzi (TcPTP2) genes. Cloning and expression of the TrPTP2 and TcPTP2 proteins allowed anti-PTP2 monoclonal antibodies to be generated in BALB/c mice. When expressed by T. rangeli epimastigotes and trypomastigotes, native TrPTP2 is detected as a ~65 kDa protein associated with the parasite's flagellum. Given that the flagellum is an important structure for cell differentiation in trypanosomatids, the presence of a protein responsible for tyrosine dephosphorylation in the T. rangeli flagellum could represent an interesting mechanism of regulation in this structure.

Searching for the role of protein phosphatases in eukaryotic microorganisms

Preference for specific protein substrates together with differential sensitivity to activators and inhibitors has allowed classification of serine/threonine protein phosphatases (PPs) into four major types designated types 1, 2A, 2B and 2C (PP1, PP2A, PP2B and PP2C, respectively). Comparison of sequences within their catalytic domains has indicated that PP1, PP2A and PP2B are members of the same gene family named PPP. On the other hand, the type 2C enzyme does not share sequence homology with the PPP members and thus represents another gene family, known as PPM. In this report we briefly summarize some of our studies about the role of serine/threonine phosphatases in growth and differentiation of three different eukaryotic models: Blastocladiella emersonii, Neurospora crassa and Dictyostelium discoideum. Our observations suggest that PP2C is the major phosphatase responsible for dephosphorylation of amidotransferase, an enzyme that controls cell wall synthesis during Blastocladiella emersonii zoospore germination. We also report the existence of a novel acid- and thermo-stable protein purified from Neurospora crassa mycelia, which specifically inhibits the PP1 activity of this fungus and mammals. Finally, we comment on our recent results demonstrating that Dictyostelium discoideum expresses a gene that codes for PP1, although this activity has never been demonstrated biochemically in this organism.

The eukaryotic Pso2/Snm1/Artemis proteins and their function as genomic and cellular caretakers

DNA double-strand breaks (DSBs) represent a major threat to the genomic stability of eukaryotic cells. DNA repair mechanisms such as non-homologous end joining (NHEJ) are responsible for the maintenance of eukaryotic genomes. Dysfunction of one or more of the many protein complexes that function in NHEJ can lead to sensitivity to DNA damaging agents, apoptosis, genomic instability, and severe combined immunodeficiency. One protein, Pso2p, was shown to participate in the repair of DSBs induced by DNA inter-strand cross-linking (ICL) agents such as cisplatin, nitrogen mustard or photo-activated bi-functional psoralens. The molecular function of Pso2p in DNA repair is unknown, but yeast and mammalian cell line mutants for PSO2 show the same cellular responses as strains with defects in NHEJ, e.g., sensitivity to ICLs and apoptosis. The Pso2p human homologue Artemis participates in V(D)J recombination. Mutations in Artemis induce a variety of immunological deficiencies, a predisposition to lymphomas, and an increase in chromosomal aberrations. In order to better understand the role of Pso2p in the repair of DSBs generated as repair intermediates of ICLs, an in silico approach was used to characterize the catalytic domain of Pso2p, which led to identification of novel Pso2p homologues in other organisms. Moreover, we found the catalytic core of Pso2p fused to different domains. In plants, a specific ATP-dependent DNA ligase I contains the catalytic core of Pso2p, constituting a new DNA ligase family, which was named LIG6. The possible functions of Pso2p/Artemis/Lig6p in NHEJ and V(D)J recombination and in other cellular metabolic reactions are discussed.

Bradysia hygida (Diptera, Sciaridae) presents two eukaryotic Elongation Factor 1A gene homologues: partial characterization of the eukaryotic Elongation Factor 1A-F1 gene

Elongation factor 1A is a highly conserved protein that participates in translation. We report the occurrence of two genes homologous to the eukaryotic Elongation Factor 1A in Bradysia hygida and describe the partial cloning and characterization of the B. hygida eukaryotic Elongation Factor 1A-F1 (BheEF1A-F1) gene. The pattern of BheEF1A-F1 expression in the salivary gland at the end of the fourth larval instar was investigated using real-time PCR. The results showed that BheEF1A-F1 expression levels are relatively constant at the time when rapid changes in protein synthesis occur in this tissue. In situ hybridization experiments coupled to Southern blot analyses showed that the BheEF1A-F1 gene is located at position 3d of the A chromosome and a second gene homologous to eEF1A is located at position 6a of the X chromosome. Southern blot analyses showed that both the BheEF1A-F1 gene and the second gene homologous to eEF1A constitute non-amplified genes. The present results contribute to the molecular characterization of a sciarid eEF1A gene.

BC047440 antisense eukaryotic expression vectors inhibited HepG2 cell proliferation and suppressed xenograft tumorigenicity

The biological functions of the BC047440 gene highly expressed by hepatocellular carcinoma (HCC) are unknown. The objective of this study was to reconstruct antisense eukaryotic expression vectors of the gene for inhibiting HepG2 cell proliferation and suppressing their xenograft tumorigenicity. The full-length BC047440 cDNA was cloned from human primary HCC by RT-PCR. BC047440 gene fragments were ligated with pMD18-T simple vectors and subsequent pcDNA3.1(+) plasmids to construct the recombinant antisense eukaryotic vector pcDNA3.1(+)BC047440AS. The endogenous BC047440 mRNA abundance in target gene-transfected, vector-transfected and naive HepG2 cells was semiquantitatively analyzed by RT-PCR and cell proliferation was measured by the MTT assay. Cell cycle distribution and apoptosis were profiled by flow cytometry. The in vivo xenograft experiment was performed on nude mice to examine the effects of antisense vector on tumorigenicity. BC047440 cDNA fragments were reversely inserted into pcDNA3.1(+) plasmids. The antisense vector significantly reduced the endogenous BC047440 mRNA abundance by 41% in HepG2 cells and inhibited their proliferation in vitro (P < 0.01). More cells were arrested by the antisense vector at the G1 phase in an apoptosis-independent manner (P = 0.014). Additionally, transfection with pcDNA3.1(+)BC047440AS significantly reduced the xenograft tumorigenicity in nude mice. As a novel cell cycle regulator associated with HCC, the BC047440 gene was involved in cell proliferation in vitro and xenograft tumorigenicity in vivo through apoptosis-independent mechanisms.

THE POWER OF THE SMALL: THE EXAMPLE OF Paracoccidioides brasiliensis CONIDIA

SUMMARYResearch on Paracoccidioides brasiliensis has centered in the yeast cell probably because of the lack of distinctive features in the mycelium. In 1942 and for the first time, lateral conidia were noticed in the fungus' hyphae. Later on, Brazilian, Venezuelan and Argentinean researchers described "aleurias" when the fungus was grown in natural substrates. In 1970 authors became interested in the conidia and were able to obtain them in large numbers and treat them as individual units. Their shape and size were defined and the presence of all the elements of a competent eukaryotic cell were demonstrated. Conidia exhibited thermal dimorphism and, additionally, when given intranasally to BALB/c male mice, they converted into yeasts in the lungs and produce progressive pulmonary lesions with further dissemination to other organs. Studies on the phagocyte-conidia interaction were revealing and showed that these versatile structures allow a better understanding of the host- P. brasiliensisinteractions.

Scanning electron microscopy of the antennal sensilla in female Culicoides paraensis (Diptera: Ceratopogonidae)

We studied by sanning electron microscopy the number, types, structure and distribution of the antennal sensilla of the medical important ceratopogonid Culicoides paraensis (Goeldi). There are about 174 sense organs on the antenmal flagellum which are classified as sensilla chaetica; sharp-tipped and blunt-tipped (type I and II) sensilla trichodea; sensilla basiconica; sensilla coeloconica; sensilla ampullacea and styloconic-type sensilla. The role of antennal sensory organs are discussed regarding the host preference of the biting midges.

On the morphology of Laevapex vazi n. sp. from Brazil (Mollusca: Pulmonata: Basommatophora: Ancylidae)

A description of Laevapex vazi n. sp. based on 8 specimens collectec in Ourinhos, state of São Paulo, is presented. Shell thin, diaphanous, with a light brown periostracum and moderately elliptical opening. Apex not pointed, smooth, situated on the right posterior region of the shell, inclined to the right often reaching the edge of the shell or extending beyond it. Concentric lines clearly visible; radial striation not visible or when perceptible very thin, here and there. Ratios: shell width/shell lenght = 0,60 - 0,67 (mean = 0,63); shell height/shell length = 0,50 - 0,61 (mean = 0,55); shell height/shell width = 0,33 - 0,40 (mean = 0,35). Body of normal ancylid type; mantle pigmentation concentrated on the left side; three muscles are seen: a round posterior one on the left side, an elliptical muscle on the right anterior side and an almost almond-shaped one on the left anterior side. Tentacles with a medium core of black pigment. Pseudobranch two-lobed and folded, the dorsal lobe smaller than the vetral one. Ovotestis with 20 unbranched diverticula, around a short collecting canal. Ovispermiduct with an enlargement with several round outpocketings constituting the seminal vesicle. Carrefour as a round sac. Albumen gland almost cylindrical with several acinous diverticula. Elongated nidamental gland continous with the galndular wall of the uterus; uterus flattened and thin-walled. Spermathecal body almost rounded. Pear-shaped prostate without diverticula. Penial complex without flagellum but with well-developed ultra-penis and penis. Jaw horseshoe shaped. Radular forma 20.1.20; raquidian tooth quadricuspid, asymmetrical. The genus Laevapex Walker, 1903 is recorded for the first time in Brazil. It is easily distinguished from South American Gundlachia Pfeiffer, 1849 by its penial complex. Laevapex vazi is dedicated to Dr. Jorge Faria Vaz, from SUCEN-SP, who have been sent to me the specimens.

Freeze-fracture study of Trichomonas vaginalis

The freeze-fracture technique was used to analyse the organization of the plasma membrane, as well as membranes of cytoplasmic organelles, of the pathogenic protozoan Trichomonas vaginalis. Rosettes formed by 4 to 14 intramembranous particles were seen on the fracture faces of the membrane lining the anterior flagella as well as in fracture faces of the plasma membrane enclosing the anterior region of the protozoan and in cytoplasmic organelles. Special organization of the membrane particles were also seen in the region of association of the recurrent flagellum to the cell body.

Immunogold labeling and cerium cytochemistry of the enzyme ecto-5'-nucleotidase in promastigote forms of Leishmania species

We have applied both enzyme cytochemistry and immunological labeling techniques to characterize the enzyme 5'-nucleotidase (5'-Nase), at the ultrastructural level, in promastigote forms of four Leishmania species: Leishmania amazonensis, Leishmania mexicana, Leishmania donovani and Leishmania chagasi. The cerium phosphate staining was localized at the surface of the cell body, the flagellum and the flagellar pocket membranes of all the parasites studied. The immunogold labelling technique confirmed these results. In this report we localized 5'-Nase in L. chagasi and L. amazonensis which have been implicated respectively in visceral and cutaneous forms of leishmaniasis. In addition, we confirmed the localization of this phosphomonoesterase in the other two species studied. The superior quality of the images, obtained with both methodologies, confirms that these parasites possess mechanisms capable of hydrolyzing nucleotide monophosphates, and that the expression of 5'-Nase is associated with the outer surface of the plasma membrane.

Gundlachia dutrae: n. sp. from northwest Brazil (Mollusca: Basommatophora: Ancylidae)

A new species of Gundlachia, Gundlachia dutrae is described from northwest Brazil. It is distinguishable from other congenerie species by characteristics of the shell, radula and internal organs. Shell relatively high. Aperture near-circular; periostracum dark brown without periostracal hairs. Apex slightly inclined to the right, projected but not hooked, with an apical depression surrounted by a sculpture of well-marked irregular punctations. Shell surface with prominent radial sculpture. No septate specimens were observed. Ratios (n= 59): shell width/shell lenght = 0,66- 0,79 (mean 0,73); shell height/shell length = 0,32- 0,45 ( mean 0,37); shell height/shell width = 0,43- 0,63 (mean 0,51). Body of normal ancylid type; mantle pigmentation dark brown or black, concentrated along the mantle collar. The dorsal surface of the right anterior muscle is elongated and medially constricted. The left anterior and the posterior muscles are almost elliptical. Adhesive area is V-shaped. Pseudobranch unpigmented bearing a very small and thin dorsal lobe. Ovotestis with more than 25 unbranched diverticula. Ovispermiduct with seminal vesicle rather developed. Elongated nidamental gland continous with the glandular wall of the uterus. Nidamental gland appendix ending into a bulbous swelling Spermathecal body almost rounded. Well-developed prostate with five long diverticula. Ejaculatory complex with long glandular flagellum, without a penis or true ultra-penis. "Penis sheath" developed. "Ultra-penis" projected as a tube inside the lumem of prepuce, with a slit between "ultra-penis" and "penis sheath". Rachidian tooth tetracuspid, with two median cusps assymmetrical and aculeated. Lateral teeth tricuspid, with a reduced endocon and a prominent mesocon. A well marked gap occurs between meso and ectocon. Marginal teeth similar to lateral ones. Jaw T-shaped, with about 28 dorsal plates.

Aspects of immunity for the AMA-1 family of molecules in humans and non-human primates malarias

The apical membrane antigen (AMA-1) family of malaria merozoite proteins is characterised by a high degree of inter-species conservation. Evidence that the protein (PK66/AMA-1) from the simian parasite Plasmodium knowlesi was protective in rhesus monkeys suggested that the 83kDa P. falciparum equivalent (PF83/AMA-1) should be investigated for protective effects in humans. Here we briefly review pertinent comparative data, and describe the use of an eukaryotic full length recombinant PF83/AMA-1 molecule to develop a sensitive ELISA for the determination of serological responses in endemic populations. The assay has revealed surprisingly high levels of humoral response to this quantitatively minor antigen. We also show that PK66/AMA-1 inhibitory mAb's are active against merozoites subsequent to release from schizont-infected red cells, further implicating AMA-1 molecules in red cell invasion.

Occurrence of "Nuages" and "Lamellae Anulata" during spermatogenesis in Dermatobia hominis (Diptera: Cuterebridae)

Various types of "nuages" and "lamellae anulata" can be found during Dermatobia hominis spermatogenesis. In spermatogonia, the "nuages" occur as granules juxtaposed to the cytoplasmic face of the nuclear envelope or as cytoplasmic granules similar to glycogen granules. In spermatocytes, in addition to the "nuages", dense spherical bodies of approximately 1.0 µm in diameter are also observed. In the spermatids the "nuages" can be of the following types: perinuclear granules, spherical granules with diameters varying in length from 0.5 to 1.0 µm, granules similar to glycogen granules, granules with variable diameters which accumulate at the flagellum base forming the centriole adjunct, or remain in the cytoplasm. "Nuages" can also be observed in these cellular types as dense masses, without a definite outline and are common to animal germinal cells in general. The "lamellae anulata" on the other hand, are observed only in spermatocytes I and in early spermatids, being always immersed in electron-dense material of indefinite outline. In spermatids, the "lamellae anulata" are close to the nuclear envelope suggesting, in spite of opposing opinions, that these cells are envolved in the synthesis and transport of material from the nucleus to the cytoplasm.

A short review on the morphology of Trypanosoma cruzi: from 1909 to 1999

The morphology of Trypanosoma cruzi is reviewed since the initial description of Giemsa-stained preparations by Carlos Chagas until the most recent micrographs obtained with freeze-fracture techniques. Special emphasis is given to structures such as the cell surface, the flagellum, the kinetoplast, the reservosomes and the endocytic pathway, and the acidocalcisomes.

Microsporidia and acquired immunodeficiency syndrome

Microsporidia is a common term that has been used to refer to a group of eukaryotic, obligate intracellular protozoan parasites belonging to the phylum Microspora. They are important agricultural parasites, contaminating commercial insects; they are also important by infecting laboratory rodents, rabbits and primates. Ever since the early cases found by Magarino Torres, who reported the presence of Encephalitozoon in a patient suffering of a meningoencephalomyelitis, some human pathology caused by microsporidia has been described. However, only after the acquired immunodeficiency syndrome outbreak have these organisms appeared as significant etiological agents in different pathologies. Even so, they remain underestimated. In the present article, the importance of microsporidia for the human pathology in immunocompromised host has been stressed.