Nucleus behavior during the closed mitosis of Tritrichomonas foetus

We present observations on the fine structure and the division process of the nucleus in the protist Tritrichomonas foetus, parasite of the urogenital tract of cattle. The nucleus was followed by immunofluorescence and electron microscopy during interphase and mitosis. Conventional karyotyping coupled to image processing and bright field Panotic staining were used to follow nucleus modifications, chromosome number and condensation pattern along the whole cell cycle. Confocal laser scanning microscopy (CLSM) using DNA fluorescent probes, followed by image processing in the SURF-Driver program, produced three-dimensional reconstruction data of the mitotic nucleus under each phase of the division process. Immunocytochemistry in thin-sections revealed the chromosome spatial arrangement after bromodeoxyuridine incorporation and immunogold labeling using anti-DNA monoclonal antibodies. Our results indicate that: (1) the nucleus assumes different size and shapes along mitosis: it appears oval in interphase, becoming lobed or concave in prophase, then undergoing torsion and constriction, displaying an 'S' shape (metaphase). Next, it becomes elongated and it is finally separated in two nuclei at the transition of anaphase to telophase; (2) T. foetus nucleus harbors five chromosomes; (3) chromosomes become condensed in a pre-mitotic phase; (4) the nucleolus persists during the mitosis.     

The mitotic spindle and associated membranes in the closed mitosis of trichomonads

In the present work, we followed the several phases of Tritrichomonas foetus and Trichomonas vaginalis cell cycles using immunofluorescence, serial thin sections, three-dimensional (3D) reconstruction, and transmission electron microscopy. In motile trichomonad cells or in pseudocyst forms, the nuclear envelope persists throughout mitosis, and the spindle is extranuclear. We found three types of spindle microtubules: pole-to-nucleus microtubules which are attached to the nuclear envelope, pole-to-pole microtubules forming a cylindrical, cytoplasmic groove on the nuclear compartment in pseudocysts of T. foetus cells, and pole-to-cytosol microtubules which extend freely into the cytoplasm. We demonstrated that: (1) in T. foetus, the spindle is assembled from an MTOC located at the base of the costa, underneath one of the basal bodies; (2) the spindle presents an unusual arc shape during the initial phases of mitosis in motile trophozoites; (3) the spindle microtubules are glutamylated, but not acetylated; (4) several membranes similar to those of the endoplasmic reticulum follow the spindle microtubules; (5) finger-like projections extend from the nucleus towards the cell poles in pseudocysts and multinucleated cells; and (6) vesicles formed in between the two nuclear membranes are seen in the course of mitosis in both trophozoite and pseudocyst forms.     

Dynamics of T cells and TCR excision circles differ after treatment of acute and chronic HIV infection

We quantified T cell proliferation and thymic function in primary HIV infection (PHI; n = 19) and chronic HIV infection (CHI; n = 14) by measuring Ki67 staining and TCR excision circle (TREC) number. After antiretroviral therapy of PHI there is a profound decrease in the number and percentage of Ki67(+) T cells (<6% Ki67(+)) with no significant increase in TREC per million cells and a transient increase in TREC per milliliter. In contrast, after antiretroviral therapy of CHI there is a reduction in the percentage but little change in the total number of Ki67(+)CD4(+) T cells associated with increases in both TREC per million cells and TREC per milliliter. Using a mathematical model that accounts for proliferation, death, and redistribution of T cells, we find that redistribution is consistent with the TREC changes observed during treatment of PHI and that an increase in thymic output is needed to explain the increase in TREC during treatment of CHI. Consideration of TREC per milliliter shows that changes in proliferation alone cannot explain the changes in TREC. In addition, although increased proliferation of memory cells in HIV infection has been established, we find no difference in TREC per million CD45RA(-) "memory" T cells between healthy and infected individuals (p = 0.154 for CD4(+); p = 0.383 for CD8(+)). Finally, although the number of TREC per million cells is always much lower in memory T cells than in naive T cells, in the setting of HIV infection, given that memory cells make up a larger proportion of total T cells, we find that 50% of TREC per milliliter in CD4(+) T cells is harbored in the CD45RA(-) "memory" subset of our infected subjects.     

Biochemical and functional characterization of recombinant Rhodnius prolixus platelet aggregation inhibitor 1 as a novel lipocalin with high affinity for adenosine diphosphate and other adenine nucleotides

The Rhodnius prolixus aggregation inhibitor 1 (RPAI-1) is a novel blood-sucking salivary molecule that binds to ADP and attenuates platelet aggregation. In this report, we determine the binding constants and specificity of RPAI-1 for adenine nucleotides and its functional significance. By the Hummel-Dreyer method of equilibrium gel filtration, we show that RPAI-1 binds ADP with a K(0.5) of 48.6 plus minus 12.2 nM. RPAI-1 also displays high-affinity binding to ATP, AMP, Ado, AP4A, and alpha,beta Met ADP; however, RPAI-1 does not bind to inosine, guanosine, uridine, or cytidine. Binding is not modified by EDTA, indicating that Ado structure but not phosphate groups or Ca(2+) is necessary for binding. By computer simulation, we show that RPAI-1 is more effective in scavenging low but not high concentrations of ADP, in contrast to R. prolixus apyrase. RPAI-1 inhibits in vitro the ADP-dependent platelet-rich plasma aggregation by collagen (COLL), TRAP, PAF, and A23187 but did not block platelet aggregation by ristocetin or phorbol myristate acetate (PMA) and only slightly attenuated that by convulxin. RPAI-1 prolongs the closure time as assessed with PFA-100, when COLL-Epi but not COLL-ADP cartridges are employed. RPAI-1 also affects platelet-mediated hemostasis time and COLL-induced thrombus formation at high shear as assessed with the Clot Signature Analyzer. We conclude that RPAI-1 exerts an antiplatelet effect due to scavenging of low concentrations of ADP in vitro and in vivo. RPAI-1 is the first lipocalin described so far with unique specificity for adenine nucleotides.     

The new bilaterally pedicled V-Y advancement flap for face reconstruction

A new bilaterally pedicled V-Y advancement flap based on two subcutaneous pedicles that vascularize the skin island through subdermal plexus lateral bridges is described for face reconstruction. It differs from traditional V-Y advancement flaps in that it does not rely on the classic subcutaneous "vertical" pedicle that is sectioned from top to bottom to improve advancement of the skin island. This technique had predictable results for 12 years in 425 consecutive patients, with infection occurring in 2.8 percent of the cases and complete necrosis in less than 1 percent of the flaps.     

Genotoxic evaluation of the organophosphorous pesticide temephos

The chemical compound temephos (0,0,0',0'-tetrametyl-0,0'-thiodi-p-phenylene phosphorothioate) is an organophosphorous pesticide that has been used in Brazil since 1967 in control campaigns against the mosquito Aedes aegypti, the vector of dengue and yellow fever. We used single cell gel electrophoresis (SCGE), SOS/umu and Ames/Salmonella assays to test the toxicity and mutagenicity of temephos. Temephos was genotoxic in the SCGE assay, inducing severe DNA lesions (type IV lesions) at doses above 1.34 micro M. It was mutagenic, but not toxic, in the SOS/umu assay to Escherichia coli strain PQ37, but not to PQ35, at concentrations above 1.33 micro M, particularly when the S9 mixture was not used in the assay. Temephos was not mutagenic in the Ames assay with S. typhimurium strains TA97, TA98, TA100 and TA102, both with and without metabolic activation. However, temephos at concentrations above 3.33 micro M was mutagenic to TA98NR, YG7104 and YG7108, both with and without metabolic activation. In conclusion, temephos was genotoxic and mutagenic in all the three tests used, and in two of them at concentrations similar to those routinely used to combat Aedes aegypti.     

Cytometric,morphologic and enzymatic characterisation of haemocytes in Anodonta cygnea

The haemocytes in bivalve mussels are involved in many processes such as lesion repair, shell repair, elimination of small particles and toxic substances. In Anodonta cygnea there are two categories of haemolymph cells, the granulocytes and hyalinocytes. Two groups of cells were identified by flow cytometry and morphological studies: one with larger size and granularity representing 75%, and another group of cells (25%) which were approximately half the size. The cytochemical reactions showed peroxidase activity in the larger cells and a weak prophenoloxidase activity in the smaller cells. These characteristics suggest that the most common haemocytes are granulocytes and hyalinocytes are less common. Enzymatic studies showed clear activities of few enzymes in different compartments of the mantle. Both haemocytes presented significant variations for alpha-manosidase and beta-glucurosidase activities depending on the acid or alkaline pH. Almost all were sensitive to the pH changes, mainly the beta-galactosidase in the haemolymph plasma. On the contrary, the same enzymatic analysis in the extrapallial elements showed more stabilised activities. The simulation of acidic and alkaline condition with the observation of significant morphological and enzymatic activity changes, allow us to speculate some functional role, mainly in the haemolymph elements. The granulocytes may be speculated to have intense involvement in the digestion of small residues with the formation of calcareous stores while the hyalinocytes are more responsible for the elimination of soluble cytotoxic compounds.     

Electrocyte (Na(+),K(+))ATPase inhibition induced by zinc is reverted by dithiothreitol

The Mg(2+)-dependent (Na(+),K(+))ATPase maintains several cellular processes and is essential for cell excitability. In view of the importance of the enzyme activity, the interaction and binding affinities to substrates and metal ions have been studied. We determined the effect of Zinc ion (Zn(2+)) on the (Na(+),K(+))ATPase activity present in both conducting (non-innervated) and post-synaptic (innervated) membranes of electrocyte from Electrophorus electricus (L.). Zn(2+) is involved in many biological functions and is present in pre-synaptic nerve terminals. This metal, which has affinity for thiol groups, acted as a potent competitive inhibitor of (Na(+),K(+))ATPase of both membrane fractions, which were obtained by differential centrifugation of the E. electricus main electric organ homogenate. We tried to recover the enzyme activity using dithiothreitol, a reducing agent. Kinetic analysis showed that dithiothreitol acted as a non-essential non-competitive activator of (Na(+),K(+))ATPase from both membrane fractions and was able to revert the Zn(2+) inhibition at mM concentrations. In the presence of dithiothreitol, this metal behaved as a competitive inhibitor of (Na(+),K(+))ATPase in the non-innervated membrane fractions and presented a non-competitive inhibition of (Na(+),K(+))ATPase in innervated membrane fractions. This difference may be attributed to formation of a Zn-dithiothreitol complex, as well as the involvement of other binding sites for both agents. The consequences of the enzyme inhibition by Zn(2+) may be considered in regard to its neurotoxic effects.     

Gap-phase regeneration in a semideciduous mesophytic forest, south–eastern Brazil

The study was carried out in ten natural canopy gaps in the SantaGenebra County Reserve (22°4945S, 47°0633W)in the county of Campinas, São Paulo State, Brazil. The size and canopyopenness of the gaps were studied using hemispherical photographs. Thevegetation survey included all shrubs and trees with height 0.50m in the gaps interiors and all the individuals with PBH(perimeterat breast height) 15 cm in a 3 m surroundingborder of the gaps. The similarity among the gaps and among their surroundingareas was assessed by the Jaccard similarity index and by cluster analyzes. Thegap size varied from 20.09 to 468 m², with apredominance of small gaps. The families with the greatest species richness inthe gaps were Rutaceae, Rubiaceae and Euphorbiaceae. The species with thegreatest number of individuals in the gaps were Coffeaarabica L., Hybanthus atropurpureus (St. Hil.)Taub. and Actinostemon klotschii (Muell. Arg.) Pax, allwidely distributed in the understorey. Shade-tolerant species (late secondaryspecies) predominated in function of the predominance of small gaps. The highnumber of species found in the gaps reflects the importance of thesedisturbances in the maintenance of species diversity in the studied forest.     

Pseudocysts in trichomonads--new insights

Tritrichomonas foetus and Trichomonas vaginalis, parasitic protists of the urogenital tract, display a trophozoite and a pseudocyst stage. The ultrastructure of the trophozoite was compared with the pseudocyst form. The latter appears under unfavorable environmental conditions when the flagella are internalized, and a true cell wall is not formed. Although some authors consider this form as a degenerate stage, the cell behaves as a resistant form. Pseudocysts were found in natural culture conditions and also under induction by hydroxyurea or cycles of cooling and warming cultures. They were studied by light and scanning and transmission electron microscopy, using immunofluorescence and videomicroscopy. This report presents evidence that the trichomonad pseudocysts appear under stress conditions and that they are competent to divide. Pseudocysts differ from trophozoites in that: (1) the flagella are located in endocytic vacuoles and remain beating; (2) the axostyle and the costa are not depolymerized but present a curved shape; (3) the axostyle does not exhibit staining with anti-tubulin antibodies when the mitotic spindle is observed; (4) the mitotic process occurs within pseudocysts but differs from that described for trophozoites; (5) a nuclear canal is formed connecting the two spindle poles; and (6) the process is reversible if the cells are transferred to fresh medium.